Single-Cell, Multiplexed Protein Detection of Rare Tumor Cells Based on a Beads-on-Barcode Antibody Microarray.

PubMed

Yang, Liu; Wang, Zhihua; Deng, Yuliang; Li, Yan; Wei, Wei; Shi, Qihui

2016-11-15

Circulating tumor cells (CTCs) shed from tumor sites and represent the molecular characteristics of the tumor. Besides genetic and transcriptional characterization, it is important to profile a panel of proteins with single-cell precision for resolving CTCs' phenotype, organ-of-origin, and drug targets. We describe a new technology that enables profiling multiple protein markers of extraordinarily rare tumor cells at the single-cell level. This technology integrates a microchip consisting of 15000 60 pL-sized microwells and a novel beads-on-barcode antibody microarray (BOBarray). The BOBarray allows for multiplexed protein detection by assigning two independent identifiers (bead size and fluorescent color) of the beads to each protein. Four bead sizes (1.75, 3, 4.5, and 6 μm) and three colors (blue, green, and yellow) are utilized to encode up to 12 different proteins. The miniaturized BOBarray can fit an array of 60 pL-sized microwells that isolate single cells for cell lysis and the subsequent detection of protein markers. An enclosed 60 pL-sized microchamber defines a high concentration of proteins released from lysed single cells, leading to single-cell resolution of protein detection. The protein markers assayed in this study include organ-specific markers and drug targets that help to characterize the organ-of-origin and drug targets of isolated rare tumor cells from blood samples. This new approach enables handling a very small number of cells and achieves single-cell, multiplexed protein detection without loss of rare but clinically important tumor cells.

Near-IR laser-triggered target cell collection using a carbon nanotube-based cell-cultured substrate.

PubMed

Sada, Takao; Fujigaya, Tsuyohiko; Niidome, Yasuro; Nakazawa, Kohji; Nakashima, Naotoshi

2011-06-28

Unique near-IR optical properties of single-walled carbon nanotube (SWNTs) are of interest in many biological applications. Here we describe the selective cell detachment and collection from an SWNT-coated cell-culture dish triggered by near-IR pulse laser irradiation. First, HeLa cells were cultured on an SWNT-coated dish prepared by a spraying of an aqueous SWNT dispersion on a glass dish. The SWNT-coated dish was found to show a good cell adhesion behavior as well as a cellular proliferation rate similar to a conventional glass dish. We discovered, by near-IR pulse laser irradiation (at the laser power over 25 mW) to the cell under optical microscopic observation, a quick single-cell detachment from the SWNT-coated surface. Shockwave generation from the irradiated SWNTs is expected to play an important role for the cell detachment. Moreover, we have succeeded in catapulting the target single cell from the cultured medium when the depth of the medium was below 150 μm and the laser power was stronger than 40 mW. The captured cell maintained its original shape. The retention of the genetic information of the cell was confirmed by the polymerase chain reaction (PCR) technique. A target single-cell collection from a culture medium under optical microscopic observation is significant in wide fields of single-cell studies in biological areas.

Surface-modified CMOS IC electrochemical sensor array targeting single chromaffin cells for highly parallel amperometry measurements.

PubMed

Huang, Meng; Delacruz, Joannalyn B; Ruelas, John C; Rathore, Shailendra S; Lindau, Manfred

2018-01-01

Amperometry is a powerful method to record quantal release events from chromaffin cells and is widely used to assess how specific drugs modify quantal size, kinetics of release, and early fusion pore properties. Surface-modified CMOS-based electrochemical sensor arrays allow simultaneous recordings from multiple cells. A reliable, low-cost technique is presented here for efficient targeting of single cells specifically to the electrode sites. An SU-8 microwell structure is patterned on the chip surface to provide insulation for the circuitry as well as cell trapping at the electrode sites. A shifted electrode design is also incorporated to increase the flexibility of the dimension and shape of the microwells. The sensitivity of the electrodes is validated by a dopamine injection experiment. Microwells with dimensions slightly larger than the cells to be trapped ensure excellent single-cell targeting efficiency, increasing the reliability and efficiency for on-chip single-cell amperometry measurements. The surface-modified device was validated with parallel recordings of live chromaffin cells trapped in the microwells. Rapid amperometric spikes with no diffusional broadening were observed, indicating that the trapped and recorded cells were in very close contact with the electrodes. The live cell recording confirms in a single experiment that spike parameters vary significantly from cell to cell but the large number of cells recorded simultaneously provides the statistical significance.

CAR-T cells are serial killers

PubMed Central

Davenport, Alexander J; Jenkins, Misty R; Ritchie, David S; Prince, H Miles; Trapani, Joseph A; Kershaw, Michael H; Darcy, Phillip K; Neeson, Paul J

2015-01-01

Chimeric antigen receptor (CAR) T cells have enjoyed unprecedented clinical success against haematological malignancies in recent years. However, several aspects of CAR T cell biology remain unknown. We recently compared CAR and T cell receptor (TCR)-based killing in the same effector cell and showed that CAR T cells can not only efficiently kill single tumor targets, they can also kill multiple tumor targets in a sequential manner. Single and serial killing events were not sustained long term due to CAR down-regulation after 20 hours. PMID:26587330

CAR-T cells are serial killers.

PubMed

Davenport, Alexander J; Jenkins, Misty R; Ritchie, David S; Prince, H Miles; Trapani, Joseph A; Kershaw, Michael H; Darcy, Phillip K; Neeson, Paul J

2015-12-01

Chimeric antigen receptor (CAR) T cells have enjoyed unprecedented clinical success against haematological malignancies in recent years. However, several aspects of CAR T cell biology remain unknown. We recently compared CAR and T cell receptor (TCR)-based killing in the same effector cell and showed that CAR T cells can not only efficiently kill single tumor targets, they can also kill multiple tumor targets in a sequential manner. Single and serial killing events were not sustained long term due to CAR down-regulation after 20 hours.

Highly Sensitive Detection of Target Biomolecules on Cell Surface Using Gold Nanoparticle Conjugated with Aptamer Probe

NASA Astrophysics Data System (ADS)

Kim, Hyonchol; Terazono, Hideyuki; Hayashi, Masahito; Takei, Hiroyuki; Yasuda, Kenji

2012-06-01

A method of gold nanoparticle (Au NP) labeling with backscattered electron (BE) imaging of field emission scanning electron microscopy (FE-SEM) was applied for specific detection of target biomolecules on a cell surface. A single-stranded DNA aptamer, which specifically binds to the target molecule on a human acute lymphoblastic leukemia cell, was conjugated with a 20 nm Au NP and used as a probe to label its target molecule on the cell. The Au NP probe was incubated with the cell, and the interaction was confirmed using BE imaging of FE-SEM through direct counting of the number of Au NPs attached on the target cell surface. Specific Au NP-aptamer probes were observed on a single cell surface and their spatial distributions including submicron-order localizations were also clearly visualized, whereas the nonspecific aptamer probes were not observed on it. The aptamer probe can be potentially dislodged from the cell surface with treatment of nucleases, indicating that Au NP-conjugated aptamer probes can be used as sensitive and reversible probes to label target biomolecules on cells.

Bispecific single-chain diabody-immunoliposomes targeting endoglin (CD105) and fibroblast activation protein (FAP) simultaneously.

PubMed

Rabenhold, Markus; Steiniger, Frank; Fahr, Alfred; Kontermann, Roland E; Rüger, Ronny

2015-03-10

Liposomes are well-established drug delivery systems with cancer chemotherapy as main focus. To increase the cellular drug delivery, liposomes can be endowed with ligands, e.g. recombinant antibody fragments, which ensure specific cell interaction. Multispecific immunoliposomes can be prepared to improve the liposome to cell interaction by targeting multiple different targets at the same time, for instance by coupling two or more different ligands to the liposomal surface, resulting in a synergistic or additive increase in binding. An alternative approach is the use of bispecific ligands to address at least two different targets. For this purpose we cloned a single-chain diabody fragment (scDb`), a bispecific molecule targeting two antigens, endoglin (CD105) and fibroblast activation protein (FAP), expressed on cells of the tumor microenvironment. As model cell system, a human fibrosarcoma cell line was used expressing endoglin and FAP simultaneously. Monospecific immunoliposomes directed either against endoglin or FAP were compared in vitro for cell binding and cytotoxic activity with bispecific dual-targeted scFv`-IL (bispecific scFv`FAP/CD105-IL) and bispecific single-chain diabody`-IL (scDb`CD105/FAP-IL) targeting endoglin and FAP simultaneously. In the underlying study, bispecific scFv`FAP/CD105-IL interacted stronger with cells expressing FAP and endoglin (both targets simultaneously) compared to the monospecific immunoliposomes. Furthermore, bispecific scDb`-immunoliposomes increased the cell interaction massively and showed enhanced cytotoxicity against target cells using doxorubicin-loaded immunoliposomes. The use of recombinant bispecific ligands as scDb`-molecules facilitates the generation of bispecific immunoliposomes by using the established post-insertion technique, enabling an extension of the ligand specificity spectrum via genetic modification. Copyright © 2015 Elsevier B.V. All rights reserved.

Selective Gene Transfection of Individual Cells In Vitro with Plasmonic Nanobubbles

PubMed Central

Lukianova-Hleb, Ekaterina; Samaniego, Adam P.; Wen, Jianguo; Metelitsa, Leonid; Chang, Chung-Che; Lapotko, Dmitri

2011-01-01

Gene delivery and transfection of eukaryotic cells is widely used for research and for developing gene cell therapy. However, the existing methods lack selectivity, efficacy and safety when heterogeneous cell systems must be treated. We report a new method that employs plasmonic nanobubbles (PNBs) for delivery and transfection. A PNB is a novel, tunable cellular agent with a dual mechanical and optical action due to the formation of the vapor nanobubble around a transiently heated gold nanoparticle upon its exposure to a laser pulse. PNBs enabled the mechanical injection of the extracellular cDNA plasmid into the cytoplasm of individual target living cells, cultured leukemia cells and human CD34+CD117+ stem cells and expression of a green fluorescent protein (GFP) in those cells. PNB generation and lifetime correlated with the expression of green fluorescent protein in PNB-treated cells. Optical scattering by PNBs additionally provided the detection of the target cells and the guidance of cDNA injection at single cell level. In both cell models PNBs demonstrated a gene transfection effect in a single pulse treatment with high selectivity, efficacy and safety. Thus, PNBs provided targeted gene delivery at the single cell level in a single pulse procedure that can be used for safe and effective gene therapy. PMID:21315120

Selective gene transfection of individual cells in vitro with plasmonic nanobubbles.

PubMed

Lukianova-Hleb, Ekaterina Y; Samaniego, Adam P; Wen, Jianguo; Metelitsa, Leonid S; Chang, Chung-Che; Lapotko, Dmitri O

2011-06-10

Gene delivery and transfection of eukaryotic cells are widely used for research and for developing gene cell therapy. However, the existing methods lack selectivity, efficacy and safety when heterogeneous cell systems must be treated. We report a new method that employs plasmonic nanobubbles (PNBs) for delivery and transfection. A PNB is a novel, tunable cellular agent with a dual mechanical and optical action due to the formation of the vapor nanobubble around a transiently heated gold nanoparticle upon its exposure to a laser pulse. PNBs enabled the mechanical injection of the extracellular cDNA plasmid into the cytoplasm of individual target living cells, cultured leukemia cells and human CD34+ CD117+ stem cells and expression of a green fluorescent protein (GFP) in those cells. PNB generation and lifetime correlated with the expression of green fluorescent protein in PNB-treated cells. Optical scattering by PNBs additionally provided the detection of the target cells and the guidance of cDNA injection at single cell level. In both cell models PNBs demonstrated a gene transfection effect in a single pulse treatment with high selectivity, efficacy and safety. Thus, PNBs provided targeted gene delivery at the single cell level in a single pulse procedure that can be used for safe and effective gene therapy. Copyright © 2011 Elsevier B.V. All rights reserved.

Evaluation of Human Adipose Tissue Stromal Heterogeneity in Metabolic Disease Using Single Cell RNA-Seq

DTIC Science & Technology

2017-09-01

1) define functional roles for individual genes and cell types in development of obesity and insulin resistance and 2) examine novel targets against...which we can design therapies to target specific pathogenic or or health-promoting cell types. 15. SUBJECT TERMS Obesity , Type 2 Diabetes Mellitus...compromised with chronic overnutrition ( obesity ). 4 KEYWORDS: Obesity , Diabetes, Insulin Resistance, Adipose, Adipocytes, Stromal Vascular Fraction, Single

Unbiased Combinatorial Genomic Approaches to Identify Alternative Therapeutic Targets within the TSC Signaling Network

DTIC Science & Technology

2014-06-01

Specifically, we combined the CRISPR genome editing system with a novel approach allowing efficient single cell cloning of Drosophila cells with the aim of...and culture these to produce cultures completely lacking wildtype sequence at the target locus. No robust methods existed to clone single Drosophila ...targeting all kinases and phosphatases (563 genes) in the Drosophila genome . 65 samples that displayed synthetic lethality (15 genes) or synthetic

Single-hit mechanism of tumour cell killing by radiation.

PubMed

Chapman, J D

2003-02-01

To review the relative importance of the single-hit mechanism of radiation killing for tumour response to 1.8-2.0 Gy day(-1) fractions and to low dose-rate brachytherapy. Tumour cell killing by ionizing radiation is well described by the linear-quadratic equation that contains two independent components distinguished by dose kinetics. Analyses of tumour cell survival curves that contain six or more dose points usually provide good estimates of the alpha- and beta-inactivation coefficients. Superior estimates of tumour cell intrinsic radiosensitivity are obtained when synchronized populations are employed. The characteristics of single-hit inactivation of tumour cells are reviewed and compared with the characteristics of beta-inactivation. Potential molecular targets associated with single-hit inactivation are discussed along with strategies for potentiating cell killing by this mechanism. The single-hit mechanism of tumour cell killing shows no dependence on dose-rate and, consequently, no evidence of sublethal damage repair. It is uniquely potentiated by high linear-energy-transfer radiation, exhibits a smaller oxygen enhancement ratio and exhibits a larger indirect effect by hydroxyl radicals than the beta-mechanism. alpha-inactivation coefficients vary slightly throughout interphase but mitotic cells exhibit extremely high alpha-coefficients in the range of those observed for lymphocytes and some repair-deficient cells. Evidence is accumulating to suggest that chromatin in compacted form could be a radiation-hypersensitive target associated with single-hit radiation killing. Analyses of tumour cell survival curves demonstrate that it is the single-hit mechanism (alpha) that determines the majority of cell killing after doses of 2Gy and that this mechanism is highly variable between tumour cell lines. The characteristics of single-hit inactivation are qualitatively and quantitatively distinct from those of beta-inactivation. Compacted chromatin in tumour cells should be further investigated as a radiation-hypersensitive target that could be modulated for therapeutic advantage.

Immuno-Oncology-The Translational Runway for Gene Therapy: Gene Therapeutics to Address Multiple Immune Targets.

PubMed

Weß, Ludger; Schnieders, Frank

2017-12-01

Cancer therapy is once again experiencing a paradigm shift. This shift is based on extensive clinical experience demonstrating that cancer cannot be successfully fought by addressing only single targets or pathways. Even the combination of several neo-antigens in cancer vaccines is not sufficient for successful, lasting tumor eradication. The focus has therefore shifted to the immune system's role in cancer and the striking abilities of cancer cells to manipulate and/or deactivate the immune system. Researchers and pharma companies have started to target the processes and cells known to support immune surveillance and the elimination of tumor cells. Immune processes, however, require novel concepts beyond the traditional "single-target-single drug" paradigm and need parallel targeting of diverse cells and mechanisms. This review gives a perspective on the role of gene therapy technologies in the evolving immuno-oncology space and identifies gene therapy as a major driver in the development and regulation of effective cancer immunotherapy. Present challenges and breakthroughs ranging from chimeric antigen receptor T-cell therapy, gene-modified oncolytic viruses, combination cancer vaccines, to RNA therapeutics are spotlighted. Gene therapy is recognized as the most prominent technology enabling effective immuno-oncology strategies.

Microfluidic platform for multiplexed detection in single cells and methods thereof

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Meiye; Singh, Anup K.

The present invention relates to a microfluidic device and platform configured to conduct multiplexed analysis within the device. In particular, the device allows multiple targets to be detected on a single-cell level. Also provided are methods of performing multiplexed analyses to detect one or more target nucleic acids, proteins, and post-translational modifications.

Development of a microprocessing-assisted cell-systematic evolution of ligands by exponential enrichment method for human umbilical vein endothelial cells

NASA Astrophysics Data System (ADS)

Terazono, Hideyuki; Kim, Hyonchol; Nomura, Fumimasa; Yasuda, Kenji

2016-06-01

We developed a microprocessing-assisted technique to select single-strand DNA aptamers that bind to unknown targets on the cell surface by modifying the conventional systematic evolution of ligands by exponential enrichment (cell-SELEX). Our technique involves 1) the specific selection of target-cell-surface-bound aptamers without leakage of intracellular components by trypsinization and 2) cloning of aptamers by microprocessing-assisted picking of single cells using magnetic beads. After cell-SELEX, the enriched aptamers were conjugated with magnetic beads. The aptamer-magnetic beads conjugates attached to target cells were collected individually by microassisted procedures using microneedles under a microscope. After that, the sequences of the collected magnetic-bead-bound aptamers were identified. As a result, a specific aptamer for the surface of target cells, e.g., human umbilical vein endothelial cells (HUVECs), was chosen and its specificity was examined using other cell types, e.g., HeLa cells. The results indicate that this microprocessing-assisted cell-SELEX method for identifying aptamers is applicable in biological research and clinical diagnostics.

SPICE-NIRS Microbeam: a focused vertical system for proton irradiation of a single cell for radiobiological research

PubMed Central

Konishi, Teruaki; Oikawa, Masakazu; Suya, Noriyoshi; Ishikawa, Takahiro; Maeda, Takeshi; Kobayashi, Alisa; Shiomi, Naoko; Kodama, Kumiko; Hamano, Tsuyoshi; Homma-Takeda, Shino; Isono, Mayu; Hieda, Kotaro; Uchihori, Yukio; Shirakawa, Yoshiyuki

2013-01-01

The Single Particle Irradiation system to Cell (SPICE) facility at the National Institute of Radiological Sciences (NIRS) is a focused vertical microbeam system designed to irradiate the nuclei of adhesive mammalian cells with a defined number of 3.4 MeV protons. The approximately 2-μm diameter proton beam is focused with a magnetic quadrupole triplet lens and traverses the cells contained in dishes from bottom to top. All procedures for irradiation, such as cell image capturing, cell recognition and position calculation, are automated. The most distinctive characteristic of the system is its stability and high throughput; i.e. 3000 cells in a 5 mm × 5 mm area in a single dish can be routinely irradiated by the 2-μm beam within 15 min (the maximum irradiation speed is 400 cells/min). The number of protons can be set as low as one, at a precision measured by CR-39 detectors to be 99.0%. A variety of targeting modes such as fractional population targeting mode, multi-position targeting mode for nucleus irradiation and cytoplasm targeting mode are available. As an example of multi-position targeting irradiation of mammalian cells, five fluorescent spots in a cell nucleus were demonstrated using the γ-H2AX immune-staining technique. The SPICE performance modes described in this paper are in routine use. SPICE is a joint-use research facility of NIRS and its beam times are distributed for collaborative research. PMID:23287773

Probing microbubble targeting with atomic force microscopy.

PubMed

Sboros, V; Glynos, E; Ross, J A; Moran, C M; Pye, S D; Butler, M; McDicken, W N; Brown, S B; Koutsos, V

2010-10-01

Microbubble science is expanding beyond ultrasound imaging applications to biological targeting and drug/gene delivery. The characteristics of molecular targeting should be tested by a measurement system that can assess targeting efficacy and strength. Atomic force microscopy (AFM) is capable of piconewton force resolution, and is reported to measure the strength of single hydrogen bonds. An in-house targeted microbubble modified using the biotin-avidin chemistry and the CD31 antibody was used to probe cultures of Sk-Hep1 hepatic endothelial cells. We report that the targeted microbubbles provide a single distribution of adhesion forces with a median of 93pN. This interaction is assigned to the CD31 antibody-antigen unbinding event. Information on the distances between the interaction forces was obtained and could be important for future microbubble fabrication. In conclusion, the capability of single microbubbles to target cell lines was shown to be feasible with AFM.

Single-Cell Sequencing Technology in Oncology: Applications for Clinical Therapies and Research.

PubMed

Ye, Baixin; Gao, Qingping; Zeng, Zhi; Stary, Creed M; Jian, Zhihong; Xiong, Xiaoxing; Gu, Lijuan

2016-01-01

Cellular heterogeneity is a fundamental characteristic of many cancers. A lack of cellular homogeneity contributes to difficulty in designing targeted oncological therapies. Therefore, the development of novel methods to determine and characterize oncologic cellular heterogeneity is a critical next step in the development of novel cancer therapies. Single-cell sequencing (SCS) technology has been recently employed for analyzing the genetic polymorphisms of individual cells at the genome-wide level. SCS requires (1) precise isolation of the single cell of interest; (2) isolation and amplification of genetic material; and (3) descriptive analysis of genomic, transcriptomic, and epigenomic data. In addition to targeted analysis of single cells isolated from tumor biopsies, SCS technology may be applied to circulating tumor cells, which may aid in predicting tumor progression and metastasis. In this paper, we provide an overview of SCS technology and review the current literature on the potential application of SCS to clinical oncology and research.

A novel cancer-targeting transporter with integrin αvβ3 monoclonal antibody functionalized single-walled carbon nanotubes

NASA Astrophysics Data System (ADS)

Ou, Zhongmin; Wu, Baoyan; Xing, Da

2009-08-01

The pursuit of efficient and highly targeting-selective transporters is an active topic in cancer-targeting therapy. In this study, a novel cancer-targeting transporter with integrin αvβ3 monoclonal antibody functionalized single-walled carbon nanotubes (SWCNTs) was developed to investigate cancer cell targeting in vitro. SWCNTs were first modified by phospholipid-bearing polyethylene glycol (PL-PEG). PL-PEG functionalized SWCNTs were then conjugated with fluorescein isothiocyanate (FITC) labeled integrin αvβ3 monoclonal antibody to construct SWCNT-integrin αvβ3 monoclonal antibody system (denoted as SWCNT-PEG-mAb). In vitro study revealed that the system had a high efficiency in cancer cell targeting in integrin αvβ3 positive U87MG cells. Moreover, the SWCNT-PEG-mAb is stable in physiological media, and can be readily transported into U87MG cells via integrin αvβ3-mediated endocytosis in cell. In summary, the integrin αvβ3 monoclonal antibody labeled SWCNT is a potential carrier-candidate for cancer-imaging and drug-delivering in cancer-targeting therapy.

A heating-superfusion platform technology for the investigation of protein function in single cells.

PubMed

Xu, Shijun; Ainla, Alar; Jardemark, Kent; Jesorka, Aldo; Jeffries, Gavin D M

2015-01-06

Here, we report on a novel approach for the study of single-cell intracellular enzyme activity at various temperatures, utilizing a localized laser heating probe in combination with a freely positionable microfluidic perfusion device. Through directed exposure of individual cells to the pore-forming agent α-hemolysin, we have controlled the membrane permeability, enabling targeted delivery of the substrate. Mildly permeabilized cells were exposed to fluorogenic substrates to monitor the activity of intracellular enzymes, while adjusting the local temperature surrounding the target cells, using an infrared laser heating system. We generated quantitative estimates for the intracellular alkaline phosphatase activity at five different temperatures in different cell lines, constructing temperature-response curves of enzymatic activity at the single-cell level. Enzymatic activity was determined rapidly after cell permeation, generating five-point temperature-response curves within just 200 s.

A novel T cell receptor single-chain signaling complex mediates antigen-specific T cell activity and tumor control

PubMed Central

Stone, Jennifer D.; Harris, Daniel T.; Soto, Carolina M.; Chervin, Adam S.; Aggen, David H.; Roy, Edward J.; Kranz, David M.

2014-01-01

Adoptive transfer of genetically modified T cells to treat cancer has shown promise in several clinical trials. Two main strategies have been applied to redirect T cells against cancer: 1) introduction of a full-length T cell receptor (TCR) specific for a tumor-associated peptide-MHC, or 2) introduction of a chimeric antigen receptor (CAR), including an antibody fragment specific for a tumor cell surface antigen, linked intracellularly to T cell signaling domains. Each strategy has advantages and disadvantages for clinical applications. Here, we present data on the in vitro and in vivo effectiveness of a single-chain signaling receptor incorporating a TCR variable fragment as the targeting element (referred to as TCR-SCS). This receptor contained a single-chain TCR (Vβ-linker-Vα) from a high-affinity TCR called m33, linked to the intracellular signaling domains of CD28 and CD3ζ. This format avoided mispairing with endogenous TCR chains, and mediated specific T cell activity when expressed in either CD4 or CD8 T cells. TCR-SCS-transduced CD8-negative cells showed an intriguing sensitivity, compared to full-length TCRs, to higher densities of less stable pepMHC targets. T cells that expressed this peptide-specific receptor persisted in vivo, and exhibited polyfunctional responses. Growth of metastatic antigen-positive tumors was significantly inhibited by T cells that expressed this receptor, and tumor cells that escaped were antigen loss variants. TCR-SCS receptors represent an alternative targeting receptor strategy that combines the advantages of single-chain expression, avoidance of TCR chain mispairing, and targeting of intracellular antigens presented in complex with MHC proteins. PMID:25082071

Targeted self-assembly of functionalized carbon nanotubes on tumors

DOEpatents

Scheinberg, David A.; McDevitt, Michael R.; Villa, Carlos H.; Mulvey, J. Justin

2018-05-22

Provided herein are methods for delivering a molecule in situ to a cell and for treating a cancer via the in situ delivery. The methods comprise contacting or administering to the cell, as two separate components, a morpholino oligonucleotide comprising a targeting moiety followed by a single wall nanotube construct comprising second morpholino oligonucleotides complementary to the first morpholino oligonucleotides and one or both of a therapeutic or diagnostic payload molecule linked to the single wall nanotube construct. Upon self-assembly of a single wall nanotube complex via hybridization of the first morpholino and second complementary morpholino oligonucleotides at the cell, the payload molecule is delivered. Also provided is the two component self-assembly single wall nanotube system and the single wall nanotube construct comprising the second component.

Single-chain antibody-delivered Livin siRNA inhibits human malignant melanoma growth in vitro and in vivo.

PubMed

Wang, Hao; Yang, Yifei; Wang, Wei; Guan, Bing; Xun, Meng; Zhang, Hai; Wang, Ziling; Zhao, Yong

2017-05-01

Although gene therapy has brought new insights into the treatment of malignant melanoma, targeting delivery of nucleic acid which targets critical oncogene/anti-oncogene in vivo is still a bottleneck in the therapeutic application. Our previous in vitro studies have found that the oncogene Livin could serve as a potential molecular target by small interfering RNA for gene therapy of malignant melanoma. However, how to transport Livin small interfering RNA into malignant melanoma cells specifically and efficiently in vivo needs further investigation. Cumulative evidence has suggested that single-chain antibody-mediated small interfering RNA targeted delivery is an effective way to silence specific genes in human cancer cells. Indeed, this study designed a protamine-single-chain antibody fusion protein, anti-MM scFv-tP, to deliver Livin small interfering RNA into LiBr cells. Further experiments confirmed the induction of cell apoptosis and suppression of cell proliferation by anti-MM scFv-tP in LiBr cells, along with efficient silence of Livin gene both in vitro and in vivo. Altogether, our findings provide a feasible approach to transport Livin small interfering RNA to malignant melanoma cells which would be a new therapeutic strategy for combating malignant melanoma.

Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids

PubMed Central

Sakuma, Tetsushi; Takenaga, Mitsumasa; Kawabe, Yoshinori; Nakamura, Takahiro; Kamihira, Masamichi; Yamamoto, Takashi

2015-01-01

Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins. PMID:26473830

Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids.

PubMed

Sakuma, Tetsushi; Takenaga, Mitsumasa; Kawabe, Yoshinori; Nakamura, Takahiro; Kamihira, Masamichi; Yamamoto, Takashi

2015-10-09

Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.

Single-cell whole exome and targeted sequencing in NPM1/FLT3 positive pediatric acute myeloid leukemia.

PubMed

Walter, Christiane; Pozzorini, Christian; Reinhardt, Katarina; Geffers, Robert; Xu, Zhenyu; Reinhardt, Dirk; von Neuhoff, Nils; Hanenberg, Helmut

2018-02-01

The small portion of leukemic stem cells (LSCs) in acute myeloid leukemia (AML) present in children and adolescents is often masked by the high background of AML blasts and normal hematopoietic cells. The aim of the current study was to establish a simple workflow for reliable genetic analysis of single LSC-enriched blasts from pediatric patients. For three AMLs with mutations in nucleophosmin 1 and/or fms-like tyrosine kinase 3, we performed whole genome amplification on sorted single-cell DNA followed by whole exome sequencing (WES). The corresponding bulk bone marrow DNAs were also analyzed by WES and by targeted sequencing (TS) that included 54 genes associated with myeloid malignancies. Analysis revealed that read coverage statistics were comparable between single-cell and bulk WES data, indicating high-quality whole genome amplification. From 102 single-cell variants, 72 single nucleotide variants and insertions or deletions (70%) were consistently found in the two bulk DNA analyses. Variants reliably detected in single cells were also present in TS. However, initial screening by WES with read counts between 50-72× failed to detect rare AML subclones in the bulk DNAs. In summary, our study demonstrated that single-cell WES combined with bulk DNA TS is a promising tool set for detecting AML subclones and possibly LSCs. © 2017 Wiley Periodicals, Inc.

Multiepitope HER2 targeting enhances photoimmunotherapy of HER2-overexpressing cancer cells with pyropheophorbide-a immunoconjugates.

PubMed

Savellano, Mark D; Pogue, Brian W; Hoopes, P Jack; Vitetta, Ellen S; Paulsen, Keith D

2005-07-15

Multi-targeting strategies improve the efficacy of antibody and immunotoxin therapies but have not yet been thoroughly explored for HER2-based cancer treatments. We investigated multi-epitope HER2 targeting to boost photosensitizer immunoconjugate uptake as a way of enhancing photoimmunotherapy. Photoimmunotherapy may allow targeted photodynamic destruction of malignancies and may also potentiate anticancer antibodies. However, one obstacle preventing its clinical use is the delivery of enough photosensitizer immunoconjugates to target cells. Anti-HER2 photosensitizer immunoconjugates were constructed from two monoclonal antibodies (mAb), HER50 and HER66, using a novel method originally developed to label photosensitizer immunoconjugates with the photosensitizer, benzoporphyrin derivative verteporfin. Photosensitizer immunoconjugates were labeled instead with a promising alternative photosensitizer, pyropheophorbide-a (PPa), which required only minor changes to the conjugation procedure. Uptake and phototoxicity experiments using human cancer cells were conducted with the photosensitizer immunoconjugates and, for comparison, with free PPa. SK-BR-3 and SK-OV-3 cells served as HER2-overexpressing target cells. MDA-MB-468 cells served as HER2-nonexpressing control cells. Photosensitizer immunoconjugates with PPa/mAb molar ratios up to approximately 10 specifically targeted and photodynamically killed HER2-overexpressing cells. On a per mole basis, photosensitizer immunoconjugates were less phototoxic than free PPa, but photosensitizer immunoconjugates were selective for target cells whereas free PPa was not. Multiepitope targeted photoimmunotherapy with a HER50 and HER66 photosensitizer immunoconjugate mixture was significantly more effective than single-epitope targeted photoimmunotherapy with a single anti-HER2 photosensitizer immunoconjugate, provided photosensitizer immunoconjugate binding was saturated. This study shows that multiepitope targeting enhances HER2-targeted photoimmunotherapy and maintains a high degree of specificity. Consequently, it seems that multitargeted photoimmunotherapy should also be useful against cancers that overexpress other receptors.

Dynamic Analysis of Human Natural Killer Cell Response at Single-Cell Resolution in B-Cell Non-Hodgkin Lymphoma.

PubMed

Sarkar, Saheli; Sabhachandani, Pooja; Ravi, Dashnamoorthy; Potdar, Sayalee; Purvey, Sneha; Beheshti, Afshin; Evens, Andrew M; Konry, Tania

2017-01-01

Natural killer (NK) cells are phenotypically and functionally diverse lymphocytes that recognize and kill cancer cells. The susceptibility of target cancer cells to NK cell-mediated cytotoxicity depends on the strength and balance of regulatory (activating/inhibitory) ligands expressed on target cell surface. We performed gene expression arrays to determine patterns of NK cell ligands associated with B-cell non-Hodgkin lymphoma (b-NHL). Microarray analyses revealed significant upregulation of a multitude of NK-activating and costimulatory ligands across varied b-NHL cell lines and primary lymphoma cells, including ULBP1, CD72, CD48, and SLAMF6. To correlate genetic signatures with functional anti-lymphoma activity, we developed a dynamic and quantitative cytotoxicity assay in an integrated microfluidic droplet generation and docking array. Individual NK cells and target lymphoma cells were co-encapsulated in picoliter-volume droplets to facilitate monitoring of transient cellular interactions and NK cell effector outcomes at single-cell level. We identified significant variability in NK-lymphoma cell contact duration, frequency, and subsequent cytolysis. Death of lymphoma cells undergoing single contact with NK cells occurred faster than cells that made multiple short contacts. NK cells also killed target cells in droplets via contact-independent mechanisms that partially relied on calcium-dependent processes and perforin secretion, but not on cytokines (interferon-γ or tumor necrosis factor-α). We extended this technique to characterize functional heterogeneity in cytolysis of primary cells from b-NHL patients. Tumor cells from two diffuse large B-cell lymphoma patients showed similar contact durations with NK cells; primary Burkitt lymphoma cells made longer contacts and were lysed at later times. We also tested the cytotoxic efficacy of NK-92, a continuously growing NK cell line being investigated as an antitumor therapy, using our droplet-based bioassay. NK-92 cells were found to be more efficient in killing b-NHL cells compared with primary NK cells, requiring shorter contacts for faster killing activity. Taken together, our combined genetic and microfluidic analysis demonstrate b-NHL cell sensitivity to NK cell-based cytotoxicity, which was associated with significant heterogeneity in the dynamic interaction at single-cell level.

Microfluidics cell sample preparation for analysis: Advances in efficient cell enrichment and precise single cell capture

PubMed Central

Bian, Shengtai; Cheng, Yinuo; Shi, Guanya; Liu, Peng; Ye, Xiongying

2017-01-01

Single cell analysis has received increasing attention recently in both academia and clinics, and there is an urgent need for effective upstream cell sample preparation. Two extremely challenging tasks in cell sample preparation—high-efficiency cell enrichment and precise single cell capture—have now entered into an era full of exciting technological advances, which are mostly enabled by microfluidics. In this review, we summarize the category of technologies that provide new solutions and creative insights into the two tasks of cell manipulation, with a focus on the latest development in the recent five years by highlighting the representative works. By doing so, we aim both to outline the framework and to showcase example applications of each task. In most cases for cell enrichment, we take circulating tumor cells (CTCs) as the target cells because of their research and clinical importance in cancer. For single cell capture, we review related technologies for many kinds of target cells because the technologies are supposed to be more universal to all cells rather than CTCs. Most of the mentioned technologies can be used for both cell enrichment and precise single cell capture. Each technology has its own advantages and specific challenges, which provide opportunities for researchers in their own area. Overall, these technologies have shown great promise and now evolve into real clinical applications. PMID:28217240

Single cell array impedance analysis in a microfluidic device

NASA Astrophysics Data System (ADS)

Altinagac, Emre; Taskin, Selen; Kizil, Huseyin

2016-10-01

Impedance analysis of single cells is presented in this paper. Following the separation of a target cell type by dielectrophoresis in our previous work, this paper focuses on capturing the cells as a single array and performing impedance analysis to point out the signature difference between each cell type. Lab-on-a-chip devices having a titanium interdigitated electrode layer on a glass substrate and a PDMS microchannel are fabricated to capture each cell in a single form and perform impedance analysis. HCT116 (homosapiens colon colorectal carcin) and HEK293 (human embryonic kidney) cells are used in our experiments.

Single-Cell Western Blotting after Whole-Cell Imaging to Assess Cancer Chemotherapeutic Response

PubMed Central

2015-01-01

Intratumor heterogeneity remains a major obstacle to effective cancer therapy and personalized medicine. Current understanding points to differential therapeutic response among subpopulations of tumor cells as a key challenge to successful treatment. To advance our understanding of how this heterogeneity is reflected in cell-to-cell variations in chemosensitivity and expression of drug-resistance proteins, we optimize and apply a new targeted proteomics modality, single-cell western blotting (scWestern), to a human glioblastoma cell line. To acquire both phenotypic and proteomic data on the same, single glioblastoma cells, we integrate high-content imaging prior to the scWestern assays. The scWestern technique supports thousands of concurrent single-cell western blots, with each assay comprised of chemical lysis of single cells seated in microwells, protein electrophoresis from those microwells into a supporting polyacrylamide (PA) gel layer, and in-gel antibody probing. We systematically optimize chemical lysis and subsequent polyacrylamide gel electrophoresis (PAGE) of the single-cell lysate. The scWestern slides are stored for months then reprobed, thus allowing archiving and later analysis as relevant to sparingly limited, longitudinal cell specimens. Imaging and scWestern analysis of single glioblastoma cells dosed with the chemotherapeutic daunomycin showed both apoptotic (cleaved caspase 8- and annexin V-positive) and living cells. Intriguingly, living glioblastoma subpopulations show up-regulation of a multidrug resistant protein, P-glycoprotein (P-gp), suggesting an active drug efflux pump as a potential mechanism of drug resistance. Accordingly, linking of phenotype with targeted protein analysis with single-cell resolution may advance our understanding of drug response in inherently heterogeneous cell populations, such as those anticipated in tumors. PMID:25226230

Quantum dot tailored to single wall carbon nanotubes: a multifunctional hybrid nanoconstruct for cellular imaging and targeted photothermal therapy.

PubMed

Nair, Lakshmi V; Nagaoka, Yutaka; Maekawa, Toru; Sakthikumar, D; Jayasree, Ramapurath S

2014-07-23

Hybrid nanomaterial based on quantum dots and SWCNTs is used for cellular imaging and photothermal therapy. Furthermore, the ligand conjugated hybrid system (FaQd@CNT) enables selective targeting in cancer cells. The imaging capability of quantum dots and the therapeutic potential of SWCNT are available in a single system with cancer targeting property. Heat generated by the system is found to be high enough to destroy cancer cells. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting.

PubMed

Chen, Xiaoyu; Janssen, Josephine M; Liu, Jin; Maggio, Ignazio; 't Jong, Anke E J; Mikkers, Harald M M; Gonçalves, Manuel A F V

2017-09-22

Precise genome editing involves homologous recombination between donor DNA and chromosomal sequences subjected to double-stranded DNA breaks made by programmable nucleases. Ideally, genome editing should be efficient, specific, and accurate. However, besides constituting potential translocation-initiating lesions, double-stranded DNA breaks (targeted or otherwise) are mostly repaired through unpredictable and mutagenic non-homologous recombination processes. Here, we report that the coordinated formation of paired single-stranded DNA breaks, or nicks, at donor plasmids and chromosomal target sites by RNA-guided nucleases based on CRISPR-Cas9 components, triggers seamless homology-directed gene targeting of large genetic payloads in human cells, including pluripotent stem cells. Importantly, in addition to significantly reducing the mutagenicity of the genome modification procedure, this in trans paired nicking strategy achieves multiplexed, single-step, gene targeting, and yields higher frequencies of accurately edited cells when compared to the standard double-stranded DNA break-dependent approach.CRISPR-Cas9-based gene editing involves double-strand breaks at target sequences, which are often repaired by mutagenic non-homologous end-joining. Here the authors use Cas9 nickases to generate coordinated single-strand breaks in donor and target DNA for precise homology-directed gene editing.

Plasmonic nanobubbles for target cell-specific gene and drug delivery and multifunctional processing of heterogeneous cell systems

NASA Astrophysics Data System (ADS)

Lukianova-Hleb, Ekaterina Y.; Huye, Leslie E.; Brenner, Malcolm K.; Lapotko, Dmitri O.

2014-03-01

Cell and gene cancer therapies require ex vivo cell processing of human grafts. Such processing requires at least three steps - cell enrichment, cell separation (destruction), and gene transfer - each of which requires the use of a separate technology. While these technologies may be satisfactory for research use, they are of limited usefulness in the clinical treatment setting because they have a low processing rate, as well as a low transfection and separation efficacy and specificity in heterogeneous human grafts. Most problematic, because current technologies are administered in multiple steps - rather than in a single, multifunctional, and simultaneous procedure - they lengthen treatment process and introduce an unnecessary level of complexity, labor, and resources into clinical treatment; all these limitations result in high losses of valuable cells. We report a universal, high-throughput, and multifunctional technology that simultaneously (1) inject free external cargo in target cells, (2) destroys unwanted cells, and (3) preserve valuable non-target cells in heterogeneous grafts. Each of these functions has single target cell specificity in heterogeneous cell system, processing rate > 45 mln cell/min, injection efficacy 90% under 96% viability of the injected cells, target cell destruction efficacy > 99%, viability of not-target cells >99% The developed technology employs novel cellular agents, called plasmonic nanobubbles (PNBs). PNBs are not particles, but transient, intracellular events, a vapor nanobubbles that expand and collapse in mere nanoseconds under optical excitation of gold nanoparticles with short picosecond laser pulses. PNBs of different, cell-specific, size (1) inject free external cargo with small PNBs, (2) Destroy other target cells mechanically with large PNBs and (3) Preserve non-target cells. The multi-functionality, precision, and high throughput of all-in-one PNB technology will tremendously impact cell and gene therapies and other clinical applications that depend on ex vivo processing of heterogeneous cell systems.

Crispr-mediated Gene Targeting of Human Induced Pluripotent Stem Cells.

PubMed

Byrne, Susan M; Church, George M

2015-01-01

CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem or induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, we describe an optimized protocol for genome engineering of human iPSCs using a simple transient transfection of plasmids and/or single-stranded oligonucleotides. With this protocol, we achieve transfection efficiencies greater than 60%, with gene disruption efficiencies from 1-25% and gene insertion/replacement efficiencies from 0.5-10% without any further selection or enrichment steps. We also describe how to design and assess optimal sgRNA target sites and donor targeting vectors; cloning individual iPSC by single cell FACS sorting, and genotyping successfully edited cells.

Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides.

PubMed

Rivera-Torres, Natalia; Banas, Kelly; Bialk, Pawel; Bloh, Kevin M; Kmiec, Eric B

2017-01-01

CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and CRISPR/Cas9 ribonucleoprotein complex.

Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides

PubMed Central

Rivera-Torres, Natalia; Bialk, Pawel; Bloh, Kevin M.; Kmiec, Eric B.

2017-01-01

CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and CRISPR/Cas9 ribonucleoprotein complex. PMID:28052104

Cell-targeted platinum nanoparticles and nanoparticle clusters.

PubMed

Papst, Stefanie; Brimble, Margaret A; Evans, Clive W; Verdon, Daniel J; Feisst, Vaughan; Dunbar, P Rod; Tilley, Richard D; Williams, David E

2015-06-21

Herein, we report the facile preparation of cell-targeted platinum nanoparticles (PtNPs), through the design of peptides that, as a single molecule added in small concentration during the synthesis, control the size of PtNP clusters during their growth, stabilise the PtNPs in aqueous suspension and enable the functionalisation of the PtNPs with a versatile range of cell-targeting ligands. Water-soluble PtNPs targeted respectively at blood group antigens and at integrin receptors are demonstrated.

Functional single-cell hybridoma screening using droplet-based microfluidics.

PubMed

El Debs, Bachir; Utharala, Ramesh; Balyasnikova, Irina V; Griffiths, Andrew D; Merten, Christoph A

2012-07-17

Monoclonal antibodies can specifically bind or even inhibit drug targets and have hence become the fastest growing class of human therapeutics. Although they can be screened for binding affinities at very high throughput using systems such as phage display, screening for functional properties (e.g., the inhibition of a drug target) is much more challenging. Typically these screens require the generation of immortalized hybridoma cells, as well as clonal expansion in microtiter plates over several weeks, and the number of clones that can be assayed is typically no more than a few thousand. We present here a microfluidic platform allowing the functional screening of up to 300,000 individual hybridoma cell clones within less than a day. This approach should also be applicable to nonimmortalized primary B-cells, as no cell proliferation is required: Individual cells are encapsulated into aqueous microdroplets and assayed directly for the release of antibodies inhibiting a drug target based on fluorescence. We used this system to perform a model screen for antibodies that inhibit angiotensin converting enzyme 1, a target for hypertension and congestive heart failure drugs. When cells expressing these antibodies were spiked into an unrelated hybridoma cell population in a ratio of 1:10,000 we observed a 9,400-fold enrichment after fluorescence activated droplet sorting. A wide variance in antibody expression levels at the single-cell level within a single hybridoma line was observed and high expressors could be successfully sorted and recultivated.

CD22 Ligands on a Natural N-Glycan Scaffold Efficiently Deliver Toxins to B-Lymphoma Cells.

PubMed

Peng, Wenjie; Paulson, James C

2017-09-13

CD22 is a sialic acid-binding immunoglobulin-like lectin (Siglec) that is highly expressed on B-cells and B cell lymphomas, and is a validated target for antibody and nanoparticle based therapeutics. However, cell targeted therapeutics are limited by their complexity, heterogeneity, and difficulties in production. We describe here a chemically defined natural N-linked glycan scaffold that displays high affinity CD22 glycan ligands and outcompetes the natural ligand for the receptor, resulting in single molecule binding to CD22 and endocytosis into cells. Binding affinity is increased by up to 1500-fold compared to the monovalent ligand, while maintaining the selectivity for hCD22 over other Siglecs. Conjugates of these multivalent ligands with auristatin and saporin toxins are efficiently internalized via hCD22 resulting in killing of B-cell lymphoma cells. This single molecule ligand targeting strategy represents an alternative to antibody- and nanoparticle-mediated approaches for delivery of agents to cells expressing CD22 and other Siglecs.

Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip

NASA Astrophysics Data System (ADS)

Li, Ren; Zhou, Mingxing; Li, Jine; Wang, Zihua; Zhang, Weikai; Yue, Chunyan; Ma, Yan; Peng, Hailin; Wei, Zewen; Hu, Zhiyuan

2018-03-01

EGFR mutations companion diagnostics have been proved to be crucial for the efficacy of tyrosine kinase inhibitor targeted cancer therapies. To uncover multiple mutations occurred in minority of EGFR-mutated cells, which may be covered by the noises from majority of un-mutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multi-mutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cells were easily identified. By in situ lysing single cells, the cell lysates of EGFR-expressed cells were retrieved without cross-contamination. Benefited from excluding the noise from cells without EGFR expression, the simple and cost-effective Sanger's sequencing, but not the expensive deep sequencing of the whole cell population, was used to discover multi-mutations. We verified the new method with precisely discovering three most important EGFR drug-related mutations from a sample in which EGFR-mutated cells only account for a small percentage of whole cell population. The microfluidic chip is capable of discovering not only the existence of specific EGFR multi-mutations, but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. This microfluidic chip constitutes a promising method to promote simple and cost-effective Sanger's sequencing to be a routine test before performing targeted cancer therapy.[Figure not available: see fulltext.

Precise Quantitation of MicroRNA in a Single Cell with Droplet Digital PCR Based on Ligation Reaction.

PubMed

Tian, Hui; Sun, Yuanyuan; Liu, Chenghui; Duan, Xinrui; Tang, Wei; Li, Zhengping

2016-12-06

MicroRNA (miRNA) analysis in a single cell is extremely important because it allows deep understanding of the exact correlation between the miRNAs and cell functions. Herein, we wish to report a highly sensitive and precisely quantitative assay for miRNA detection based on ligation-based droplet digital polymerase chain reaction (ddPCR), which permits the quantitation of miRNA in a single cell. In this ligation-based ddPCR assay, two target-specific oligonucleotide probes can be simply designed to be complementary to the half-sequence of the target miRNA, respectively, which avoids the sophisticated design of reverse transcription and provides high specificity to discriminate a single-base difference among miRNAs with simple operations. After the miRNA-templated ligation, the ddPCR partitions individual ligated products into a water-in-oil droplet and digitally counts the fluorescence-positive and negative droplets after PCR amplification for quantification of the target molecules, which possesses the power of precise quantitation and robustness to variation in PCR efficiency. By integrating the advantages of the precise quantification of ddPCR and the simplicity of the ligation-based PCR, the proposed method can sensitively measure let-7a miRNA with a detection limit of 20 aM (12 copies per microliter), and even a single-base difference can be discriminated in let-7 family members. More importantly, due to its high selectivity and sensitivity, the proposed method can achieve precise quantitation of miRNAs in single-cell lysate. Therefore, the ligation-based ddPCR assay may serve as a useful tool to exactly reveal the miRNAs' actions in a single cell, which is of great importance for the study of miRNAs' biofunction as well as for the related biomedical studies.

Visualization and Enumeration of Bacteria Carrying a Specific Gene Sequence by In Situ Rolling Circle Amplification

PubMed Central

Maruyama, Fumito; Kenzaka, Takehiko; Yamaguchi, Nobuyasu; Tani, Katsuji; Nasu, Masao

2005-01-01

Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count single Escherichia coli cells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes inside E. coli cells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA. E. coli cells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 106-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria. PMID:16332770

Gold Nano Popcorn Attached SWCNT Hybrid Nanomaterial for Targeted Diagnosis and Photothermal Therapy of Human Breast Cancer Cells

PubMed Central

Beqa, Lule; Fan, Zhen; Singh, Anant Kumar; Senapati, Dulal; Ray, Paresh Chandra

2011-01-01

Breast cancer presents greatest challenge in health care in today’s world. The key to ultimately successful treatment of breast cancer disease is an early and accurate diagnosis. Current breast cancer treatments are often associated with severe side effects. Driven by the need, we report the design of novel hybrid nanomaterial using gold nano popcorn-attached single wall carbon nanotube for targeted diagnosis and selective photothermal treatment. Targeted SK-BR-3 human breast cancer cell sensing have been performed in 10 cancer cells/mL level, using surface enhanced Raman scattering of single walls carbon nanotube’s D and G bands. Our data show that S6 aptamer attached hybrid nanomaterial based SERS assay is highly sensitive to targeted human breast cancer SK-BR-3 cell line and it will be able to distinguish it from other non targeted MDA-MB breast cancer cell line and HaCaT normal skin cell line. Our results also show that 10 minutes of photothermal therapy treatment by 1.5 W/cm2 power, 785 nm laser is enough to kill cancer cells very effectively using S6 aptamer attached hybrid nanomaterials. Possible mechanisms for targeted sensing and operating principle for highly efficient photothermal therapy have been discussed. Our experimental results reported here open up a new possibility for using aptamers modified hybrid nanomaterial for reliable diagnosis and targeted therapy of cancer cell lines quickly. PMID:21842867

Gold nano-popcorn attached SWCNT hybrid nanomaterial for targeted diagnosis and photothermal therapy of human breast cancer cells.

PubMed

Beqa, Lule; Fan, Zhen; Singh, Anant Kumar; Senapati, Dulal; Ray, Paresh Chandra

2011-09-01

Breast cancer presents greatest challenge in health care in today's world. The key to ultimately successful treatment of breast cancer disease is an early and accurate diagnosis. Current breast cancer treatments are often associated with severe side effects. Driven by the need, we report the design of novel hybrid nanomaterial using gold nano popcorn-attached single wall carbon nanotube for targeted diagnosis and selective photothermal treatment. Targeted SK-BR-3 human breast cancer cell sensing have been performed in 10 cancer cells/mL level, using surface enhanced Raman scattering of single walls carbon nanotube's D and G bands. Our data show that S6 aptamer attached hybrid nanomaterial based SERS assay is highly sensitive to targeted human breast cancer SK-BR-3 cell line and it will be able to distinguish it from other non targeted MDA-MB breast cancer cell line and HaCaT normal skin cell line. Our results also show that 10 min of photothermal therapy treatment by 1.5 W/cm(2) power, 785 nm laser is enough to kill cancer cells very effectively using S6 aptamer attached hybrid nanomaterials. Possible mechanisms for targeted sensing and operating principle for highly efficient photothermal therapy have been discussed. Our experimental results reported here open up a new possibility for using aptamers modified hybrid nanomaterial for reliable diagnosis and targeted therapy of cancer cell lines quickly.

Enzymatic single-chain antibody tagging: a universal approach to targeted molecular imaging and cell homing in cardiovascular disease.

PubMed

Ta, H T; Prabhu, S; Leitner, E; Jia, F; von Elverfeldt, D; Jackson, Katherine E; Heidt, T; Nair, A K N; Pearce, H; von Zur Muhlen, C; Wang, X; Peter, K; Hagemeyer, C E

2011-08-05

Antibody-targeted delivery of imaging agents can enhance the sensitivity and accuracy of current imaging techniques. Similarly, homing of effector cells to disease sites increases the efficacy of regenerative cell therapy while reducing the number of cells required. Currently, targeting can be achieved via chemical conjugation to specific antibodies, which typically results in the loss of antibody functionality and in severe cell damage. An ideal conjugation technique should ensure retention of antigen-binding activity and functionality of the targeted biological component. To develop a biochemically robust, highly reproducible, and site-specific coupling method using the Staphylococcus aureus sortase A enzyme for the conjugation of a single-chain antibody (scFv) to nanoparticles and cells for molecular imaging and cell homing in cardiovascular diseases. This scFv specifically binds to activated platelets, which play a pivotal role in thrombosis, atherosclerosis, and inflammation. The conjugation procedure involves chemical and enzyme-mediated coupling steps. The scFv was successfully conjugated to iron oxide particles (contrast agents for magnetic resonance imaging) and to model cells. Conjugation efficiency ranged between 50% and 70%, and bioactivity of the scFv after coupling was preserved. The targeting of scFv-coupled cells and nanoparticles to activated platelets was strong and specific as demonstrated in in vitro static adhesion assays, in a flow chamber system, in mouse intravital microscopy, and in in vivo magnetic resonance imaging of mouse carotid arteries. This unique biotechnological approach provides a versatile and broadly applicable tool for procuring targeted regenerative cell therapy and targeted molecular imaging in cardiovascular and inflammatory diseases and beyond.

Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.

PubMed

Pollen, Alex A; Nowakowski, Tomasz J; Shuga, Joe; Wang, Xiaohui; Leyrat, Anne A; Lui, Jan H; Li, Nianzhen; Szpankowski, Lukasz; Fowler, Brian; Chen, Peilin; Ramalingam, Naveen; Sun, Gang; Thu, Myo; Norris, Michael; Lebofsky, Ronald; Toppani, Dominique; Kemp, Darnell W; Wong, Michael; Clerkson, Barry; Jones, Brittnee N; Wu, Shiquan; Knutsson, Lawrence; Alvarado, Beatriz; Wang, Jing; Weaver, Lesley S; May, Andrew P; Jones, Robert C; Unger, Marc A; Kriegstein, Arnold R; West, Jay A A

2014-10-01

Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.

Functional single-walled carbon nanotubes based on an integrin αvβ3 monoclonal antibody for highly efficient cancer cell targeting

NASA Astrophysics Data System (ADS)

Ou, Zhongmin; Wu, Baoyan; Xing, Da; Zhou, Feifan; Wang, Huiying; Tang, Yonghong

2009-03-01

The application of single-walled carbon nanotubes (SWNTs) in the field of biomedicine is becoming an entirely new and exciting topic. In this study, a novel functional SWNT based on an integrin αvβ3 monoclonal antibody was developed and was used for cancer cell targeting in vitro. SWNTs were first modified by phospholipid-bearing polyethylene glycol (PL-PEG). The PL-PEG functionalized SWNTs were then conjugated with protein A. A SWNT-integrin αvβ3 monoclonal antibody system (SWNT-PEG-mAb) was thus constructed by conjugating protein A with the fluorescein labeled integrin αvβ3 monoclonal antibody. In vitro study revealed that SWNT-PEG-mAb presented a high targeting efficiency on integrin αvβ3-positive U87MG cells with low cellular toxicity, while for integrin αvβ3-negative MCF-7 cells, the system had a low targeting efficiency, indicating that the high targeting to U87MG cells was due to the specific integrin targeting of the monoclonal antibody. In conclusion, SWNT-PEG-mAb developed in this research is a potential candidate for cancer imaging and drug delivery in cancer targeting therapy.

Selective uptake of single-walled carbon nanotubes by circulating monocytes for enhanced tumour delivery

NASA Astrophysics Data System (ADS)

Smith, Bryan Ronain; Ghosn, Eliver Eid Bou; Rallapalli, Harikrishna; Prescher, Jennifer A.; Larson, Timothy; Herzenberg, Leonore A.; Gambhir, Sanjiv Sam

2014-06-01

In cancer imaging, nanoparticle biodistribution is typically visualized in living subjects using `bulk' imaging modalities such as magnetic resonance imaging, computerized tomography and whole-body fluorescence. Accordingly, nanoparticle influx is observed only macroscopically, and the mechanisms by which they target cancer remain elusive. Nanoparticles are assumed to accumulate via several targeting mechanisms, particularly extravasation (leakage into tumour). Here, we show that, in addition to conventional nanoparticle-uptake mechanisms, single-walled carbon nanotubes are almost exclusively taken up by a single immune cell subset, Ly-6Chi monocytes (almost 100% uptake in Ly-6Chi monocytes, below 3% in all other circulating cells), and delivered to the tumour in mice. We also demonstrate that a targeting ligand (RGD) conjugated to nanotubes significantly enhances the number of single-walled carbon nanotube-loaded monocytes reaching the tumour (P < 0.001, day 7 post-injection). The remarkable selectivity of this tumour-targeting mechanism demonstrates an advanced immune-based delivery strategy for enhancing specific tumour delivery with substantial penetration.

A streamlined workflow for single-cells genome-wide copy-number profiling by low-pass sequencing of LM-PCR whole-genome amplification products.

PubMed

Ferrarini, Alberto; Forcato, Claudio; Buson, Genny; Tononi, Paola; Del Monaco, Valentina; Terracciano, Mario; Bolognesi, Chiara; Fontana, Francesca; Medoro, Gianni; Neves, Rui; Möhlendick, Birte; Rihawi, Karim; Ardizzoni, Andrea; Sumanasuriya, Semini; Flohr, Penny; Lambros, Maryou; de Bono, Johann; Stoecklein, Nikolas H; Manaresi, Nicolò

2018-01-01

Chromosomal instability and associated chromosomal aberrations are hallmarks of cancer and play a critical role in disease progression and development of resistance to drugs. Single-cell genome analysis has gained interest in latest years as a source of biomarkers for targeted-therapy selection and drug resistance, and several methods have been developed to amplify the genomic DNA and to produce libraries suitable for Whole Genome Sequencing (WGS). However, most protocols require several enzymatic and cleanup steps, thus increasing the complexity and length of protocols, while robustness and speed are key factors for clinical applications. To tackle this issue, we developed a single-tube, single-step, streamlined protocol, exploiting ligation mediated PCR (LM-PCR) Whole Genome Amplification (WGA) method, for low-pass genome sequencing with the Ion Torrent™ platform and copy number alterations (CNAs) calling from single cells. The method was evaluated on single cells isolated from 6 aberrant cell lines of the NCI-H series. In addition, to demonstrate the feasibility of the workflow on clinical samples, we analyzed single circulating tumor cells (CTCs) and white blood cells (WBCs) isolated from the blood of patients affected by prostate cancer or lung adenocarcinoma. The results obtained show that the developed workflow generates data accurately representing whole genome absolute copy number profiles of single cell and allows alterations calling at resolutions down to 100 Kbp with as few as 200,000 reads. The presented data demonstrate the feasibility of the Ampli1™ WGA-based low-pass workflow for detection of CNAs in single tumor cells which would be of particular interest for genome-driven targeted therapy selection and for monitoring of disease progression.

Fast targeted gene transfection and optogenetic modification of single neurons using femtosecond laser irradiation

PubMed Central

Antkowiak, Maciej; Torres-Mapa, Maria Leilani; Witts, Emily C.; Miles, Gareth B.; Dholakia, Kishan; Gunn-Moore, Frank J.

2013-01-01

A prevailing problem in neuroscience is the fast and targeted delivery of DNA into selected neurons. The development of an appropriate methodology would enable the transfection of multiple genes into the same cell or different genes into different neighboring cells as well as rapid cell selective functionalization of neurons. Here, we show that optimized femtosecond optical transfection fulfills these requirements. We also demonstrate successful optical transfection of channelrhodopsin-2 in single selected neurons. We extend the functionality of this technique for wider uptake by neuroscientists by using fast three-dimensional laser beam steering enabling an image-guided “point-and-transfect” user-friendly transfection of selected cells. A sub-second transfection timescale per cell makes this method more rapid by at least two orders of magnitude when compared to alternative single-cell transfection techniques. This novel technology provides the ability to carry out large-scale cell selective genetic studies on neuronal ensembles and perform rapid genetic programming of neural circuits. PMID:24257461

Fast targeted gene transfection and optogenetic modification of single neurons using femtosecond laser irradiation.

PubMed

Antkowiak, Maciej; Torres-Mapa, Maria Leilani; Witts, Emily C; Miles, Gareth B; Dholakia, Kishan; Gunn-Moore, Frank J

2013-11-21

A prevailing problem in neuroscience is the fast and targeted delivery of DNA into selected neurons. The development of an appropriate methodology would enable the transfection of multiple genes into the same cell or different genes into different neighboring cells as well as rapid cell selective functionalization of neurons. Here, we show that optimized femtosecond optical transfection fulfills these requirements. We also demonstrate successful optical transfection of channelrhodopsin-2 in single selected neurons. We extend the functionality of this technique for wider uptake by neuroscientists by using fast three-dimensional laser beam steering enabling an image-guided "point-and-transfect" user-friendly transfection of selected cells. A sub-second transfection timescale per cell makes this method more rapid by at least two orders of magnitude when compared to alternative single-cell transfection techniques. This novel technology provides the ability to carry out large-scale cell selective genetic studies on neuronal ensembles and perform rapid genetic programming of neural circuits.

Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands.

PubMed

Luo, Yan; Wang, Yongsheng; Liu, Jun; Cui, Chenchen; Wu, Yongyan; Lan, Hui; Chen, Qi; Liu, Xu; Quan, Fusheng; Guo, Zekun; Zhang, Yong

2016-02-08

Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins. Transcription-activator-like effector (TALE) nucleases (TALENs) are an efficient genome-editing tool. However, the off-target effects may lead to unintended gene mutations. In this study, we constructed TALENs and TALE nickases directed against exon 2 of the bovine β-lactoglobulin (BLG) locus. The nickases can induce a site-specific DNA single-strand break, without inducing double-strand break and nonhomologous end joining mediated gene mutation, and lower cell apoptosis rate than TALENs. After co-transfecting the bovine fetal fibroblasts with human serum albumin (HSA) gene-targeting vector and TALE nickase expression vectors, approximately 4.8% (40/835) of the cell clones contained HSA at BLG locus. Unexpectedly, one homozygous gene-targeted cell clone (1/835, 0.1%) was obtained by targeting both alleles of BLG in a single round of transfection. The recombinant protein mimicking the endogenous BLG was highly expressed and correctly folded in the mammary glands of the targeted cows, and the expression level of HSA was significantly increased in the homozygous targeted cows. Results suggested that the combination of TALE nickase-mediated gene targeting and somatic cell nuclear transfer is a feasible and safe approach in producing gene-targeted livestock.

Potentials of single-cell biology in identification and validation of disease biomarkers.

PubMed

Niu, Furong; Wang, Diane C; Lu, Jiapei; Wu, Wei; Wang, Xiangdong

2016-09-01

Single-cell biology is considered a new approach to identify and validate disease-specific biomarkers. However, the concern raised by clinicians is how to apply single-cell measurements for clinical practice, translate the message of single-cell systems biology into clinical phenotype or explain alterations of single-cell gene sequencing and function in patient response to therapies. This study is to address the importance and necessity of single-cell gene sequencing in the identification and development of disease-specific biomarkers, the definition and significance of single-cell biology and single-cell systems biology in the understanding of single-cell full picture, the development and establishment of whole-cell models in the validation of targeted biological function and the figure and meaning of single-molecule imaging in single cell to trace intra-single-cell molecule expression, signal, interaction and location. We headline the important role of single-cell biology in the discovery and development of disease-specific biomarkers with a special emphasis on understanding single-cell biological functions, e.g. mechanical phenotypes, single-cell biology, heterogeneity and organization of genome function. We have reason to believe that such multi-dimensional, multi-layer, multi-crossing and stereoscopic single-cell biology definitely benefits the discovery and development of disease-specific biomarkers. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Drop-on-Demand Single Cell Isolation and Total RNA Analysis

PubMed Central

Moon, Sangjun; Kim, Yun-Gon; Dong, Lingsheng; Lombardi, Michael; Haeggstrom, Edward; Jensen, Roderick V.; Hsiao, Li-Li; Demirci, Utkan

2011-01-01

Technologies that rapidly isolate viable single cells from heterogeneous solutions have significantly contributed to the field of medical genomics. Challenges remain both to enable efficient extraction, isolation and patterning of single cells from heterogeneous solutions as well as to keep them alive during the process due to a limited degree of control over single cell manipulation. Here, we present a microdroplet based method to isolate and pattern single cells from heterogeneous cell suspensions (10% target cell mixture), preserve viability of the extracted cells (97.0±0.8%), and obtain genomic information from isolated cells compared to the non-patterned controls. The cell encapsulation process is both experimentally and theoretically analyzed. Using the isolated cells, we identified 11 stem cell markers among 1000 genes and compare to the controls. This automated platform enabling high-throughput cell manipulation for subsequent genomic analysis employs fewer handling steps compared to existing methods. PMID:21412416

Phosphatidylserine-targeting antibodies augment the anti-tumorigenic activity of anti-PD-1 therapy by enhancing immune activation and downregulating pro-oncogenic factors induced by T-cell checkpoint inhibition in murine triple-negative breast cancers.

PubMed

Gray, Michael J; Gong, Jian; Hatch, Michaela M S; Nguyen, Van; Hughes, Christopher C W; Hutchins, Jeff T; Freimark, Bruce D

2016-05-11

The purpose of this study was to investigate the potential of antibody-directed immunotherapy targeting the aminophospholipid phosphatidylserine, which promotes immunosuppression when exposed in the tumor microenvironment, alone and in combination with antibody treatment towards the T-cell checkpoint inhibitor PD-1 in breast carcinomas, including triple-negative breast cancers. Immune-competent mice bearing syngeneic EMT-6 or E0771 tumors were subjected to treatments comprising of a phosphatidylserine-targeting and an anti-PD-1 antibody either as single or combinational treatments. Anti-tumor effects were determined by tumor growth inhibition and changes in overall survival accompanying each treatment. The generation of a tumor-specific immune response in animals undergoing complete tumor regression was assessed by secondary tumor cell challenge and splenocyte-produced IFNγ in the presence or absence of irradiated tumor cells. Changes in the presence of tumor-infiltrating lymphocytes were assessed by flow cytometry, while mRNA-based immune profiling was determined using NanoString PanCancer Immune Profiling Panel analysis. Treatment by a phosphatidylserine-targeting antibody inhibits in-vivo growth and significantly enhances the anti-tumor activity of antibody-mediated PD-1 therapy, including providing a distinct survival advantage over treatment by either single agent. Animals in which complete tumor regression occurred with combination treatments were resistant to secondary tumor challenge and presented heightened expression levels of splenocyte-produced IFNγ. Combinational treatment by a phosphatidylserine-targeting antibody with anti-PD-1 therapy increased the number of tumor-infiltrating lymphocytes more than that observed with single-arm therapies. Finally, immunoprofiling analysis revealed that the combination of anti-phosphatidylserine targeting antibody and anti-PD-1 therapy enhanced tumor-infiltrating lymphocytes, and increased expression of pro-immunosurveillance-associated cytokines while significantly decreasing expression of pro-tumorigenic cytokines that were induced by single anti-PD-1 therapy. Our data suggest that antibody therapy targeting phosphatidylserine-associated immunosuppression, which has activity as a single agent, can significantly enhance immunotherapies targeting the PD-1 pathway in murine breast neoplasms, including triple-negative breast cancers.

A generalized target theory and its applications.

PubMed

Zhao, Lei; Mi, Dong; Hu, Bei; Sun, Yeqing

2015-09-28

Different radiobiological models have been proposed to estimate the cell-killing effects, which are very important in radiotherapy and radiation risk assessment. However, most applied models have their own scopes of application. In this work, by generalizing the relationship between "hit" and "survival" in traditional target theory with Yager negation operator in Fuzzy mathematics, we propose a generalized target model of radiation-induced cell inactivation that takes into account both cellular repair effects and indirect effects of radiation. The simulation results of the model and the rethinking of "the number of targets in a cell" and "the number of hits per target" suggest that it is only necessary to investigate the generalized single-hit single-target (GSHST) in the present theoretical frame. Analysis shows that the GSHST model can be reduced to the linear quadratic model and multitarget model in the low-dose and high-dose regions, respectively. The fitting results show that the GSHST model agrees well with the usual experimental observations. In addition, the present model can be used to effectively predict cellular repair capacity, radiosensitivity, target size, especially the biologically effective dose for the treatment planning in clinical applications.

Gene targeting and cloning in pigs using fetal liver derived cells.

PubMed

Waghmare, Sanjeev K; Estrada, Jose; Reyes, Luz; Li, Ping; Ivary, Bess; Sidner, Richard A; Burlak, Chris; Tector, A Joseph

2011-12-01

Since there are no pig embryonic stem cells, pig genetic engineering is done in fetal fibroblasts that remain totipotent for only 3 to 5 wk. Nuclear donor cells that remain totipotent for longer periods of time would facilitate complicated genetic engineering in pigs. The goal of this study was to test the feasibility of using fetal liver-derived cells (FLDC) to perform gene targeting, and create a genetic knockout pig. FLDC were isolated and processed using a human liver stem cell protocol. Single copy α-1,3-galactosyl transferase knockout (GTKO) FLDCs were created using electroporation and neomycin resistant colonies were screened using PCR. Homozygous GTKO cells were created through loss of heterozygosity mutations in single GTKO FLDCs. Double GTKO FLDCs were used in somatic cell nuclear transfer (SCNT) to create GTKO pigs. FLDCs grew for more than 80 population doublings, maintaining normal karyotype. Gene targeting and loss of heterozygosity mutations produced homozygous GTKO FLDCs. FLDCs used in SCNT gave rise to homozygous GTKO pigs. FDLCs can be used in gene targeting and SCNT to produce genetically modified pigs. The increased life span in culture compared to fetal fibroblasts may facilitate genetic engineering in the pig. Copyright © 2011 Elsevier Inc. All rights reserved.

Stable Gene Targeting in Human Cells Using Single-Strand Oligonucleotides with Modified Bases

PubMed Central

Rios, Xavier; Briggs, Adrian W.; Christodoulou, Danos; Gorham, Josh M.; Seidman, Jonathan G.; Church, George M.

2012-01-01

Recent advances allow multiplexed genome engineering in E. coli, employing easily designed oligonucleotides to edit multiple loci simultaneously. A similar technology in human cells would greatly expedite functional genomics, both by enhancing our ability to test how individual variants such as single nucleotide polymorphisms (SNPs) are related to specific phenotypes, and potentially allowing simultaneous mutation of multiple loci. However, oligo-mediated targeting of human cells is currently limited by low targeting efficiencies and low survival of modified cells. Using a HeLa-based EGFP-rescue reporter system we show that use of modified base analogs can increase targeting efficiency, in part by avoiding the mismatch repair machinery. We investigate the effects of oligonucleotide toxicity and find a strong correlation between the number of phosphorothioate bonds and toxicity. Stably EGFP-corrected cells were generated at a frequency of ~0.05% with an optimized oligonucleotide design combining modified bases and reduced number of phosphorothioate bonds. We provide evidence from comparative RNA-seq analysis suggesting cellular immunity induced by the oligonucleotides might contribute to the low viability of oligo-corrected cells. Further optimization of this method should allow rapid and scalable genome engineering in human cells. PMID:22615794

Single-cell optoporation and transfection using femtosecond laser and optical tweezers.

PubMed

Waleed, Muhammad; Hwang, Sun-Uk; Kim, Jung-Dae; Shabbir, Irfan; Shin, Sang-Mo; Lee, Yong-Gu

2013-01-01

In this paper, we demonstrate a new single-cell optoporation and transfection technique using a femtosecond Gaussian laser beam and optical tweezers. Tightly focused near-infrared (NIR) femtosecond laser pulse was employed to transiently perforate the cellular membrane at a single point in MCF-7 cancer cells. A distinct technique was developed by trapping the microparticle using optical tweezers to focus the femtosecond laser precisely on the cell membrane to puncture it. Subsequently, an external gene was introduced in the cell by trapping and inserting the same plasmid-coated microparticle into the optoporated cell using optical tweezers. Various experimental parameters such as femtosecond laser exposure power, exposure time, puncture hole size, exact focusing of the femtosecond laser on the cell membrane, and cell healing time were closely analyzed to create the optimal conditions for cell viability. Following the insertion of plasmid-coated microparticles in the cell, the targeted cells exhibited green fluorescent protein (GFP) under the fluorescent microscope, hence confirming successful transfection into the cell. This new optoporation and transfection technique maximizes the level of selectivity and control over the targeted cell, and this may be a breakthrough method through which to induce controllable genetic changes in the cell.

Self-targeted salinomycin-loaded DSPE-PEG-methotrexate nanomicelles for targeting both head and neck squamous cell carcinoma cancer cells and cancer stem cells.

PubMed

Zhu, Minhui; Chen, Shicai; Hua, Libo; Zhang, Caiyun; Chen, Mengjie; Chen, Donghui; Dong, Yinmei; Zhang, Yingying; Li, Meng; Song, Xianmin; Chen, Huaiwen; Zheng, Hongliang

2017-02-01

To target both head and neck squamous cell carcinoma (HNSCC) cells and cancer stem cells (CSCs) by salinomycin-loaded DSPE-PEG-MTX (synthesized using DSPE-PEG2000-NH2 and methotrexate) nanomicelles (M-SAL-MTX). The characterization, antitumor activity and mechanism of M-SAL-MTX were evaluated. M-SAL-MTX showed enhanced inhibitory effect toward both HNSCC CSCs and non-CSCs compared with a single treatment of methotrexate and salinomycin. In nude mice-bearing HNSCC xenografts, M-SAL-MTX suppressed tumor growth more effectively than other controls including combination of methotrexate and salinomycin. Therefore, M-SAL-MTX may provide a strategy for treating HNSCC by targeting both HNSCC CSCs and HNSCC cells.

Identification and genetic analysis of cancer cells with PCR-activated cell sorting

PubMed Central

Eastburn, Dennis J.; Sciambi, Adam; Abate, Adam R.

2014-01-01

Cell sorting is a central tool in life science research for analyzing cellular heterogeneity or enriching rare cells out of large populations. Although methods like FACS and FISH-FC can characterize and isolate cells from heterogeneous populations, they are limited by their reliance on antibodies, or the requirement to chemically fix cells. We introduce a new cell sorting technology that robustly sorts based on sequence-specific analysis of cellular nucleic acids. Our approach, PCR-activated cell sorting (PACS), uses TaqMan PCR to detect nucleic acids within single cells and trigger their sorting. With this method, we identified and sorted prostate cancer cells from a heterogeneous population by performing >132 000 simultaneous single-cell TaqMan RT-PCR reactions targeting vimentin mRNA. Following vimentin-positive droplet sorting and downstream analysis of recovered nucleic acids, we found that cancer-specific genomes and transcripts were significantly enriched. Additionally, we demonstrate that PACS can be used to sort and enrich cells via TaqMan PCR reactions targeting single-copy genomic DNA. PACS provides a general new technical capability that expands the application space of cell sorting by enabling sorting based on cellular information not amenable to existing approaches. PMID:25030902

Planar optical waveguide based sandwich assay sensors and processes for the detection of biological targets including protein markers, pathogens and cellular debris

DOEpatents

Martinez, Jennifer S [Santa Fe, NM; Swanson, Basil I [Los Alamos, NM; Grace, Karen M [Los Alamos, NM; Grace, Wynne K [Los Alamos, NM; Shreve, Andrew P [Santa Fe, NM

2009-06-02

An assay element is described including recognition ligands bound to a film on a single mode planar optical waveguide, the film from the group of a membrane, a polymerized bilayer membrane, and a self-assembled monolayer containing polyethylene glycol or polypropylene glycol groups therein and an assay process for detecting the presence of a biological target is described including injecting a biological target-containing sample into a sensor cell including the assay element, with the recognition ligands adapted for binding to selected biological targets, maintaining the sample within the sensor cell for time sufficient for binding to occur between selected biological targets within the sample and the recognition ligands, injecting a solution including a reporter ligand into the sensor cell; and, interrogating the sample within the sensor cell with excitation light from the waveguide, the excitation light provided by an evanescent field of the single mode penetrating into the biological target-containing sample to a distance of less than about 200 nanometers from the waveguide thereby exciting the fluorescent-label in any bound reporter ligand within a distance of less than about 200 nanometers from the waveguide and resulting in a detectable signal.

A single-molecule view of gene regulation in cancer

NASA Astrophysics Data System (ADS)

Larson, Daniel

2013-03-01

Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. Steroid receptors coordinate a diverse range of responses in higher eukaryotes and are involved in a wide range of human diseases, including cancer. Steroid receptor response elements are present throughout the human genome and modulate chromatin remodeling and transcription in both a local and long-range fashion. As such, steroid receptor-mediated transcription is a paradigm of genetic control in the metazoan nucleus. Moreover, the ligand-dependent nature of these transcription factors makes them appealing targets for therapeutic intervention, necessitating a quantitative understanding of how receptors control output from target genes. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single gene and follow dynamic synthesis of RNA from the activated locus. The response delay is a measure of time required for chromatin remodeling at a single gene.

Control of Established Colon Cancer Xenografts Using a Novel Humanized Single Chain Antibody-Streptococcal Superantigen Fusion Protein Targeting the 5T4 Oncofetal Antigen

PubMed Central

Patterson, Kelcey G.; Dixon Pittaro, Jennifer L.; Bastedo, Peter S.; Hess, David A.; Haeryfar, S. M. Mansour; McCormick, John K.

2014-01-01

Superantigens (SAgs) are microbial toxins that cross-link T cell receptors with major histocompatibility class II (MHC-II) molecules leading to the activation of large numbers of T cells. Herein, we describe the development and preclinical testing of a novel tumor-targeted SAg (TTS) therapeutic built using the streptococcal pyrogenic exotoxin C (SpeC) SAg and targeting cancer cells expressing the 5T4 tumor-associated antigen (TAA). To inhibit potentially harmful widespread immune cell activation, a SpeC mutation within the high-affinity MHC-II binding interface was generated (SpeCD203A) that demonstrated a pronounced reduction in mitogenic activity, yet this mutant could still induce immune cell-mediated cancer cell death in vitro. To target 5T4+ cancer cells, we engineered a humanized single chain variable fragment (scFv) antibody to recognize 5T4 (scFv5T4). Specific targeting of scFv5T4 was verified. SpeCD203A fused to scFv5T4 maintained the ability to activate and induce immune cell-mediated cytotoxicity of colorectal cancer cells. Using a xenograft model of established human colon cancer, we demonstrated that the SpeC-based TTS was able to control the growth and spread of large tumors in vivo. This required both TAA targeting by scFv5T4 and functional SAg activity. These studies lay the foundation for the development of streptococcal SAgs as ‘next-generation’ TTSs for cancer immunotherapy. PMID:24736661

A Single Multiplex crRNA Array for FnCpf1-Mediated Human Genome Editing.

PubMed

Sun, Huihui; Li, Fanfan; Liu, Jie; Yang, Fayu; Zeng, Zhenhai; Lv, Xiujuan; Tu, Mengjun; Liu, Yeqing; Ge, Xianglian; Liu, Changbao; Zhao, Junzhao; Zhang, Zongduan; Qu, Jia; Song, Zongming; Gu, Feng

2018-06-15

Cpf1 has been harnessed as a tool for genome manipulation in various species because of its simplicity and high efficiency. Our recent study demonstrated that FnCpf1 could be utilized for human genome editing with notable advantages for target sequence selection due to the flexibility of the protospacer adjacent motif (PAM) sequence. Multiplex genome editing provides a powerful tool for targeting members of multigene families, dissecting gene networks, modeling multigenic disorders in vivo, and applying gene therapy. However, there are no reports at present that show FnCpf1-mediated multiplex genome editing via a single customized CRISPR RNA (crRNA) array. In the present study, we utilize a single customized crRNA array to simultaneously target multiple genes in human cells. In addition, we also demonstrate that a single customized crRNA array to target multiple sites in one gene could be achieved. Collectively, FnCpf1, a powerful genome-editing tool for multiple genomic targets, can be harnessed for effective manipulation of the human genome. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Single-Cell in Situ RNA Analysis With Switchable Fluorescent Oligonucleotides.

PubMed

Xiao, Lu; Guo, Jia

2018-01-01

Comprehensive RNA analyses in individual cells in their native spatial contexts promise to transform our understanding of normal physiology and disease pathogenesis. Here we report a single-cell in situ RNA analysis approach using switchable fluorescent oligonucleotides (SFO). In this method, transcripts are first hybridized by pre-decoding oligonucleotides. These oligonucleotides subsequently recruit SFO to stain their corresponding RNA targets. After fluorescence imaging, all the SFO in the whole specimen are simultaneously removed by DNA strand displacement reactions. Through continuous cycles of target staining, fluorescence imaging, and SFO removal, a large number of different transcripts can be identified by unique fluorophore sequences and visualized at the optical resolution. To demonstrate the feasibility of this approach, we show that the hybridized SFO can be efficiently stripped by strand displacement reactions within 30 min. We also demonstrate that this SFO removal process maintains the integrity of the RNA targets and the pre-decoding oligonucleotides, and keeps them hybridized. Applying this approach, we show that transcripts can be restained in at least eight hybridization cycles with high analysis accuracy, which theoretically would enable the whole transcriptome to be quantified at the single molecule sensitivity in individual cells. This in situ RNA analysis technology will have wide applications in systems biology, molecular diagnosis, and targeted therapies.

Retargeting of adenovirus vectors through genetic fusion of a single-chain or single-domain antibody to capsid protein IX.

PubMed

Poulin, Kathy L; Lanthier, Robert M; Smith, Adam C; Christou, Carin; Risco Quiroz, Milagros; Powell, Karen L; O'Meara, Ryan W; Kothary, Rashmi; Lorimer, Ian A; Parks, Robin J

2010-10-01

Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions.

Real time observation of the ultrasound stimulated disintegration of optically trapped microbubbles in proximity to biological cells

NASA Astrophysics Data System (ADS)

Prentice, Paul; MacDonald, Michael P.; Cuschieri, Alfred; Dholakia, Kishan; Campbell, Paul

2005-08-01

Cells that are exposed to varying amounts of ultrasonic energy in the presence of ultrasound contrast agent (UCA) may undergo either permanent cell membrane damage (lethal sonoporation), or a transient enhancement of membrane permeability (reversible or non lethal sonoporation). The merits of each mode are clear; lethal sonoporation constitutes a significant tumour therapy weapon, whilst its less intrusive counterpart, reversible sonoporation, represents an effective non-invasive targeted drug delivery technique. Our working hypothesis for understanding this problem was that the root cause and effect in sonoporation involves the interaction of individual cells with single microbubbles, and to that end we devised an experiment that facilitates video rate observation of this specific scenario under well defined optical control. Specifically, we have constructed an innovative hybridization apparatus involving holographic optical trapping of single and multiple UCA microbubbles, together with the facility to irradiate with MHz pulsed ultrasound energy in the presence cancerous cells. This approach allows the isolation of a target microbubble from a resident population and the relocation to a [controllable] predetermined position relative to a cell within a monolayer. Frame extraction from standard framing rate video microscopy demonstrates the individuality of single microbubble-cell interactions. We describe a fluorescence microscopy protocol that will allow future study of the potential to deliver molecular species to cells, the dependence of the delivery on the initial microbubble-cell distance and to determine the targeted cell survival.

Diverse Class 2 CRISPR-Cas Effector Proteins for Genome Engineering Applications.

PubMed

Pyzocha, Neena K; Chen, Sidi

2018-02-16

CRISPR-Cas genome editing technologies have revolutionized modern molecular biology by making targeted DNA edits simple and scalable. These technologies are developed by domesticating naturally occurring microbial adaptive immune systems that display wide diversity of functionality for targeted nucleic acid cleavage. Several CRISPR-Cas single effector enzymes have been characterized and engineered for use in mammalian cells. The unique properties of the single effector enzymes can make a critical difference in experimental use or targeting specificity. This review describes known single effector enzymes and discusses their use in genome engineering applications.

Single-cell multimodal profiling reveals cellular epigenetic heterogeneity.

PubMed

Cheow, Lih Feng; Courtois, Elise T; Tan, Yuliana; Viswanathan, Ramya; Xing, Qiaorui; Tan, Rui Zhen; Tan, Daniel S W; Robson, Paul; Loh, Yuin-Han; Quake, Stephen R; Burkholder, William F

2016-10-01

Sample heterogeneity often masks DNA methylation signatures in subpopulations of cells. Here, we present a method to genotype single cells while simultaneously interrogating gene expression and DNA methylation at multiple loci. We used this targeted multimodal approach, implemented on an automated, high-throughput microfluidic platform, to assess primary lung adenocarcinomas and human fibroblasts undergoing reprogramming by profiling epigenetic variation among cell types identified through genotyping and transcriptional analysis.

Small molecule targeting of the actin associating protein tropomyosin Tpm3.1 increases neuroblastoma cell response to Rac inhibition of multicellular invasion.

PubMed

Mitchell, Camilla B; Stehn, Justine R; O'Neill, Geraldine M

2018-05-12

The migration and invasion of cells through tissues in the body is facilitated by a dynamic actin cytoskeleton. The actin-associating protein, tropomyosin Tpm3.1 has emerged to play important roles in cell migration and invasion. To date, investigations have focused on single cell migration and invasion where Tpm3.1 expression is inversely associated with Rac GTPase-mediated cell invasion. While single cell and collective cell invasion have many features in common, collective invasion is additionally impacted by cell-cell adhesion, and the role of Tpm3.1 in collective invasion has not been established. In the present study we have modelled multicellular invasion using neuroblastoma spheroids embedded in 3D collagen and analysed the function of Tpm3.1 using recently established compounds that target the Tpm3.1 C-terminus. The major findings from our study reveal that combined Rac inhibition and Tpm3.1 targeting result in greater inhibition of multicellular invasion than either treatment alone. Together, the data suggest that Tpm3.1 disruption sensitizes neuroblastoma cells to Rac inhibition of multicellular invasion. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Development of Cell-SELEX Technology and Its Application in Cancer Diagnosis and Therapy.

PubMed

Chen, Man; Yu, Yuanyuan; Jiang, Feng; Zhou, Junwei; Li, Yongshu; Liang, Chao; Dang, Lei; Lu, Aiping; Zhang, Ge

2016-12-10

SELEX (systematic evolution of ligands by exponential enrichment) is a process involving the progressive isolation of high selective ssDNA/RNA from a combinatorial single-stranded oligonucleotide library through repeated rounds of binding, partitioning and amplification. SELEX-derived single-stranded DNA/RNA molecules, called aptamers, are selected against a wide range of targets, including purified proteins, live cells, tissues, microorganisms, small molecules and so on. With the development of SELEX technology over the last two decades, various modified SELEX processes have been arisen. A majority of aptamers are selected against purified proteins through traditional SELEX. Unfortunately, more and more evidence showed aptamers selected against purified membrane proteins failed to recognize their targets in live cells. Cell-SELEX could develop aptamers against a particular target cell line to discriminate this cell line from others. Therefore, cell-SELEX has been widely used to select aptamers for the application of both diagnosis and therapy of various diseases, especially for cancer. In this review, the advantages and limitations of cell-SELEX and SELEX against purified protein will be compared. Various modified cell-SELEX techniques will be summarized, and application of cell-SELEX in cancer diagnosis and therapy will be discussed.

Development of Cell-SELEX Technology and Its Application in Cancer Diagnosis and Therapy

PubMed Central

Chen, Man; Yu, Yuanyuan; Jiang, Feng; Zhou, Junwei; Li, Yongshu; Liang, Chao; Dang, Lei; Lu, Aiping; Zhang, Ge

2016-01-01

SELEX (systematic evolution of ligands by exponential enrichment) is a process involving the progressive isolation of high selective ssDNA/RNA from a combinatorial single-stranded oligonucleotide library through repeated rounds of binding, partitioning and amplification. SELEX-derived single-stranded DNA/RNA molecules, called aptamers, are selected against a wide range of targets, including purified proteins, live cells, tissues, microorganisms, small molecules and so on. With the development of SELEX technology over the last two decades, various modified SELEX processes have been arisen. A majority of aptamers are selected against purified proteins through traditional SELEX. Unfortunately, more and more evidence showed aptamers selected against purified membrane proteins failed to recognize their targets in live cells. Cell-SELEX could develop aptamers against a particular target cell line to discriminate this cell line from others. Therefore, cell-SELEX has been widely used to select aptamers for the application of both diagnosis and therapy of various diseases, especially for cancer. In this review, the advantages and limitations of cell-SELEX and SELEX against purified protein will be compared. Various modified cell-SELEX techniques will be summarized, and application of cell-SELEX in cancer diagnosis and therapy will be discussed. PMID:27973403

Pulsed Irradiation Improves Target Selectivity of Infrared Laser-Evoked Gene Operator for Single-Cell Gene Induction in the Nematode C. elegans

PubMed Central

Suzuki, Motoshi; Toyoda, Naoya; Takagi, Shin

2014-01-01

Methods for turning on/off gene expression at the experimenter’s discretion would be useful for various biological studies. Recently, we reported on a novel microscope system utilizing an infrared laser-evoked gene operator (IR-LEGO) designed for inducing heat shock response efficiently in targeted single cells in living organisms without cell damage, thereby driving expression of a transgene under the control of a heat shock promoter. Although the original IR-LEGO can be successfully used for gene induction, several limitations hinder its wider application. Here, using the nematode Caenorhabditis elegans (C. elegans) as a subject, we have made improvements in IR-LEGO. For better spatial control of heating, a pulsed irradiation method using an optical chopper was introduced. As a result, single cells of C. elegans embryos as early as the 2-cell stage and single neurons in ganglia can be induced to express genes selectively. In addition, the introduction of site-specific recombination systems to IR-LEGO enables the induction of gene expression controlled by constitutive and cell type-specific promoters. The strategies adopted here will be useful for future applications of IR-LEGO to other organisms. PMID:24465705

Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells.

PubMed

Agasti, Sarit S; Liong, Monty; Peterson, Vanessa M; Lee, Hakho; Weissleder, Ralph

2012-11-14

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.

Cell-specific transmembrane injection of molecular cargo with gold nanoparticle-generated transient plasmonic nanobubbles

PubMed Central

Lukianova-Hleb, Ekaterina Y.; Wagner, Daniel S.; Brenner, Malcolm K.; Lapotko, Dmitri O.

2012-01-01

Optimal cell therapies require efficient, selective and rapid delivery of molecular cargo into target cells without compromising their viability. Achieving these goals ex vivo in bulk heterogeneous multi-cell systems such as human grafts is impeded by low selectivity and speed of cargo delivery and by significant damage to target and non-target cells. We have developed a cell level approach for selective and guided trans-membrane injection of extracellular cargo into specific target cells using transient plasmonic nanobubbles (PNB) as cell-specific nano-injectors. As a technical platform for this method we developed a laser flow cell processing system. The PNB injection method and flow system were tested in heterogeneous cell suspensions of target and non-target cells for delivery of Dextran-FITC dye into squamous cell carcinoma HN31 cells and transfection of human T-cells with a green fluorescent protein-encoding plasmid. In both models the method demonstrated single cell type selectivity, high efficacy of delivery (96% both for HN31 cells T-cells), speed of delivery (nanoseconds) and viability of treated target cells (96% for HN31 cells and 75% for T-cells). The PNB injection method may therefore be beneficial for real time processing of human grafts without removal of physiologically important cells. PMID:22521612

Cell-specific transmembrane injection of molecular cargo with gold nanoparticle-generated transient plasmonic nanobubbles.

PubMed

Lukianova-Hleb, Ekaterina Y; Wagner, Daniel S; Brenner, Malcolm K; Lapotko, Dmitri O

2012-07-01

Optimal cell therapies require efficient, selective and rapid delivery of molecular cargo into target cells without compromising their viability. Achieving these goals ex vivo in bulk heterogeneous multi-cell systems such as human grafts is impeded by low selectivity and speed of cargo delivery and by significant damage to target and non-target cells. We have developed a cell level approach for selective and guided transmembrane injection of extracellular cargo into specific target cells using transient plasmonic nanobubbles (PNB) as cell-specific nano-injectors. As a technical platform for this method we developed a laser flow cell processing system. The PNB injection method and flow system were tested in heterogeneous cell suspensions of target and non-target cells for delivery of Dextran-FITC dye into squamous cell carcinoma HN31 cells and transfection of human T-cells with a green fluorescent protein-encoding plasmid. In both models the method demonstrated single cell type selectivity, high efficacy of delivery (96% both for HN31 cells T-cells), speed of delivery (nanoseconds) and viability of treated target cells (96% for HN31 cells and 75% for T-cells). The PNB injection method may therefore be beneficial for real time processing of human grafts without removal of physiologically important cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher

NASA Astrophysics Data System (ADS)

Normanno, Davide; Boudarène, Lydia; Dugast-Darzacq, Claire; Chen, Jiji; Richter, Christian; Proux, Florence; Bénichou, Olivier; Voituriez, Raphaël; Darzacq, Xavier; Dahan, Maxime

2015-07-01

Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein-DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells.

Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii.

PubMed

Jiang, Wenzhi; Brueggeman, Andrew J; Horken, Kempton M; Plucinak, Thomas M; Weeks, Donald P

2014-11-01

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a powerful and precise tool for targeted gene modification (e.g., gene knockout and gene replacement) in numerous eukaryotic organisms. Initial attempts to apply this technology to a model, the single-cell alga, Chlamydomonas reinhardtii, failed to yield cells containing edited genes. To determine if the Cas9 and single guide RNA (sgRNA) genes were functional in C. reinhardtii, we tested the ability of a codon-optimized Cas9 gene along with one of four different sgRNAs to cause targeted gene disruption during a 24-h period immediately following transformation. All three exogenously supplied gene targets as well as the endogenous FKB12 (rapamycin sensitivity) gene of C. reinhardtii displayed distinct Cas9/sgRNA-mediated target site modifications as determined by DNA sequencing of cloned PCR amplicons of the target site region. Success in transient expression of Cas9 and sgRNA genes contrasted with the recovery of only a single rapamycin-resistant colony bearing an appropriately modified FKB12 target site in 16 independent transformation experiments involving >10(9) cells. Failure to recover transformants with intact or expressed Cas9 genes following transformation with the Cas9 gene alone (or even with a gene encoding a Cas9 lacking nuclease activity) provided strong suggestive evidence for Cas9 toxicity when Cas9 is produced constitutively in C. reinhardtii. The present results provide compelling evidence that Cas9 and sgRNA genes function properly in C. reinhardtii to cause targeted gene modifications and point to the need for a focus on development of methods to properly stem Cas9 production and/or activity following gene editing. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Integrated single-cell genetic and transcriptional analysis suggests novel drivers of chronic lymphocytic leukemia

PubMed Central

Wang, Lili; Fan, Jean; Francis, Joshua M.; Georghiou, George; Hergert, Sarah; Li, Shuqiang; Gambe, Rutendo; Zhou, Chensheng W.; Yang, Chunxiao; Xiao, Sheng; Cin, Paola Dal; Bowden, Michaela; Kotliar, Dylan; Shukla, Sachet A.; Brown, Jennifer R.; Neuberg, Donna; Alessi, Dario R.; Zhang, Cheng-Zhong; Kharchenko, Peter V.; Livak, Kenneth J.; Wu, Catherine J.

2017-01-01

Intra-tumoral genetic heterogeneity has been characterized across cancers by genome sequencing of bulk tumors, including chronic lymphocytic leukemia (CLL). In order to more accurately identify subclones, define phylogenetic relationships, and probe genotype–phenotype relationships, we developed methods for targeted mutation detection in DNA and RNA isolated from thousands of single cells from five CLL samples. By clearly resolving phylogenic relationships, we uncovered mutated LCP1 and WNK1 as novel CLL drivers, supported by functional evidence demonstrating their impact on CLL pathways. Integrative analysis of somatic mutations with transcriptional states prompts the idea that convergent evolution generates phenotypically similar cells in distinct genetic branches, thus creating a cohesive expression profile in each CLL sample despite the presence of genetic heterogeneity. Our study highlights the potential for single-cell RNA-based targeted analysis to sensitively determine transcriptional and mutational profiles of individual cancer cells, leading to increased understanding of driving events in malignancy. PMID:28679620

Therapeutically targeting glypican-2 via single-domain antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma

PubMed Central

Li, Nan; Fu, Haiying; Hewitt, Stephen M.; Dimitrov, Dimiter S.

2017-01-01

Neuroblastoma is a childhood cancer that is fatal in almost half of patients despite intense multimodality treatment. This cancer is derived from neuroendocrine tissue located in the sympathetic nervous system. Glypican-2 (GPC2) is a cell surface heparan sulfate proteoglycan that is important for neuronal cell adhesion and neurite outgrowth. In this study, we find that GPC2 protein is highly expressed in about half of neuroblastoma cases and that high GPC2 expression correlates with poor overall survival compared with patients with low GPC2 expression. We demonstrate that silencing of GPC2 by CRISPR-Cas9 or siRNA results in the inhibition of neuroblastoma tumor cell growth. GPC2 silencing inactivates Wnt/β-catenin signaling and reduces the expression of the target gene N-Myc, an oncogenic driver of neuroblastoma tumorigenesis. We have isolated human single-domain antibodies specific for GPC2 by phage display technology and found that the single-domain antibodies can inhibit active β-catenin signaling by disrupting the interaction of GPC2 and Wnt3a. To explore GPC2 as a potential target in neuroblastoma, we have developed two forms of antibody therapeutics, immunotoxins and chimeric antigen receptor (CAR) T cells. Immunotoxin treatment was demonstrated to inhibit neuroblastoma growth in mice. CAR T cells targeting GPC2 eliminated tumors in a disseminated neuroblastoma mouse model where tumor metastasis had spread to multiple clinically relevant sites, including spine, skull, legs, and pelvis. This study suggests GPC2 as a promising therapeutic target in neuroblastoma. PMID:28739923

Therapeutically targeting glypican-2 via single-domain antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma.

PubMed

Li, Nan; Fu, Haiying; Hewitt, Stephen M; Dimitrov, Dimiter S; Ho, Mitchell

2017-08-08

Neuroblastoma is a childhood cancer that is fatal in almost half of patients despite intense multimodality treatment. This cancer is derived from neuroendocrine tissue located in the sympathetic nervous system. Glypican-2 (GPC2) is a cell surface heparan sulfate proteoglycan that is important for neuronal cell adhesion and neurite outgrowth. In this study, we find that GPC2 protein is highly expressed in about half of neuroblastoma cases and that high GPC2 expression correlates with poor overall survival compared with patients with low GPC2 expression. We demonstrate that silencing of GPC2 by CRISPR-Cas9 or siRNA results in the inhibition of neuroblastoma tumor cell growth. GPC2 silencing inactivates Wnt/β-catenin signaling and reduces the expression of the target gene N-Myc, an oncogenic driver of neuroblastoma tumorigenesis. We have isolated human single-domain antibodies specific for GPC2 by phage display technology and found that the single-domain antibodies can inhibit active β-catenin signaling by disrupting the interaction of GPC2 and Wnt3a. To explore GPC2 as a potential target in neuroblastoma, we have developed two forms of antibody therapeutics, immunotoxins and chimeric antigen receptor (CAR) T cells. Immunotoxin treatment was demonstrated to inhibit neuroblastoma growth in mice. CAR T cells targeting GPC2 eliminated tumors in a disseminated neuroblastoma mouse model where tumor metastasis had spread to multiple clinically relevant sites, including spine, skull, legs, and pelvis. This study suggests GPC2 as a promising therapeutic target in neuroblastoma.

Development of Ca2+ hotspots between Lymnaea neurons during synaptogenesis

PubMed Central

Feng, Zhong-Ping; Grigoriev, Nikita; Munno, David; Lukowiak, Ken; MacVicar, Brian A; Goldberg, Jeffrey I; Syed, Naweed I

2002-01-01

Calcium (Ca2+) channel clustering at specific presynaptic sites is a hallmark of mature synapses. However, the spatial distribution patterns of Ca2+ channels at newly formed synapses have not yet been demonstrated. Similarly, it is unclear whether Ca2+ ‘hotspots’ often observed at the presynaptic sites are indeed target cell contact specific and represent a specialized mechanism by which Ca2+ channels are targeted to select synaptic sites. Utilizing both soma–soma paired (synapsed) and single neurons from the mollusk Lymnaea, we have tested the hypothesis that differential gradients of voltage-dependent Ca2+ signals develop in presynaptic neuron at its contact point with the postsynaptic neuron; and that these Ca2+ hotspots are target cell contact specific. Fura-2 imaging, or two-photon laser scanning microscopy of Calcium Green, was coupled with electrophysiological techniques to demonstrate that voltage-induced Ca2+ gradients (hotspots) develop in the presynaptic cell at its contact point with the postsynaptic neuron, but not in unpaired single cells. The incidence of Ca2+ hotspots coincided with the appearance of synaptic transmission between the paired cells, and these gradients were target cell contact specific. In contrast, the voltage-induced Ca2+ signal in unpaired neurons was uniformly distributed throughout the somata; a similar pattern of Ca2+ gradient was observed in the presynaptic neuron when it was soma–soma paired with a non-synaptic partner cell. Moreover, voltage clamp recording techniques, in conjunction with a fast, optical differential perfusion system, were used to demonstrate that the total whole-cell Ca2+ (or Ba2+) current density in single and paired cells was not significantly different. However, the amplitude of Ba2+ current was significantly higher in the presynaptic cell at its contact side with the postsynaptic neurons, compared with non-contacted regions. In summary, this study demonstrates that voltage-induced Ca2+ hotspots develop in the presynaptic cell, concomitant with the appearance of synaptic transmission between the soma–soma paired cells. The appearance of Ca2+ gradients in presynaptic neurons is target cell contact specific and is probably due to a spatial redistribution of existing channels during synaptogenesis. PMID:11850501

Dual HER2\\PIK3CA targeting overcomes single-agent acquired resistance in HER2 amplified uterine serous carcinoma cell lines in vitro and in vivo

PubMed Central

Lopez, Salvatore; Cocco, Emiliano; Black, Jonathan; Bellone, Stefania; Bonazzoli, Elena; Predolini, Federica; Ferrari, Francesca; Schwab, Carlton L.; English, Diana P.; Ratner, Elena; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E.; Terranova, Corrado; Angioli, Roberto; Santin, Alessandro D.

2015-01-01

HER2/neu gene amplification and PIK3CA driver mutations are common in uterine serous carcinoma (USC), and may represent ideal therapeutic targets against this aggressive variant of endometrial cancer. We examined the sensitivity to neratinib, taselisib and the combination of the two compounds in in vitro and in vivo experiments using PIK3CA mutated and PIK3CA-wild type HER2/neu amplified USC cell lines. Cell viability and cell cycle distribution were assessed using flow-cytometry assays. Downstream signaling was assessed by immunoblotting. Preclinical efficacy of single versus dual inhibition was evaluated in vivo using two USC-xenografts. We found both single agent neratinib and taselisib to be active but only transiently effective in controlling the in vivo growth of USC xenografts harboring HER2/neu gene amplification with or without oncogenic PIK3CA mutations. In contrast, the combination of the two inhibitors caused a stronger and long lasting growth inhibition in both USC xenografts when compared to single agent therapy. Combined targeting of HER2 and PIK3CA was associated with a significant and dose-dependent increase in the percentage of cells in the G0/G1 phase of the cell cycle and a dose-dependent decline in the phosphorylation of S6. Importantly, dual inhibition therapy initiated after tumor progression in single agent-treated mice was still remarkably effective at inducing tumor regression in both large PIK3CA or pan-ErbB inhibitor-resistant USC xenografts. Dual HER2/PIK3CA blockade may represent a novel therapeutic option for USC patients harboring tumors with HER2/neu gene amplification and mutated or wild type PIK3CA resistant to chemotherapy. PMID:26333383

Number of infection events per cell during HIV-1 cell-free infection.

PubMed

Ito, Yusuke; Remion, Azaria; Tauzin, Alexandra; Ejima, Keisuke; Nakaoka, Shinji; Iwasa, Yoh; Iwami, Shingo; Mammano, Fabrizio

2017-07-26

HIV-1 accumulates changes in its genome through both recombination and mutation during the course of infection. For recombination to occur, a single cell must be infected by two HIV strains. These coinfection events were experimentally demonstrated to occur more frequently than would be expected for independent infection events and do not follow a random distribution. Previous mathematical modeling approaches demonstrated that differences in target cell susceptibility can explain the non-randomness, both in the context of direct cell-to-cell transmission, and in the context of free virus transmission (Q. Dang et al., Proc. Natl. Acad. Sci. USA 101:632-7, 2004: K. M. Law et al., Cell reports 15:2711-83, 2016). Here, we build on these notions and provide a more detailed and extensive quantitative framework. We developed a novel mathematical model explicitly considering the heterogeneity of target cells and analysed datasets of cell-free HIV-1 single and double infection experiments in cell culture. Particularly, in contrast to the previous studies, we took into account the different susceptibility of the target cells as a continuous distribution. Interestingly, we showed that the number of infection events per cell during cell-free HIV-1 infection follows a negative-binomial distribution, and our model reproduces these datasets.

Tumor-targeting CTL expressing a single-chain Fv specific for VEGFR2.

PubMed

Kanagawa, Naoko; Yanagawa, Tatsuya; Mukai, Yohei; Yoshioka, Yasuo; Okada, Naoki; Nakagawa, Shinsaku

2010-03-26

Cytotoxic T lymphocytes (CTL) are critical effector cells in tumor immunity. Adoptive transfer therapy with in vitro-expanded tumor-specific CTL is a promising approach for preventing cancer metastasis and recurrence. Transferred CTL are not effective in clinical trials, however, due to inadequate tumor-infiltration. Therefore, the development of functionally modified CTL, such as tumor-targeting CTL, is widely desired. Here, we designed the tumor-targeting CTL expressing a single-chain antibody fragment (scFv-CTL) specific for vascular endothelial growth factor receptor 2 (VEGFR2/flk1) by transducing the CTL with a retroviral vector. The scFv-CTL bound to VEGFR2/flk1-expressing cells and retained their cytotoxic activity against tumor cells. In addition, adoptive transfer of scFv-CTL into tumor-bearing mice effectively suppressed tumor growth due to the augmented accumulation of the transferred CTL in the tumor tissue. These findings indicate that the creation of CTL capable of targeting tumor vascular endothelial cells by scFv-expression technique is considerably promising for improvement of efficacy in adoptive immunotherapy. Copyright (c) 2010 Elsevier Inc. All rights reserved.

A generic, cost-effective, and scalable cell lineage analysis platform

PubMed Central

Biezuner, Tamir; Spiro, Adam; Raz, Ofir; Amir, Shiran; Milo, Lilach; Adar, Rivka; Chapal-Ilani, Noa; Berman, Veronika; Fried, Yael; Ainbinder, Elena; Cohen, Galit; Barr, Haim M.; Halaban, Ruth; Shapiro, Ehud

2016-01-01

Advances in single-cell genomics enable commensurate improvements in methods for uncovering lineage relations among individual cells. Current sequencing-based methods for cell lineage analysis depend on low-resolution bulk analysis or rely on extensive single-cell sequencing, which is not scalable and could be biased by functional dependencies. Here we show an integrated biochemical-computational platform for generic single-cell lineage analysis that is retrospective, cost-effective, and scalable. It consists of a biochemical-computational pipeline that inputs individual cells, produces targeted single-cell sequencing data, and uses it to generate a lineage tree of the input cells. We validated the platform by applying it to cells sampled from an ex vivo grown tree and analyzed its feasibility landscape by computer simulations. We conclude that the platform may serve as a generic tool for lineage analysis and thus pave the way toward large-scale human cell lineage discovery. PMID:27558250

SCPortalen: human and mouse single-cell centric database

PubMed Central

Noguchi, Shuhei; Böttcher, Michael; Hasegawa, Akira; Kouno, Tsukasa; Kato, Sachi; Tada, Yuhki; Ura, Hiroki; Abe, Kuniya; Shin, Jay W; Plessy, Charles; Carninci, Piero

2018-01-01

Abstract Published single-cell datasets are rich resources for investigators who want to address questions not originally asked by the creators of the datasets. The single-cell datasets might be obtained by different protocols and diverse analysis strategies. The main challenge in utilizing such single-cell data is how we can make the various large-scale datasets to be comparable and reusable in a different context. To challenge this issue, we developed the single-cell centric database ‘SCPortalen’ (http://single-cell.clst.riken.jp/). The current version of the database covers human and mouse single-cell transcriptomics datasets that are publicly available from the INSDC sites. The original metadata was manually curated and single-cell samples were annotated with standard ontology terms. Following that, common quality assessment procedures were conducted to check the quality of the raw sequence. Furthermore, primary data processing of the raw data followed by advanced analyses and interpretation have been performed from scratch using our pipeline. In addition to the transcriptomics data, SCPortalen provides access to single-cell image files whenever available. The target users of SCPortalen are all researchers interested in specific cell types or population heterogeneity. Through the web interface of SCPortalen users are easily able to search, explore and download the single-cell datasets of their interests. PMID:29045713

Direct and sustained intracellular delivery of exogenous molecules using acoustic-transfection with high frequency ultrasound

PubMed Central

Yoon, Sangpil; Kim, Min Gon; Chiu, Chi Tat; Hwang, Jae Youn; Kim, Hyung Ham; Wang, Yingxiao; Shung, K. Kirk

2016-01-01

Controlling cell functions for research and therapeutic purposes may open new strategies for the treatment of many diseases. An efficient and safe introduction of membrane impermeable molecules into target cells will provide versatile means to modulate cell fate. We introduce a new transfection technique that utilizes high frequency ultrasound without any contrast agents such as microbubbles, bringing a single-cell level targeting and size-dependent intracellular delivery of macromolecules. The transfection apparatus consists of an ultrasonic transducer with the center frequency of over 150 MHz and an epi-fluorescence microscope, entitled acoustic-transfection system. Acoustic pulses, emitted from an ultrasonic transducer, perturb the lipid bilayer of the cell membrane of a targeted single-cell to induce intracellular delivery of exogenous molecules. Simultaneous live cell imaging using HeLa cells to investigate the intracellular concentration of Ca2+ and propidium iodide (PI) and the delivery of 3 kDa dextran labeled with Alexa 488 were demonstrated. Cytosolic delivery of 3 kDa dextran induced via acoustic-transfection was manifested by diffused fluorescence throughout whole cells. Short-term (6 hr) cell viability test and long-term (40 hr) cell tracking confirmed that the proposed approach has low cell cytotoxicity. PMID:26843283

Direct and sustained intracellular delivery of exogenous molecules using acoustic-transfection with high frequency ultrasound

NASA Astrophysics Data System (ADS)

Yoon, Sangpil; Kim, Min Gon; Chiu, Chi Tat; Hwang, Jae Youn; Kim, Hyung Ham; Wang, Yingxiao; Shung, K. Kirk

2016-02-01

Controlling cell functions for research and therapeutic purposes may open new strategies for the treatment of many diseases. An efficient and safe introduction of membrane impermeable molecules into target cells will provide versatile means to modulate cell fate. We introduce a new transfection technique that utilizes high frequency ultrasound without any contrast agents such as microbubbles, bringing a single-cell level targeting and size-dependent intracellular delivery of macromolecules. The transfection apparatus consists of an ultrasonic transducer with the center frequency of over 150 MHz and an epi-fluorescence microscope, entitled acoustic-transfection system. Acoustic pulses, emitted from an ultrasonic transducer, perturb the lipid bilayer of the cell membrane of a targeted single-cell to induce intracellular delivery of exogenous molecules. Simultaneous live cell imaging using HeLa cells to investigate the intracellular concentration of Ca2+ and propidium iodide (PI) and the delivery of 3 kDa dextran labeled with Alexa 488 were demonstrated. Cytosolic delivery of 3 kDa dextran induced via acoustic-transfection was manifested by diffused fluorescence throughout whole cells. Short-term (6 hr) cell viability test and long-term (40 hr) cell tracking confirmed that the proposed approach has low cell cytotoxicity.

Advances in single-cell RNA sequencing and its applications in cancer research.

PubMed

Zhu, Sibo; Qing, Tao; Zheng, Yuanting; Jin, Li; Shi, Leming

2017-08-08

Unlike population-level approaches, single-cell RNA sequencing enables transcriptomic analysis of an individual cell. Through the combination of high-throughput sequencing and bioinformatic tools, single-cell RNA-seq can detect more than 10,000 transcripts in one cell to distinguish cell subsets and dynamic cellular changes. After several years' development, single-cell RNA-seq can now achieve massively parallel, full-length mRNA sequencing as well as in situ sequencing and even has potential for multi-omic detection. One appealing area of single-cell RNA-seq is cancer research, and it is regarded as a promising way to enhance prognosis and provide more precise target therapy by identifying druggable subclones. Indeed, progresses have been made regarding solid tumor analysis to reveal intratumoral heterogeneity, correlations between signaling pathways, stemness, drug resistance, and tumor architecture shaping the microenvironment. Furthermore, through investigation into circulating tumor cells, many genes have been shown to promote a propensity toward stemness and the epithelial-mesenchymal transition, to enhance anchoring and adhesion, and to be involved in mechanisms of anoikis resistance and drug resistance. This review focuses on advances and progresses of single-cell RNA-seq with regard to the following aspects: 1. Methodologies of single-cell RNA-seq 2. Single-cell isolation techniques 3. Single-cell RNA-seq in solid tumor research 4. Single-cell RNA-seq in circulating tumor cell research 5.

Advances in single-cell RNA sequencing and its applications in cancer research

PubMed Central

Zhu, Sibo; Qing, Tao; Zheng, Yuanting; Jin, Li; Shi, Leming

2017-01-01

Unlike population-level approaches, single-cell RNA sequencing enables transcriptomic analysis of an individual cell. Through the combination of high-throughput sequencing and bioinformatic tools, single-cell RNA-seq can detect more than 10,000 transcripts in one cell to distinguish cell subsets and dynamic cellular changes. After several years’ development, single-cell RNA-seq can now achieve massively parallel, full-length mRNA sequencing as well as in situ sequencing and even has potential for multi-omic detection. One appealing area of single-cell RNA-seq is cancer research, and it is regarded as a promising way to enhance prognosis and provide more precise target therapy by identifying druggable subclones. Indeed, progresses have been made regarding solid tumor analysis to reveal intratumoral heterogeneity, correlations between signaling pathways, stemness, drug resistance, and tumor architecture shaping the microenvironment. Furthermore, through investigation into circulating tumor cells, many genes have been shown to promote a propensity toward stemness and the epithelial-mesenchymal transition, to enhance anchoring and adhesion, and to be involved in mechanisms of anoikis resistance and drug resistance. This review focuses on advances and progresses of single-cell RNA-seq with regard to the following aspects: 1. Methodologies of single-cell RNA-seq 2. Single-cell isolation techniques 3. Single-cell RNA-seq in solid tumor research 4. Single-cell RNA-seq in circulating tumor cell research 5. Perspectives PMID:28881849

Effects of in vivo hydrocortisone on lymphocyte-mediated cytotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Katz, P.; Zaytoun, A.M.; Lee, J.H. Jr.

To examine the effects of in vivo hydrocortisone sodium succinate (HC) on natural killer (NK) cell and antibody-dependent cellular cytotoxicity (ADCC), 11 normal adults received a single intravenous bolus of 400 mg hydrocortisone. Lymphocytes were tested for NK activity and ADCC using 51chromium (51Cr)-release and single cell cytotoxicity assays against Molt-4 and sensitized RL O leads to target cells, respectively. Four hours after injection, both NK and ADCC activity were transiently increased in the 51Cr-release system. At 4 hours, there was a twofold increase in the relative frequency of potentially cytotoxic target binding cells but the absolute number of thesemore » cells did not change. However, the percentage lysis of bound targets at 4 hours was not altered. These data suggest that: 1) lymphocytes participating in NK and ADCC reactions are refractory to the kinetic and functional effects of HC; 2) the increased lytic activity observed at 4 hours is due to a selective depletion of noncytotoxic cells from the circulation; and 3) NK and ADCC activity did not differ in their responses to HC.« less

Tumor-targeting domains for chimeric antigen receptor T cells.

PubMed

Bezverbnaya, Ksenia; Mathews, Ashish; Sidhu, Jesse; Helsen, Christopher W; Bramson, Jonathan L

2017-01-01

Immunotherapy with chimeric antigen receptor (CAR) T cells has been advancing steadily in clinical trials. Since the ability of engineered T cells to recognize intended tumor-associated targets is crucial for the therapeutic success, antigen-binding domains play an important role in shaping T-cell responses. Single-chain antibody and T-cell receptor fragments, natural ligands, repeat proteins, combinations of the above and universal tag-specific domains have all been used in the antigen-binding moiety of chimeric receptors. Here we outline the advantages and disadvantages of different domains, discuss the concepts of affinity and specificity, and highlight the recent progress of each targeting strategy.

Gene transfer of Hodgkin cell lines via multivalent anti-CD30 scFv displaying bacteriophage.

PubMed

Chung, Yoon-Suk A; Sabel, Katja; Krönke, Martin; Klimka, Alexander

2008-04-16

The display of binding ligands, such as recombinant antibody fragments, on the surface of filamentous phage makes it possible to specifically attach these phage particles to target cells. After uptake of the phage, their internal single-stranded DNA is processed by the host cell, which allows transient expression of an encoded eukaryotic gene cassette. This opens the possibility to use bacteriophage as vectors for targeted gene therapy, although the transduction efficiency is very low. Here we demonstrate the display of an anti-CD30 single chain variable fragment fused to the major coat protein pVIII on the surface of bacteriophage. These phage particles showed an improved binding and transduction efficiency of CD30 positive Hodgkin-lymphoma cells, compared to bacteriophage with the anti-CD30 single chain variable fragment fused to the minor coat protein pIII. We can conclude from the results that the postulated multivalency of the anti-CD30-pVIII displaying bacteriophage combined with disseminated display of the anti-CD30 scFv on the whole particle surface is responsible for the improved gene transfer rate. These results mark an important step towards the use of phage particles as a cheap and safe gene transfer vehicle for the gene delivery of the desired target cells via their specific surface receptors.

Microplasma Induced Cell Morphological Changes and Apoptosis of Ex Vivo Cultured Human Anterior Lens Epithelial Cells – Relevance to Capsular Opacification

PubMed Central

Hojnik, Nataša; Filipič, Gregor; Lazović, Saša; Vesel, Alenka; Primc, Gregor; Mozetič, Miran; Hawlina, Marko; Petrovski, Goran; Cvelbar, Uroš

2016-01-01

Inducing selective or targeted cell apoptosis without affecting large number of neighbouring cells remains a challenge. A plausible method for treatment of posterior capsular opacification (PCO) due to remaining lens epithelial cells (LECs) by reactive chemistry induced by localized single electrode microplasma discharge at top of a needle-like glass electrode with spot size ~3 μm is hereby presented. The focused and highly-localized atmospheric pressure microplasma jet with electrode discharge could induce a dose-dependent apoptosis in selected and targeted individual LECs, which could be confirmed by real-time monitoring of the morphological and structural changes at cellular level. Direct cell treatment with microplasma inside the medium appeared more effective in inducing apoptosis (caspase 8 positivity and DNA fragmentation) at a highly targeted cell level compared to treatment on top of the medium (indirect treatment). Our results show that single cell specific micropipette plasma can be used to selectively induce demise in LECs which remain in the capsular bag after cataract surgery and thus prevent their migration (CXCR4 positivity) to the posterior lens capsule and PCO formation. PMID:27832099

Dissecting the human microbiome with single-cell genomics.

PubMed

Tolonen, Andrew C; Xavier, Ramnik J

2017-06-14

Recent advances in genome sequencing of single microbial cells enable the assignment of functional roles to members of the human microbiome that cannot currently be cultured. This approach can reveal the genomic basis of phenotypic variation between closely related strains and can be applied to the targeted study of immunogenic bacteria in disease.

Octa-ammonium POSS-conjugated single-walled carbon nanotubes as vehicles for targeted delivery of paclitaxel

PubMed Central

Naderi, Naghmeh; Madani, Seyed Y.; Mosahebi, Afshin; Seifalian, Alexander M.

2015-01-01

Background Carbon nanotubes (CNTs) have unique physical and chemical properties. Furthermore, novel properties can be developed by attachment or encapsulation of functional groups. These unique properties facilitate the use of CNTs in drug delivery. We developed a new nanomedicine consisting of a nanocarrier, cell-targeting molecule, and chemotherapeutic drug and assessed its efficacy in vitro. Methods The efficacy of a single-walled carbon nanotubes (SWCNTs)-based nanoconjugate system is assessed in the targeted delivery of paclitaxel (PTX) to cancer cells. SWCNTs were oxidized and reacted with octa-ammonium polyhedral oligomeric silsesquioxanes (octa-ammonium POSS) to render them biocompatible and water dispersable. The functionalized SWCNTs were loaded with PTX, a chemotherapeutic agent toxic to cancer cells, and Tn218 antibodies for cancer cell targeting. The nanohybrid composites were characterized with transmission electron microscopy (TEM), Fourier transform infrared (FTIR), and ultraviolet–visible–near-infrared (UV–Vis–NIR). Additionally, their cytotoxic effects on Colon cancer cell (HT-29) and Breast cancer cell (MCF-7) lines were assessed in vitro. Results TEM, FTIR, and UV–Vis–NIR studies confirmed side-wall functionalization of SWCNT with COOH-groups, PTX, POSS, and antibodies. Increased cell death was observed with PTX–POSS–SWCNT, PTX–POSS–Ab–SWCNT, and free PTX compared to functionalized-SWCNT (f-SWCNT), POSS–SWCNT, and cell-only controls at 48 and 72 h time intervals in both cell lines. At all time intervals, there was no significant cell death in the POSS–SWCNT samples compared to cell-only controls. Conclusion The PTX-based nanocomposites were shown to be as cytotoxic as free PTX. This important finding indicates successful release of PTX from the nanocomposites and further reiterates the potential of SWCNTs to deliver drugs directly to targeted cells and tissues. PMID:26356347

megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering.

PubMed

Boissel, Sandrine; Jarjour, Jordan; Astrakhan, Alexander; Adey, Andrew; Gouble, Agnès; Duchateau, Philippe; Shendure, Jay; Stoddard, Barry L; Certo, Michael T; Baker, David; Scharenberg, Andrew M

2014-02-01

Rare-cleaving endonucleases have emerged as important tools for making targeted genome modifications. While multiple platforms are now available to generate reagents for research applications, each existing platform has significant limitations in one or more of three key properties necessary for therapeutic application: efficiency of cleavage at the desired target site, specificity of cleavage (i.e. rate of cleavage at 'off-target' sites), and efficient/facile means for delivery to desired target cells. Here, we describe the development of a single-chain rare-cleaving nuclease architecture, which we designate 'megaTAL', in which the DNA binding region of a transcription activator-like (TAL) effector is used to 'address' a site-specific meganuclease adjacent to a single desired genomic target site. This architecture allows the generation of extremely active and hyper-specific compact nucleases that are compatible with all current viral and nonviral cell delivery methods.

Single-Cell RNA-Seq Reveals Transcriptional Heterogeneity in Latent and Reactivated HIV-Infected Cells.

PubMed

Golumbeanu, Monica; Cristinelli, Sara; Rato, Sylvie; Munoz, Miguel; Cavassini, Matthias; Beerenwinkel, Niko; Ciuffi, Angela

2018-04-24

Despite effective treatment, HIV can persist in latent reservoirs, which represent a major obstacle toward HIV eradication. Targeting and reactivating latent cells is challenging due to the heterogeneous nature of HIV-infected cells. Here, we used a primary model of HIV latency and single-cell RNA sequencing to characterize transcriptional heterogeneity during HIV latency and reactivation. Our analysis identified transcriptional programs leading to successful reactivation of HIV expression. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Droplet electric separator microfluidic device for cell sorting

NASA Astrophysics Data System (ADS)

Guo, Feng; Ji, Xing-Hu; Liu, Kan; He, Rong-Xiang; Zhao, Li-Bo; Guo, Zhi-Xiao; Liu, Wei; Guo, Shi-Shang; Zhao, Xing-Zhong

2010-05-01

A simple and effective droplet electric separator microfluidic device was developed for cell sorting. The aqueous droplet without precharging operation was influenced to move a distance in the channel along the electric field direction by applying dc voltage on the electrodes beside the channel, which made the target droplet flowing to the collector. Single droplet can be isolated in a sorting rate of ˜100 Hz with microelectrodes under a required pulse. Single or multiple mammalian cell (HePG2) encapsulated in the surfactant free alginate droplet could be sorted out respectively. This method may be used for single cell operation or analysis.

Tools for Genomic and Transcriptomic Analysis of Microbes at Single-Cell Level

PubMed Central

Chen, Zixi; Chen, Lei; Zhang, Weiwen

2017-01-01

Microbiologists traditionally study population rather than individual cells, as it is generally assumed that the status of individual cells will be similar to that observed in the population. However, the recent studies have shown that the individual behavior of each single cell could be quite different from that of the whole population, suggesting the importance of extending traditional microbiology studies to single-cell level. With recent technological advances, such as flow cytometry, next-generation sequencing (NGS), and microspectroscopy, single-cell microbiology has greatly enhanced the understanding of individuality and heterogeneity of microbes in many biological systems. Notably, the application of multiple ‘omics’ in single-cell analysis has shed light on how individual cells perceive, respond, and adapt to the environment, how heterogeneity arises under external stress and finally determines the fate of the whole population, and how microbes survive under natural conditions. As single-cell analysis involves no axenic cultivation of target microorganism, it has also been demonstrated as a valuable tool for dissecting the microbial ‘dark matter.’ In this review, current state-of-the-art tools and methods for genomic and transcriptomic analysis of microbes at single-cell level were critically summarized, including single-cell isolation methods and experimental strategies of single-cell analysis with NGS. In addition, perspectives on the future trends of technology development in the field of single-cell analysis was also presented. PMID:28979258

Targeted salinomycin delivery with EGFR and CD133 aptamers based dual-ligand lipid-polymer nanoparticles to both osteosarcoma cells and cancer stem cells.

PubMed

Chen, Fangyi; Zeng, Yibin; Qi, Xiaoxia; Chen, Yanchao; Ge, Zhe; Jiang, Zengxin; Zhang, Xinchao; Dong, Yinmei; Chen, Huaiwen; Yu, Zuochong

2018-06-10

We previously developed salinomycin (sali)-entrapped nanoparticles labeled with CD133 aptamers which could efficiently eliminate CD133 + osteosarcoma cancer stem cells (CSCs). However, sufficient evidences suggest that the simultaneous targeting both CSCs and cancer cells is pivotal in achieving preferable cancer therapeutic efficacy, due to the spontaneous conversion between cancer cells and CSCs. We hereby constructed sali-entrapped lipid-polymer nanoparticles labeled with CD133 and EGFR aptamers (CESP) to target both osteosarcoma cells and CSCs. The cytotoxicity of CESP in osteosarcoma cells and CSCs was superior to that of single targeting or nontargeted sali-loaded nanoparticles. Administration of CESP in vivo showed the best efficacy in inhibiting tumor growth than other controls in osteosarcoma-bearing mice. Thus, CESP was demonstrated to be capable of efficiently targeting both osteosarcoma CSCs and cancer cells, and it represents an effective potential approach to treat osteosarcoma. Copyright © 2018 Elsevier Inc. All rights reserved.

Oxygen Nanobubble Tracking by Light Scattering in Single Cells and Tissues.

PubMed

Bhandari, Pushpak; Wang, Xiaolei; Irudayaraj, Joseph

2017-03-28

Oxygen nanobubbles (ONBs) have significant potential in targeted imaging and treatment in cancer diagnosis and therapy. Precise localization and tracking of single ONBs is demonstrated based on hyperspectral dark-field microscope (HSDFM) to image and track single oxygen nanobubbles in single cells. ONBs were proposed as promising contrast-generating imaging agents due to their strong light scattering generated from nonuniformity of refractive index at the interface. With this powerful platform, we have revealed the trajectories and quantities of ONBs in cells, and demonstrated the relation between the size and diffusion coefficient. We have also evaluated the presence of ONBs in the nucleus with respect to an increase in incubation time and have quantified the uptake in single cells in ex vivo tumor tissues. Our results demonstrate that HSDFM can be a versatile platform to detect and measure cellulosic nanoparticles at the single-cell level and to assess the dynamics and trajectories of this delivery system.

Microchip-Based Single-Cell Functional Proteomics for Biomedical Applications

PubMed Central

Lu, Yao; Yang, Liu; Wei, Wei; Shi, Qihui

2017-01-01

Cellular heterogeneity has been widely recognized but only recently have single cell tools become available that allow characterizing heterogeneity at the genomic and proteomic levels. We review the technological advances in microchip-based toolkits for single-cell functional proteomics. Each of these tools has distinct advantages and limitations, and a few have advanced toward being applied to address biological or clinical problems that fail to be addressed by traditional population-based methods. High-throughput single-cell proteomic assays generate high-dimensional data sets that contain new information and thus require developing new analytical framework to extract new biology. In this review article, we highlight a few biological and clinical applications in which the microchip-based single-cell proteomic tools provide unique advantages. The examples include resolving functional heterogeneity and dynamics of immune cells, dissecting cell-cell interaction by creating well-contolled on-chip microenvironment, capturing high-resolution snapshots of immune system functions in patients for better immunotherapy and elucidating phosphoprotein signaling networks in cancer cells for guiding effective molecularly targeted therapies. PMID:28280819

Targeted gene expression without a tissue-specific promoter: creating mosaic embryos using laser-induced single-cell heat shock

NASA Technical Reports Server (NTRS)

Halfon, M. S.; Kose, H.; Chiba, A.; Keshishian, H.

1997-01-01

We have developed a method to target gene expression in the Drosophila embryo to a specific cell without having a promoter that directs expression in that particular cell. Using a digitally enhanced imaging system to identify single cells within the living embryo, we apply a heat shock to each cell individually by using a laser microbeam. A 1- to 2-min laser treatment is sufficient to induce a heat-shock response but is not lethal to the heat-shocked cells. Induction of heat shock was measured in a variety of cell types, including neurons and somatic muscles, by the expression of beta-galactosidase from an hsp26-lacZ reporter construct or by expression of a UAS target gene after induction of hsGAL4. We discuss the applicability of this technique to ectopic gene expression studies, lineage tracing, gene inactivation studies, and studies of cells in vitro. Laser heat shock is a versatile technique that can be adapted for use in a variety of research organisms and is useful for any studies in which it is desirable to express a given gene in only a distinct cell or clone of cells, either transiently or constitutively, at a time point of choice.

Targeting of phage particles towards endothelial cells by antibodies selected through a multi-parameter selection strategy.

PubMed

Mandrup, Ole A; Lykkemark, Simon; Kristensen, Peter

2017-02-10

One of the hallmarks of cancer is sustained angiogenesis. Here, normal endothelial cells are activated, and their formation of new blood vessels leads to continued tumour growth. An improved patient condition is often observed when angiogenesis is prevented or normalized through targeting of these genomically stable endothelial cells. However, intracellular targets constitute a challenge in therapy, as the agents modulating these targets have to be delivered and internalized specifically to the endothelial cells. Selection of antibodies binding specifically to certain cell types is well established. It is nonetheless a challenge to ensure that the binding of antibodies to the target cell will mediate internalization. Previously selection of such antibodies has been performed targeting cancer cell lines; most often using either monovalent display or polyvalent display. In this article, we describe selections that isolate internalizing antibodies by sequential combining monovalent and polyvalent display using two types of helper phages, one which increases display valence and one which reduces background. One of the selected antibodies was found to mediate internalization into human endothelial cells, although our results confirms that the single stranded nature of the DNA packaged into phage particles may limit applications aimed at targeting nucleic acids in mammalian cells.

Diadenosine tetraphosphate-gating of recombinant pancreatic ATP-sensitive K(+) channels.

PubMed

Jovanovic, S; Jovanovic, A

2001-02-01

Diadenosine tetraphosphate (Ap4A) has been recently discovered in the pancreatic beta cells where targets ATP-sensitive K(+) (K(ATP)) channels, depolarizes the cell membrane and induces insulin secretion. However, whether Ap4A inhibit pancreatic K(ATP) channels by targeting protein channel complex itself was unknown. Therefore, we coexpressed pancreatic K(ATP) channel subunits, Kir6.2 and SUR1, in COS-7 cells and examined the effect of Ap4A on the single channel behavior using the inside-out configuration of the patch-clamp technique. Ap4A inhibited channel opening in a concentration-dependent manner. Analysis of single channels demonstrated that Ap4A did not change intraburst kinetic behavior of K(ATP) channels, but rather decreased burst duration and increased between-burst duration. It is concluded that Ap4A antagonizes K(ATP) channel opening by targeting channel subunits themselves and by keeping channels longer in closed interburst states.

Mechanical Strains Induced in Osteoblasts by Use of Point Femtosecond Laser Targeting

PubMed Central

Bomzon, Ze'ev; Day, Daniel; Gu, Min; Cartmell, Sarah

2006-01-01

A study demonstrating how ultrafast laser radiation stimulates osteoblasts is presented. The study employed a custom made optical system that allowed for simultaneous confocal cell imaging and targeted femtosecond pulse laser irradiation. When femtosecond laser light was focused onto a single cell, a rise in intracellular Ca2+ levels was observed followed by contraction of the targeted cell. This contraction caused deformation of neighbouring cells leading to a heterogeneous strain field throughout the monolayer. Quantification of the strain fields in the monolayer using digital image correlation revealed local strains much higher than threshold values typically reported to stimulate extracellular bone matrix production in vitro. This use of point targeting with femtosecond pulse lasers could provide a new method for stimulating cell activity in orthopaedic tissue engineering. PMID:23165014

Cell-specific optoporation with near-infrared ultrafast laser and functionalized gold nanoparticles

NASA Astrophysics Data System (ADS)

Bergeron, Eric; Boutopoulos, Christos; Martel, Rosalie; Torres, Alexandre; Rodriguez, Camille; Niskanen, Jukka; Lebrun, Jean-Jacques; Winnik, Françoise M.; Sapieha, Przemyslaw; Meunier, Michel

2015-10-01

Selective targeting of diseased cells can increase therapeutic efficacy and limit off-target adverse effects. We developed a new tool to selectively perforate living cells with functionalized gold nanoparticles (AuNPs) and near-infrared (NIR) femtosecond (fs) laser. The receptor CD44 strongly expressed by cancer stem cells was used as a model for selective targeting. Citrate-capped AuNPs (100 nm in diameter) functionalized with 0.01 orthopyridyl-disulfide-poly(ethylene glycol) (5 kDa)-N-hydroxysuccinimide (OPSS-PEG-NHS) conjugated to monoclonal antibodies per nm2 and 5 μM HS-PEG (5 kDa) were colloidally stable in cell culture medium containing serum proteins. These AuNPs attached mostly as single particles 115 times more to targeted CD44+ MDA-MB-231 and CD44+ ARPE-19 cells than to non-targeted CD44- 661W cells. Optimally functionalized AuNPs enhanced the fs laser (800 nm, 80-100 mJ cm-2 at 250 Hz or 60-80 mJ cm-2 at 500 Hz) to selectively perforate targeted cells without affecting surrounding non-targeted cells in co-culture. This novel highly versatile treatment paradigm can be adapted to target and perforate other cell populations by adapting to desired biomarkers. Since living biological tissues absorb energy very weakly in the NIR range, the developed non-invasive tool may provide a safe, cost-effective clinically relevant approach to ablate pathologically deregulated cells and limit complications associated with surgical interventions.Selective targeting of diseased cells can increase therapeutic efficacy and limit off-target adverse effects. We developed a new tool to selectively perforate living cells with functionalized gold nanoparticles (AuNPs) and near-infrared (NIR) femtosecond (fs) laser. The receptor CD44 strongly expressed by cancer stem cells was used as a model for selective targeting. Citrate-capped AuNPs (100 nm in diameter) functionalized with 0.01 orthopyridyl-disulfide-poly(ethylene glycol) (5 kDa)-N-hydroxysuccinimide (OPSS-PEG-NHS) conjugated to monoclonal antibodies per nm2 and 5 μM HS-PEG (5 kDa) were colloidally stable in cell culture medium containing serum proteins. These AuNPs attached mostly as single particles 115 times more to targeted CD44+ MDA-MB-231 and CD44+ ARPE-19 cells than to non-targeted CD44- 661W cells. Optimally functionalized AuNPs enhanced the fs laser (800 nm, 80-100 mJ cm-2 at 250 Hz or 60-80 mJ cm-2 at 500 Hz) to selectively perforate targeted cells without affecting surrounding non-targeted cells in co-culture. This novel highly versatile treatment paradigm can be adapted to target and perforate other cell populations by adapting to desired biomarkers. Since living biological tissues absorb energy very weakly in the NIR range, the developed non-invasive tool may provide a safe, cost-effective clinically relevant approach to ablate pathologically deregulated cells and limit complications associated with surgical interventions. Electronic supplementary information (ESI) available: Characterization of functionalized gold nanoparticles by UV-visible-NIR spectroscopy and zeta potential measurements; selectivity of cell targeting with functionalized gold nanoparticles by immunofluorescence, flow cytometry and scanning electron microscopy; selective treatment of targeted cells with functionalized gold nanoparticles and ultrafast laser. See DOI: 10.1039/c5nr05650k

Bi-specific splice-switching PMO oligonucleotides conjugated via a single peptide active in a mouse model of Duchenne muscular dystrophy

PubMed Central

Shabanpoor, Fazel; McClorey, Graham; Saleh, Amer F.; Järver, Peter; Wood, Matthew J.A.; Gait, Michael J.

2015-01-01

The potential for therapeutic application of splice-switching oligonucleotides (SSOs) to modulate pre-mRNA splicing is increasingly evident in a number of diseases. However, the primary drawback of this approach is poor cell and in vivo oligonucleotide uptake efficacy. Biological activities can be significantly enhanced through the use of synthetically conjugated cationic cell penetrating peptides (CPPs). Studies to date have focused on the delivery of a single SSO conjugated to a CPP, but here we describe the conjugation of two phosphorodiamidate morpholino oligonucleotide (PMO) SSOs to a single CPP for simultaneous delivery and pre-mRNA targeting of two separate genes, exon 23 of the Dmd gene and exon 5 of the Acvr2b gene, in a mouse model of Duchenne muscular dystrophy. Conjugations of PMOs to a single CPP were carried out through an amide bond in one case and through a triazole linkage (‘click chemistry’) in the other. The most active bi-specific CPP–PMOs demonstrated comparable exon skipping levels for both pre-mRNA targets when compared to individual CPP–PMO conjugates both in cell culture and in vivo in the mdx mouse model. Thus, two SSOs with different target sequences conjugated to a single CPP are biologically effective and potentially suitable for future therapeutic exploitation. PMID:25468897

Multiplexed Single Intact Cell Droplet Digital PCR (MuSIC ddPCR) Method for Specific Detection of Enterohemorrhagic E. coli (EHEC) in Food Enrichment Cultures

PubMed Central

McMahon, Tanis C.; Blais, Burton W.; Wong, Alex; Carrillo, Catherine D.

2017-01-01

Foodborne illness attributed to enterohemorrhagic E. coli (EHEC), a highly pathogenic subset of Shiga toxin-producing E. coli (STEC), is increasingly recognized as a significant public health issue. Current microbiological methods for identification of EHEC in foods often use PCR-based approaches to screen enrichment broth cultures for characteristic gene markers [i.e., Shiga toxin (stx) and intimin (eae)]. However, false positives arise when complex food matrices, such as beef, contain mixtures of eae-negative STEC and eae-positive E. coli, but no EHEC with both markers in a single cell. To reduce false-positive detection of EHEC in food enrichment samples, a Multiplexed, Single Intact Cell droplet digital PCR (MuSIC ddPCR) assay capable of detecting the co-occurrence of the stx and eae genes in a single bacterial cell was developed. This method requires: (1) dispersal of intact bacteria into droplets; (2) release of genomic DNA (gDNA) by heat lysis; and (3) amplification and detection of genetic targets (stx and eae) using standard TaqMan chemistries with ddPCR. Performance of the method was tested with panels of EHEC and non-target E. coli. By determining the linkage (i.e., the proportion of droplets in which stx and eae targets were both amplified), samples containing EHEC (typically greater than 20% linkage) could be distinguished from samples containing mixtures of eae-negative STEC and eae-positive E. coli (0–2% linkage). The use of intact cells was necessary as this linkage was not observed with gDNA extracts. EHEC could be accurately identified in enrichment broth cultures containing excess amounts of background E. coli and in enrichment cultures derived from ground beef/pork and leafy-green produce samples. To our knowledge, this is the first report of dual-target detection in single bacterial cells using ddPCR. The application of MuSIC ddPCR to enrichment-culture screening would reduce false-positives, thereby improving the cost, speed, and accuracy of current methods for EHEC detection in foods. PMID:28303131

Multiplexed Single Intact Cell Droplet Digital PCR (MuSIC ddPCR) Method for Specific Detection of Enterohemorrhagic E. coli (EHEC) in Food Enrichment Cultures.

PubMed

McMahon, Tanis C; Blais, Burton W; Wong, Alex; Carrillo, Catherine D

2017-01-01

Foodborne illness attributed to enterohemorrhagic E. coli (EHEC), a highly pathogenic subset of Shiga toxin-producing E. coli (STEC), is increasingly recognized as a significant public health issue. Current microbiological methods for identification of EHEC in foods often use PCR-based approaches to screen enrichment broth cultures for characteristic gene markers [i.e., Shiga toxin ( stx ) and intimin ( eae )]. However, false positives arise when complex food matrices, such as beef, contain mixtures of eae -negative STEC and eae -positive E. coli , but no EHEC with both markers in a single cell. To reduce false-positive detection of EHEC in food enrichment samples, a Multiplexed, Single Intact Cell droplet digital PCR (MuSIC ddPCR) assay capable of detecting the co-occurrence of the stx and eae genes in a single bacterial cell was developed. This method requires: (1) dispersal of intact bacteria into droplets; (2) release of genomic DNA (gDNA) by heat lysis; and (3) amplification and detection of genetic targets ( stx and eae ) using standard TaqMan chemistries with ddPCR. Performance of the method was tested with panels of EHEC and non-target E. coli . By determining the linkage (i.e., the proportion of droplets in which stx and eae targets were both amplified), samples containing EHEC (typically greater than 20% linkage) could be distinguished from samples containing mixtures of eae -negative STEC and eae -positive E. coli (0-2% linkage). The use of intact cells was necessary as this linkage was not observed with gDNA extracts. EHEC could be accurately identified in enrichment broth cultures containing excess amounts of background E. coli and in enrichment cultures derived from ground beef/pork and leafy-green produce samples. To our knowledge, this is the first report of dual-target detection in single bacterial cells using ddPCR. The application of MuSIC ddPCR to enrichment-culture screening would reduce false-positives, thereby improving the cost, speed, and accuracy of current methods for EHEC detection in foods.

Dual targeting of gene delivery by genetic modification of adenovirus serotype 5 fibers and cell-selective transcriptional control.

PubMed

Work, L M; Ritchie, N; Nicklin, S A; Reynolds, P N; Baker, A H

2004-08-01

Adenovirus (Ad)-mediated gene delivery is a promising approach for genetic manipulation of the vasculature and is being used in both preclinical models and clinical trials. However, safety concerns relating to infection of nontarget tissue and the poor infectivity of vascular cells compared to other cell types necessitates Ad vector refinement. Here, we combine a transductional targeting approach to improve vascular cell infectivity through RGD peptide insertion into adenovirus fibers, combined with transcriptional targeting to endothelial cells using a approximately 1 kb fragment of the fms-like tyrosine kinase receptor-1 (FLT-1) promoter. Single- and double-modified vectors were characterized in human cell lines that either support or have silenced FLT-1 expression. In rat hepatocytes and endothelial cells, the double modification substantially shifted transduction profiles toward vascular endothelial cells. Furthermore, in intact aortae derived from spontaneously hypertensive rats that display enhanced alphav integrin expression on dysfunctional endothelium, enhanced levels of transduction were observed using the double-modified vector but not in aortae derived from normotensive control rats. Our data indicate that Ad-mediated transduction can be beneficially modified in vitro and in vivo by combining fiber modification and a cell-selective promoter within a single-component vector system.

Nonviral RNA transfection to transiently modify T cells with chimeric antigen receptors for adoptive therapy.

PubMed

Riet, Tobias; Holzinger, Astrid; Dörrie, Jan; Schaft, Niels; Schuler, Gerold; Abken, Hinrich

2013-01-01

Redirecting T cells with a chimeric antigen receptor (CAR) of predefined specificity showed remarkable efficacy in the adoptive therapy trials of malignant diseases. The CAR consists of a single chain fragment of variable region (scFv) antibody targeting domain covalently linked to the CD3ζ signalling domain of the T cell receptor complex to mediate T cell activation upon antigen engagement. By using an antibody-derived targeting domain a CAR can potentially redirect T cells towards any target expressed on the cell surface as long as a binding domain is available. Antibody-mediated targeting moreover circumvents MHC restriction of the targeted antigen, thereby broadening the potential of applicability of adoptive T cell therapy. While T cells were so far genetically modified by viral transduction, transient modification with a CAR by RNA transfection gained increasing interest during the last years. This chapter focuses on methods to modify human T cells from peripheral blood with a CAR by electroporation of in vitro transcribed RNA and to test modified T cells for function for use in adoptive immunotherapy.

Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin

PubMed Central

Zhen, Chao Yu; Tatavosian, Roubina; Huynh, Thao Ngoc; Duc, Huy Nguyen; Das, Raibatak; Kokotovic, Marko; Grimm, Jonathan B; Lavis, Luke D; Lee, Jun; Mejia, Frances J; Li, Yang; Yao, Tingting; Ren, Xiaojun

2016-01-01

The Polycomb PRC1 plays essential roles in development and disease pathogenesis. Targeting of PRC1 to chromatin is thought to be mediated by the Cbx family proteins (Cbx2/4/6/7/8) binding to histone H3 with a K27me3 modification (H3K27me3). Despite this prevailing view, the molecular mechanisms of targeting remain poorly understood. Here, by combining live-cell single-molecule tracking (SMT) and genetic engineering, we reveal that H3K27me3 contributes significantly to the targeting of Cbx7 and Cbx8 to chromatin, but less to Cbx2, Cbx4, and Cbx6. Genetic disruption of the complex formation of PRC1 facilitates the targeting of Cbx7 to chromatin. Biochemical analyses uncover that the CD and AT-hook-like (ATL) motif of Cbx7 constitute a functional DNA-binding unit. Live-cell SMT of Cbx7 mutants demonstrates that Cbx7 is targeted to chromatin by co-recognizing of H3K27me3 and DNA. Our data suggest a novel hierarchical cooperation mechanism by which histone modifications and DNA coordinate to target chromatin regulatory complexes. DOI: http://dx.doi.org/10.7554/eLife.17667.001 PMID:27723458

[Construction, expression and characterization of the fusion gene of super-antigen SEA and single chain Fv of the ND-1 monoclonal antibody against human colorectal cancer].

PubMed

Chen, Hang; Li, Li; Fang, Jin

2012-04-01

To construct and express the recombinant ND-1-scFv/SEA, a fusion protein of superantigen (staphylococcal enterotoxinA, SEA) and single-chain variable fragment of monoclonal antibody ND-1 against human clolorectal carcinoma, and to enhance the targeted killing effect of SEA. The expression of the fusion protein was induced in E.coli M15 by IPTG. Ni-NTA resin affinity chromatography was used to separate and purify the expressed product. The specific binding activity of the purified ND-1-scFv/SEA protein was examined by indirect immunofluorescence assay and the targeted-cytotoxicity was determined using MTT assay. The expressing vector of fusion gene ND-1scFv/SEA was constructed successfully. ND-1-scFv/SEA protein retained a high binding affinity to antigen-positive human colorectal cancer cell CCL-187 and had a stronger capability to activate PBMC and kill the target cells compared to SEA alone, with a killing rate of 91% at 4 μg/mL. ND-1-scFv/SEA fusion protein could specifically target colorectal cancer cell, enhance the activity of kill tumor cell and has potential applications in the targeted therapy of colorectal cancer.

Programmable oligonucleotide probes design and applications for in situ and in vivo RNA imaging in cells

NASA Astrophysics Data System (ADS)

Cheglakov, Zoya

Unequal spreading of mRNA is a frequent experience observed in varied cell lines. The study of cellular processes dynamics and precise localization of mRNAs offers a vital toolbox to target specific proteins in precise cytoplasmic areas and provides a convenient instrument to uncover their mechanisms and functions. Latest methodological innovations have allowed imaging of a single mRNA molecule in situ and in vivo. Today, Fluorescent In Situ Hybridization (FISH) methods allow the studying of mRNA expression and offer a vital toolbox for accurate biological models. Studies enable analysis of the dynamics of an individual mRNA, have uncovered the multiplex RNA transport systems. With all current approaches, a single mRNA tracking in the mammalian cells is still challenging. This thesis describes mRNA detection methods based on programmable fluorophore-labeled DNA structures complimentary to native targets providing an accurate mRNA imaging in mammalian cells. First method represents beta-actin (ACTB) transcripts in situ detection in human cells, the technique strategy is based on programmable DNA probes, amplified by rolling circle amplification (RCA). The method reports precise localization of molecule of interest with an accuracy of a single-cell. Visualization and localization of specific endogenous mRNA molecules in real-time in vivo has the promising to innovate cellular biology studies, medical analysis and to provide a vital toolbox in drugs invention area. Second method described in this thesis represents miR-21 miRNA detection within a single live-cell resolution. The method using fluorophore-labeled short synthetic DNAs probes forming a stem-loop shape and generating Fluorescent Resonance Energy Transfer (FRET) as a result of target-probes hybridization. Catalytic nucleic acid (DNAzymes) probes are cooperative tool for precise detection of different mRNA targets. With assistance of a complementary fluorophore-quencher labeled substrate, the DNAzymes provide a beneficial strategy for simultaneous tracking readily accomplished by multicolor imaging with diverse fluorescent tags. The third method in this thesis will demonstrate the advantage of DNAzymes probes amplification, and offers potential strategy to monitor miRNAs in mammalian live cells.

A high content, high throughput cellular thermal stability assay for measuring drug-target engagement in living cells.

PubMed

Massey, Andrew J

2018-01-01

Determining and understanding drug target engagement is critical for drug discovery. This can be challenging within living cells as selective readouts are often unavailable. Here we describe a novel method for measuring target engagement in living cells based on the principle of altered protein thermal stabilization / destabilization in response to ligand binding. This assay (HCIF-CETSA) utilizes high content, high throughput single cell immunofluorescent detection to determine target protein levels following heating of adherent cells in a 96 well plate format. We have used target engagement of Chk1 by potent small molecule inhibitors to validate the assay. Target engagement measured by this method was subsequently compared to target engagement measured by two alternative methods (autophosphorylation and CETSA). The HCIF-CETSA method appeared robust and a good correlation in target engagement measured by this method and CETSA for the selective Chk1 inhibitor V158411 was observed. However, these EC50 values were 23- and 12-fold greater than the autophosphorylation IC50. The described method is therefore a valuable advance in the CETSA method allowing the high throughput determination of target engagement in adherent cells.

Human Single-Chain Fv Immunoconjugates Targeted to a Melanoma-Associated Chondroitin Sulfate Proteoglycan Mediate Specific Lysis of Human Melanoma Cells by Natural Killer Cells and Complement

NASA Astrophysics Data System (ADS)

Wang, Baiyang; Chen, Yi-Bin; Ayalon, Oran; Bender, Jeffrey; Garen, Alan

1999-02-01

Two antimelanoma immunoconjugates containing a human single-chain Fv (scFv) targeting domain conjugated to the Fc effector domain of human IgG1 were synthesized as secreted two-chain molecules in Chinese hamster ovary and Drosophila S2 cells, and purified by affinity chromatography on protein A. The scFv targeting domains originally were isolated as melanoma-specific clones from a scFv fusion-phage library, derived from the antibody repertoire of a vaccinated melanoma patient. The purified immunoconjugates showed similar binding specificity as did the fusion-phage clones. Binding occurred to human melanoma cells but not to human melanocytes or to several other types of normal cells and tumor cells. A 250-kDa melanoma protein was immunoprecipitated by the immunoconjugates and analyzed by mass spectrometry, using two independent procedures. A screen of protein sequence databases showed an exact match of several peptide masses between the immunoprecipitated protein and the core protein of a chondroitin sulfate proteoglycan, which is expressed on the surface of most human melanoma cells. The Fc effector domain of the immunoconjugates binds natural killer (NK) cells and also the C1q protein that initiates the complement cascade; both NK cells and complement can activate powerful cytolytic responses against the targeted tumor cells. An in vitro cytolysis assay was used to test for an immunoconjugate-dependent specific cytolytic response against cultured human melanoma cells by NK cells and complement. The melanoma cells, but not the human fibroblast cells used as the control, were efficiently lysed by both NK cells and complement in the presence of the immunoconjugates. The in vitro results suggest that the immunoconjugates also could activate a specific cytolytic immune response against melanoma tumors in vivo.

Dysregulated microRNA Activity in Shwachman-Diamond Syndrome

DTIC Science & Technology

2016-09-01

define transcriptional signatures of bone marrow failure in SDS using single cell RNA -seq of patient cells. We will analyze these datasets to test the...microRNA expression profiles from HSPCs to be overlaid onto mRNA profiles. 15. SUBJECT TERMS Single cell RNA -seq; bone marrow failure; hematopoiesis...myelopoiesis; targeted RNA -seq 16. SECURITY CLASSIFICATION OF: U 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON

Properties of V1 Neurons Tuned to Conjunctions of Visual Features: Application of the V1 Saliency Hypothesis to Visual Search behavior

PubMed Central

Zhaoping, Li; Zhe, Li

2012-01-01

From a computational theory of V1, we formulate an optimization problem to investigate neural properties in the primary visual cortex (V1) from human reaction times (RTs) in visual search. The theory is the V1 saliency hypothesis that the bottom-up saliency of any visual location is represented by the highest V1 response to it relative to the background responses. The neural properties probed are those associated with the less known V1 neurons tuned simultaneously or conjunctively in two feature dimensions. The visual search is to find a target bar unique in color (C), orientation (O), motion direction (M), or redundantly in combinations of these features (e.g., CO, MO, or CM) among uniform background bars. A feature singleton target is salient because its evoked V1 response largely escapes the iso-feature suppression on responses to the background bars. The responses of the conjunctively tuned cells are manifested in the shortening of the RT for a redundant feature target (e.g., a CO target) from that predicted by a race between the RTs for the two corresponding single feature targets (e.g., C and O targets). Our investigation enables the following testable predictions. Contextual suppression on the response of a CO-tuned or MO-tuned conjunctive cell is weaker when the contextual inputs differ from the direct inputs in both feature dimensions, rather than just one. Additionally, CO-tuned cells and MO-tuned cells are often more active than the single feature tuned cells in response to the redundant feature targets, and this occurs more frequently for the MO-tuned cells such that the MO-tuned cells are no less likely than either the M-tuned or O-tuned neurons to be the most responsive neuron to dictate saliency for an MO target. PMID:22719829

Properties of V1 neurons tuned to conjunctions of visual features: application of the V1 saliency hypothesis to visual search behavior.

PubMed

Zhaoping, Li; Zhe, Li

2012-01-01

From a computational theory of V1, we formulate an optimization problem to investigate neural properties in the primary visual cortex (V1) from human reaction times (RTs) in visual search. The theory is the V1 saliency hypothesis that the bottom-up saliency of any visual location is represented by the highest V1 response to it relative to the background responses. The neural properties probed are those associated with the less known V1 neurons tuned simultaneously or conjunctively in two feature dimensions. The visual search is to find a target bar unique in color (C), orientation (O), motion direction (M), or redundantly in combinations of these features (e.g., CO, MO, or CM) among uniform background bars. A feature singleton target is salient because its evoked V1 response largely escapes the iso-feature suppression on responses to the background bars. The responses of the conjunctively tuned cells are manifested in the shortening of the RT for a redundant feature target (e.g., a CO target) from that predicted by a race between the RTs for the two corresponding single feature targets (e.g., C and O targets). Our investigation enables the following testable predictions. Contextual suppression on the response of a CO-tuned or MO-tuned conjunctive cell is weaker when the contextual inputs differ from the direct inputs in both feature dimensions, rather than just one. Additionally, CO-tuned cells and MO-tuned cells are often more active than the single feature tuned cells in response to the redundant feature targets, and this occurs more frequently for the MO-tuned cells such that the MO-tuned cells are no less likely than either the M-tuned or O-tuned neurons to be the most responsive neuron to dictate saliency for an MO target.

Global preamplification simplifies targeted mRNA quantification

PubMed Central

Kroneis, Thomas; Jonasson, Emma; Andersson, Daniel; Dolatabadi, Soheila; Ståhlberg, Anders

2017-01-01

The need to perform gene expression profiling using next generation sequencing and quantitative real-time PCR (qPCR) on small sample sizes and single cells is rapidly expanding. However, to analyse few molecules, preamplification is required. Here, we studied global and target-specific preamplification using 96 optimised qPCR assays. To evaluate the preamplification strategies, we monitored the reactions in real-time using SYBR Green I detection chemistry followed by melting curve analysis. Next, we compared yield and reproducibility of global preamplification to that of target-specific preamplification by qPCR using the same amount of total RNA. Global preamplification generated 9.3-fold lower yield and 1.6-fold lower reproducibility than target-specific preamplification. However, the performance of global preamplification is sufficient for most downstream applications and offers several advantages over target-specific preamplification. To demonstrate the potential of global preamplification we analysed the expression of 15 genes in 60 single cells. In conclusion, we show that global preamplification simplifies targeted gene expression profiling of small sample sizes by a flexible workflow. We outline the pros and cons for global preamplification compared to target-specific preamplification. PMID:28332609

Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strauch, Eva-Maria; Bernard, Steffen M.; La, David

Many viral surface glycoproteins and cell surface receptors are homo-oligomers1, 2, 3, 4, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites5, 6, 7, 8. In the first step, a small protein ismore » designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.« less

Targeting foreign major histocompatibility complex molecules to tumors by tumor cell specific single chain antibody (scFv).

PubMed

Li, Jinhua; Franek, Karl J; Patterson, Andrea L; Holmes, Lillia M; Burgin, Kelly E; Ji, Jianfei; Yu, Xianzhong; Wagner, Thomas E; Wei, Yanzhang

2003-11-01

Down-regulation of the major histocompatibility complex (MHC) is one of the major mechanisms that tumor cells adopted to escape immunosurveillance. Therefore, specifically coating tumor cells with foreign MHC may make tumor cells a better target for immune recognition and surveillance. In this study, we designed and generated a fusion protein, H2Kd/scPSMA, consisting of a single chain antibody against human prostate specific membrane antigen (PSMA) and the extracellular domain of mouse H-2Kd. The expression of this fusion protein in B16F0 mouse melanoma cells was confirmed by RT-PCR and fluorescent activated cell sorting (FACS). Our animal study showed that the expression of H2Kd/scPSMA in B16F0/PSMA5, a B16F0 cell line expressing human PSMA, significantly inhibited tumor growth as demonstrated in the pulmonary metastasis assay and tumor growth study and improved overall survival.

Microcinematographic and electron microscopic analysis of target cell lysis induced by cytotoxic T lymphocytes.

PubMed Central

Matter, A

1979-01-01

A study was carried out to determine the sequence of events of T-cell mediated target cell lysis in microcinematography and electron microscopy. Highly efficient cytotoxic T lymphocytes (CTL) were generated in vivo and in vitro using preimmunized spleen cells and purification procedures. Such CTL were highly specific. This specificity correlated well with the number of adhesions formed between CTL and targets and this criterion was used to study killer-target cell interaction. Microcinematography showed that target cell lysis at the single cell level, despite time variations, could be clearly separated into three phases: (a) a recognition phase, visible by random crawling of CTL over the target cell surface until firm contact was established; (b) a post-recognition phase, during which firm contact between CTL and target was maintained without gross modification of either cell; (c) a phase of target cell disintegration, mainly characterized by vigorous blebbing of the cell membrane resulting in a motionless carcass of the target cell but not in its total dissolution. Only later this carcass decayed and formed a necrotic ghost. Electron microscopic observations were put into sequence according to microcinematography. Post-recognition phase was characterized by a tight apposition of the membranes of CTL and target cell. No gap junctions could be observed. During target cell disintegration, profound cytoplasmic and nuclear changes occurred simultaneous with surface blebbing. Most noticeable were extensive internal vacuolization, mitochondrial swelling, nuclear pycnosis and dissolution of the nucleolus. These observations suggested that target cell lysis does not start with a surface phenomenon similar to complement lysis, but a process involving practically the whole cell simultaneously. It is conceivable, therefore, that the signal from the CTL is transmitted across the target cell, and that the switch to sudden cell death is manipulated deep inside the cell. Images Figure 3 Figures 4-7 Figures 8-11 Figure 12 Figures 13-14 Figure 15 PMID:312256

The Fate of a Normal Human Cell Traversed by a Single Charged Particle

NASA Astrophysics Data System (ADS)

Fournier, C.; Zahnreich, S.; Kraft, D.; Friedrich, T.; Voss, K.-O.; Durante, M.; Ritter, S.

2012-09-01

The long-term ``fate'' of normal human cells after single hits of charged particles is one of the oldest unsolved issues in radiation protection and cellular radiobiology. Using a high-precision heavy-ion microbeam we could target normal human fibroblasts with exactly one or five carbon ions and measured the early cytogenetic damage and the late behaviour using single-cell cloning. Around 70% of the first cycle cells presented visible aberrations in mFISH after a single ion traversal, and about 5% of the cells were still able to form colonies. In one third of selected high-proliferative colonies we observed clonal (radiation-induced) aberrations. Terminal differentiation and markers of senescence (PCNA, p16) in the descendants of cells traversed by one carbon ion occurred earlier than in controls, but no evidence of radiation-induced chromosomal instability was found. We conclude that cells surviving single-ion traversal, often carrying clonal chromosome aberrations, undergo accelerated senescence but maintain chromosomal stability.

The Fate of a Normal Human Cell Traversed by a Single Charged Particle

PubMed Central

Fournier, C.; Zahnreich, S.; Kraft, D.; Friedrich, T.; Voss, K.-O.; Durante, M.; Ritter, S.

2012-01-01

The long-term “fate” of normal human cells after single hits of charged particles is one of the oldest unsolved issues in radiation protection and cellular radiobiology. Using a high-precision heavy-ion microbeam we could target normal human fibroblasts with exactly one or five carbon ions and measured the early cytogenetic damage and the late behaviour using single-cell cloning. Around 70% of the first cycle cells presented visible aberrations in mFISH after a single ion traversal, and about 5% of the cells were still able to form colonies. In one third of selected high-proliferative colonies we observed clonal (radiation-induced) aberrations. Terminal differentiation and markers of senescence (PCNA, p16) in the descendants of cells traversed by one carbon ion occurred earlier than in controls, but no evidence of radiation-induced chromosomal instability was found. We conclude that cells surviving single-ion traversal, often carrying clonal chromosome aberrations, undergo accelerated senescence but maintain chromosomal stability. PMID:22966418

Application of single-cell sequencing in human cancer.

PubMed

Rantalainen, Mattias

2017-11-02

Precision medicine is emerging as a cornerstone of future cancer care with the objective of providing targeted therapies based on the molecular phenotype of each individual patient. Traditional bulk-level molecular phenotyping of tumours leads to significant information loss, as the molecular profile represents an average phenotype over large numbers of cells, while cancer is a disease with inherent intra-tumour heterogeneity at the cellular level caused by several factors, including clonal evolution, tissue hierarchies, rare cells and dynamic cell states. Single-cell sequencing provides means to characterize heterogeneity in a large population of cells and opens up opportunity to determine key molecular properties that influence clinical outcomes, including prognosis and probability of treatment response. Single-cell sequencing methods are now reliable enough to be used in many research laboratories, and we are starting to see applications of these technologies for characterization of human primary cancer cells. In this review, we provide an overview of studies that have applied single-cell sequencing to characterize human cancers at the single-cell level, and we discuss some of the current challenges in the field. © The Author 2017. Published by Oxford University Press.

A Versatile Microarray Platform for Capturing Rare Cells

NASA Astrophysics Data System (ADS)

Brinkmann, Falko; Hirtz, Michael; Haller, Anna; Gorges, Tobias M.; Vellekoop, Michael J.; Riethdorf, Sabine; Müller, Volkmar; Pantel, Klaus; Fuchs, Harald

2015-10-01

Analyses of rare events occurring at extremely low frequencies in body fluids are still challenging. We established a versatile microarray-based platform able to capture single target cells from large background populations. As use case we chose the challenging application of detecting circulating tumor cells (CTCs) - about one cell in a billion normal blood cells. After incubation with an antibody cocktail, targeted cells are extracted on a microarray in a microfluidic chip. The accessibility of our platform allows for subsequent recovery of targets for further analysis. The microarray facilitates exclusion of false positive capture events by co-localization allowing for detection without fluorescent labelling. Analyzing blood samples from cancer patients with our platform reached and partly outreached gold standard performance, demonstrating feasibility for clinical application. Clinical researchers free choice of antibody cocktail without need for altered chip manufacturing or incubation protocol, allows virtual arbitrary targeting of capture species and therefore wide spread applications in biomedical sciences.

Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells.

PubMed

Darmanis, Spyros; Gallant, Caroline Julie; Marinescu, Voichita Dana; Niklasson, Mia; Segerman, Anna; Flamourakis, Georgios; Fredriksson, Simon; Assarsson, Erika; Lundberg, Martin; Nelander, Sven; Westermark, Bengt; Landegren, Ulf

2016-01-12

Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell's phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Single-Cell Imaging Using Radioluminescence Microscopy Reveals Unexpected Binding Target for [18F]HFB.

PubMed

Kiru, Louise; Kim, Tae Jin; Shen, Bin; Chin, Frederick T; Pratx, Guillem

2018-06-01

Cell-based therapies are showing great promise for a variety of diseases, but remain hindered by the limited information available regarding the biological fate, migration routes and differentiation patterns of infused cells in trials. Previous studies have demonstrated the feasibility of using positron emission tomography (PET) to track single cells utilising an approach known as positron emission particle tracking (PEPT). The radiolabel hexadecyl-4-[ 18 F]fluorobenzoate ([ 18 F]HFB) was identified as a promising candidate for PEPT, due to its efficient and long-lasting labelling capabilities. The purpose of this work was to characterise the labelling efficiency of [ 18 F]HFB in vitro at the single-cell level prior to in vivo studies. The binding efficiency of [ 18 F]HFB to MDA-MB-231 and Jurkat cells was verified in vitro using bulk gamma counting. The measurements were subsequently repeated in single cells using a new method known as radioluminescence microscopy (RLM) and binding of the radiolabel to the single cells was correlated with various fluorescent dyes. Similar to previous reports, bulk cell labelling was significantly higher with [ 18 F]HFB (18.75 ± 2.47 dpm/cell, n = 6) than 2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]FDG) (7.59 ± 0.73 dpm/cell, n = 7; p ≤ 0.01). However, single-cell imaging using RLM revealed that [ 18 F]HFB accumulation in live cells (8.35 ± 1.48 cpm/cell, n = 9) was not significantly higher than background levels (4.83 ± 0.52 cpm/cell, n = 12; p > 0.05) and was 1.7-fold lower than [ 18 F]FDG uptake in the same cell line (14.09 ± 1.90 cpm/cell, n = 13; p < 0.01). Instead, [ 18 F]HFB was found to bind significantly to fragmented membranes associated with dead cell nuclei, suggesting an alternative binding target for [ 18 F]HFB. This study demonstrates that bulk analysis alone does not always accurately portray the labelling efficiency, therefore highlighting the need for more routine screening of radiolabels using RLM to identify heterogeneity at the single-cell level.

Single-Cell Isolation of Circulating Tumor Cells from Whole Blood by Lateral Magnetophoretic Microseparation and Microfluidic Dispensing.

PubMed

Kim, Jinho; Cho, Hyungseok; Han, Song-I; Han, Ki-Ho

2016-05-03

This paper introduces a single-cell isolation technology for circulating tumor cells (CTCs) using a microfluidic device (the "SIM-Chip"). The SIM-Chip comprises a lateral magnetophoretic microseparator and a microdispenser as a two-step cascade platform. First, CTCs were enriched from whole blood by the lateral magnetophoretic microseparator based on immunomagnetic nanobeads. Next, the enriched CTCs were electrically identified by single-cell impedance cytometer and isolated as single cells using the microshooter. Using 200 μL of whole blood spiked with 50 MCF7 breast cancer cells, the analysis demonstrated that the single-cell isolation efficiency of the SIM-Chip was 82.4%, and the purity of the isolated MCF7 cells with respect to WBCs was 92.45%. The data also showed that the WBC depletion rate of the SIM-Chip was 2.5 × 10(5) (5.4-log). The recovery rates were around 99.78% for spiked MCF7 cells ranging in number from 10 to 90. The isolated single MCF7 cells were intact and could be used for subsequent downstream genetic assays, such as RT-PCR. Single-cell culture evaluation of the proliferation of MCF7 cells isolated by the SIM-Chip showed that 84.1% of cells at least doubled in 5 days. Consequently, the SIM-Chip could be used for single-cell isolation of rare target cells from whole blood with high purity and recovery without cell damage.

Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia.

PubMed

Giustacchini, Alice; Thongjuea, Supat; Barkas, Nikolaos; Woll, Petter S; Povinelli, Benjamin J; Booth, Christopher A G; Sopp, Paul; Norfo, Ruggiero; Rodriguez-Meira, Alba; Ashley, Neil; Jamieson, Lauren; Vyas, Paresh; Anderson, Kristina; Segerstolpe, Åsa; Qian, Hong; Olsson-Strömberg, Ulla; Mustjoki, Satu; Sandberg, Rickard; Jacobsen, Sten Eirik W; Mead, Adam J

2017-06-01

Recent advances in single-cell transcriptomics are ideally placed to unravel intratumoral heterogeneity and selective resistance of cancer stem cell (SC) subpopulations to molecularly targeted cancer therapies. However, current single-cell RNA-sequencing approaches lack the sensitivity required to reliably detect somatic mutations. We developed a method that combines high-sensitivity mutation detection with whole-transcriptome analysis of the same single cell. We applied this technique to analyze more than 2,000 SCs from patients with chronic myeloid leukemia (CML) throughout the disease course, revealing heterogeneity of CML-SCs, including the identification of a subgroup of CML-SCs with a distinct molecular signature that selectively persisted during prolonged therapy. Analysis of nonleukemic SCs from patients with CML also provided new insights into cell-extrinsic disruption of hematopoiesis in CML associated with clinical outcome. Furthermore, we used this single-cell approach to identify a blast-crisis-specific SC population, which was also present in a subclone of CML-SCs during the chronic phase in a patient who subsequently developed blast crisis. This approach, which might be broadly applied to any malignancy, illustrates how single-cell analysis can identify subpopulations of therapy-resistant SCs that are not apparent through cell-population analysis.

Dual HER2/PIK3CA Targeting Overcomes Single-Agent Acquired Resistance in HER2-Amplified Uterine Serous Carcinoma Cell Lines In Vitro and In Vivo.

PubMed

Lopez, Salvatore; Cocco, Emiliano; Black, Jonathan; Bellone, Stefania; Bonazzoli, Elena; Predolini, Federica; Ferrari, Francesca; Schwab, Carlton L; English, Diana P; Ratner, Elena; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E; Terranova, Corrado; Angioli, Roberto; Santin, Alessandro D

2015-11-01

HER2/neu gene amplification and PIK3CA driver mutations are common in uterine serous carcinoma (USC) and may represent ideal therapeutic targets against this aggressive variant of endometrial cancer. We examined the sensitivity to neratinib, taselisib, and the combination of the two compounds in in vitro and in vivo experiments using PIK3CA-mutated and PIK3CA wild-type HER2/neu-amplified USC cell lines. Cell viability and cell-cycle distribution were assessed using flow-cytometry assays. Downstream signaling was assessed by immunoblotting. Preclinical efficacy of single versus dual inhibition was evaluated in vivo using two USC xenografts. We found both single-agent neratinib and taselisib to be active but only transiently effective in controlling the in vivo growth of USC xenografts harboring HER2/neu gene amplification with or without oncogenic PIK3CA mutations. In contrast, the combination of the two inhibitors caused a stronger and long-lasting growth inhibition in both USC xenografts when compared with single-agent therapy. Combined targeting of HER2 and PIK3CA was associated with a significant and dose-dependent increase in the percentage of cells in the G0-G1 phase of the cell cycle and a dose-dependent decline in the phosphorylation of S6. Importantly, dual inhibition therapy initiated after tumor progression in single-agent-treated mice was still remarkably effective at inducing tumor regression in both large PIK3CA and pan-ErbB inhibitor-resistant USC xenografts. Dual HER2/PIK3CA blockade may represent a novel therapeutic option for USC patients harboring tumors with HER2/neu gene amplification and mutated or wild-type PIK3CA resistant to chemotherapy. ©2015 American Association for Cancer Research.

Single α-particle irradiation permits real-time visualization of RNF8 accumulation at DNA damaged sites

NASA Astrophysics Data System (ADS)

Muggiolu, Giovanna; Pomorski, Michal; Claverie, Gérard; Berthet, Guillaume; Mer-Calfati, Christine; Saada, Samuel; Devès, Guillaume; Simon, Marina; Seznec, Hervé; Barberet, Philippe

2017-01-01

As well as being a significant source of environmental radiation exposure, α-particles are increasingly considered for use in targeted radiation therapy. A better understanding of α-particle induced damage at the DNA scale can be achieved by following their tracks in real-time in targeted living cells. Focused α-particle microbeams can facilitate this but, due to their low energy (up to a few MeV) and limited range, α-particles detection, delivery, and follow-up observations of radiation-induced damage remain difficult. In this study, we developed a thin Boron-doped Nano-Crystalline Diamond membrane that allows reliable single α-particles detection and single cell irradiation with negligible beam scattering. The radiation-induced responses of single 3 MeV α-particles delivered with focused microbeam are visualized in situ over thirty minutes after irradiation by the accumulation of the GFP-tagged RNF8 protein at DNA damaged sites.

Functionalized Single-Walled Carbon Nanotube-Based Fuel Cell Benchmarked Against US DOE 2017 Technical Targets

PubMed Central

Jha, Neetu; Ramesh, Palanisamy; Bekyarova, Elena; Tian, Xiaojuan; Wang, Feihu; Itkis, Mikhail E.; Haddon, Robert C.

2013-01-01

Chemically modified single-walled carbon nanotubes (SWNTs) with varying degrees of functionalization were utilized for the fabrication of SWNT thin film catalyst support layers (CSLs) in polymer electrolyte membrane fuel cells (PEMFCs), which were suitable for benchmarking against the US DOE 2017 targets. Use of the optimum level of SWNT -COOH functionality allowed the construction of a prototype SWNT-based PEMFC with total Pt loading of 0.06 mgPt/cm2 - well below the value of 0.125 mgPt/cm2 set as the US DOE 2017 technical target for total Pt group metals (PGM) loading. This prototype PEMFC also approaches the technical target for the total Pt content per kW of power (<0.125 gPGM/kW) at cell potential 0.65 V: a value of 0.15 gPt/kW was achieved at 80°C/22 psig testing conditions, which was further reduced to 0.12 gPt/kW at 35 psig back pressure. PMID:23877112

Functionalized single-walled carbon nanotube-based fuel cell benchmarked against US DOE 2017 technical targets.

PubMed

Jha, Neetu; Ramesh, Palanisamy; Bekyarova, Elena; Tian, Xiaojuan; Wang, Feihu; Itkis, Mikhail E; Haddon, Robert C

2013-01-01

Chemically modified single-walled carbon nanotubes (SWNTs) with varying degrees of functionalization were utilized for the fabrication of SWNT thin film catalyst support layers (CSLs) in polymer electrolyte membrane fuel cells (PEMFCs), which were suitable for benchmarking against the US DOE 2017 targets. Use of the optimum level of SWNT -COOH functionality allowed the construction of a prototype SWNT-based PEMFC with total Pt loading of 0.06 mg(Pt)/cm²--well below the value of 0.125 mg(Pt)/cm² set as the US DOE 2017 technical target for total Pt group metals (PGM) loading. This prototype PEMFC also approaches the technical target for the total Pt content per kW of power (<0.125 g(PGM)/kW) at cell potential 0.65 V: a value of 0.15 g(Pt)/kW was achieved at 80°C/22 psig testing conditions, which was further reduced to 0.12 g(Pt)/kW at 35 psig back pressure.

Production of genome-edited pluripotent stem cells and mice by CRISPR/Cas.

PubMed

Horii, Takuro; Hatada, Izuho

2016-01-01

Clustered regularly at interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nucleases, so-called CRISPR/Cas, was recently developed as an epoch-making genome engineering technology. This system only requires Cas9 nuclease and single-guide RNA complementary to a target locus. CRISPR/Cas enables the generation of knockout cells and animals in a single step. This system can also be used to generate multiple mutations and knockin in a single step, which is not possible using other methods. In this review, we provide an overview of genome editing by CRISPR/Cas in pluripotent stem cells and mice.

Filopodia Conduct Target Selection in Cortical Neurons Using Differences in Signal Kinetics of a Single Kinase.

PubMed

Mao, Yu-Ting; Zhu, Julia X; Hanamura, Kenji; Iurilli, Giuliano; Datta, Sandeep Robert; Dalva, Matthew B

2018-05-16

Dendritic filopodia select synaptic partner axons by interviewing the cell surface of potential targets, but how filopodia decipher the complex pattern of adhesive and repulsive molecular cues to find appropriate contacts is unknown. Here, we demonstrate in cortical neurons that a single cue is sufficient for dendritic filopodia to reject or select specific axonal contacts for elaboration as synaptic sites. Super-resolution and live-cell imaging reveals that EphB2 is located in the tips of filopodia and at nascent synaptic sites. Surprisingly, a genetically encoded indicator of EphB kinase activity, unbiased classification, and a photoactivatable EphB2 reveal that simple differences in the kinetics of EphB kinase signaling at the tips of filopodia mediate the choice between retraction and synaptogenesis. This may enable individual filopodia to choose targets based on differences in the activation rate of a single tyrosine kinase, greatly simplifying the process of partner selection and suggesting a general principle. Copyright © 2018 Elsevier Inc. All rights reserved.

Killing of intrafamilial leukocytes by earthworm effector cells.

PubMed

Suzuki, M M; Cooper, E L

1995-01-01

When Lumbricus and Eisenia coelomocytes are cultured together in intrafamilial xenogeneic combinations, significant cytotoxicity occurs at 24 h but not at 5 nor 72 h, as shown by trypan blue assay. In a 4.5-h assay, measuring 51Cr release, using an effector/target ratio of 25:1, unpooled cells from a single Lumbricus killed Eisenia cells at levels of 6% and 14%. However, Eisenia coelomocyte survival was high and identical in either cell-free xenogeneic (Lumbricus) coelomic fluid or in artificial medium. In this 1-way assay, earthworm (Lumbricus) coelomocytes act as effector cells that kill non-self target cells, even those of other earthworms. Comparisons with previous results reveal greater reliability and consistently repeatable results when the 51Cr release assay is used to measure cytotoxicity regardless of the targets.

Laser-assisted patch clamping: a methodology

NASA Technical Reports Server (NTRS)

Henriksen, G. H.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

1997-01-01

Laser microsurgery can be used to perform both cell biological manipulations, such as targeted cell ablation, and molecular genetic manipulations, such as genetic transformation and chromosome dissection. In this report, we describe a laser microsurgical method that can be used either to ablate single cells or to ablate a small area (1-3 microns diameter) of the extracellular matrix. In plants and microorganisms, the extracellular matrix consists of the cell wall. While conventional patch clamping of these cells, as well as of many animal cells, requires enzymatic digestion of the extracellular matrix, we illustrate that laser microsurgery of a portion of the wall enables patch clamp access to the plasma membrane of higher plant cells remaining situated in their tissue environment. What follows is a detailed description of the construction and use of an economical laser microsurgery system, including procedures for single cell and targeted cell wall ablation. This methodology will be of interest to scientists wishing to perform cellular or subcellular ablation with a high degree of accuracy, or wishing to study how the extracellular matrix affects ion channel function.

In-situ coupling between kinase activities and protein dynamics within single focal adhesions

NASA Astrophysics Data System (ADS)

Wu, Yiqian; Zhang, Kaiwen; Seong, Jihye; Fan, Jason; Chien, Shu; Wang, Yingxiao; Lu, Shaoying

2016-07-01

The dynamic activation of oncogenic kinases and regulation of focal adhesions (FAs) are crucial molecular events modulating cell adhesion in cancer metastasis. However, it remains unclear how these events are temporally coordinated at single FA sites. Therefore, we targeted fluorescence resonance energy transfer (FRET)-based biosensors toward subcellular FAs to report local molecular events during cancer cell adhesion. Employing single FA tracking and cross-correlation analysis, we quantified the dynamic coupling characteristics between biochemical kinase activities and structural FA within single FAs. We show that kinase activations and FA assembly are strongly and sequentially correlated, with the concurrent FA assembly and Src activation leading focal adhesion kinase (FAK) activation by 42.6 ± 12.6 sec. Strikingly, the temporal coupling between kinase activation and individual FA assembly reflects the fate of FAs at later stages. The FAs with a tight coupling tend to grow and mature, while the less coupled FAs likely disassemble. During FA disassembly, however, kinase activations lead the disassembly, with FAK being activated earlier than Src. Therefore, by integrating subcellularly targeted FRET biosensors and computational analysis, our study reveals intricate interplays between Src and FAK in regulating the dynamic life of single FAs in cancer cells.

Dissecting and Targeting Latent Metastasis

DTIC Science & Technology

2014-09-01

metastasis of breast cancer (LMBC). These cells retain the potential to form overt metastasis for years. Targeting LMBC with new drugs offers an...cancer cell extravasation through the BBB in experimental models and predict brain metastasis in the clinic (16). Once inside the brain parenchyma... drugs that perturb gap junction activity (Chen et al submitted for publication). In our experiments, both drugs as single agents were effective

Automated profiling of individual cell-cell interactions from high-throughput time-lapse imaging microscopy in nanowell grids (TIMING).

PubMed

Merouane, Amine; Rey-Villamizar, Nicolas; Lu, Yanbin; Liadi, Ivan; Romain, Gabrielle; Lu, Jennifer; Singh, Harjeet; Cooper, Laurence J N; Varadarajan, Navin; Roysam, Badrinath

2015-10-01

There is a need for effective automated methods for profiling dynamic cell-cell interactions with single-cell resolution from high-throughput time-lapse imaging data, especially, the interactions between immune effector cells and tumor cells in adoptive immunotherapy. Fluorescently labeled human T cells, natural killer cells (NK), and various target cells (NALM6, K562, EL4) were co-incubated on polydimethylsiloxane arrays of sub-nanoliter wells (nanowells), and imaged using multi-channel time-lapse microscopy. The proposed cell segmentation and tracking algorithms account for cell variability and exploit the nanowell confinement property to increase the yield of correctly analyzed nanowells from 45% (existing algorithms) to 98% for wells containing one effector and a single target, enabling automated quantification of cell locations, morphologies, movements, interactions, and deaths without the need for manual proofreading. Automated analysis of recordings from 12 different experiments demonstrated automated nanowell delineation accuracy >99%, automated cell segmentation accuracy >95%, and automated cell tracking accuracy of 90%, with default parameters, despite variations in illumination, staining, imaging noise, cell morphology, and cell clustering. An example analysis revealed that NK cells efficiently discriminate between live and dead targets by altering the duration of conjugation. The data also demonstrated that cytotoxic cells display higher motility than non-killers, both before and during contact. broysam@central.uh.edu or nvaradar@central.uh.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Single-domain antibody bioconjugated near-IR quantum dots for targeted cellular imaging of pancreatic cancer.

PubMed

Zaman, Md Badruz; Baral, Toya Nath; Jakubek, Zygmunt J; Zhang, Jianbing; Wu, Xiaohua; Lai, Edward; Whitfield, Dennis; Yu, Kui

2011-05-01

Successful targeted imaging of BxPC3 human pancreatic cancer cells is feasible with near-IR CdTeSe/CdS quantum dots (QDs) functionalized with single-domain antibody (sdAb) 2A3. For specific targeting, sdAbs are superior to conventional antibodies, especially in terms of stability, aggregation, and production cost. The bright CdTeSe/CdS QDs were synthesized to emit in the diagnostic window of 650-900 nm with a narrow emission band. 2A3 was derived from llama and is small in size of 13 kDa, but with fully-functional recognition to the target carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), a possible biomarker as a therapeutic target of pancreatic cancer. For compelling imaging, optical may be the most sensible among the various imaging modalities, regarding the sensitivity and cost. This first report on sdAb-conjugated near-IR QDs with high signal to background sensitivity for targeted cellular imaging brings insights into the development of optical molecular imaging for early stage cancer diagnosis.

Evaluation of the genotoxic potential of 3-monochloropropane-1,2-diol (3-MCPD) and its metabolites, glycidol and beta-chlorolactic acid, using the single cell gel/comet assay.

PubMed

El Ramy, R; Ould Elhkim, M; Lezmi, S; Poul, J M

2007-01-01

3-monochloropropane-1,2-diol (3-MCPD) is a member of a group of chemicals known as chloropropanols. It is found in many foods and food ingredients as a result of food processing. 3-MCPD is regarded as a rat carcinogen known to induce Leydig-cell and mammary gland tumours in males and kidney tumours in both genders. The aim of our study was to clarify the possible involvement of genotoxic mechanisms in 3-MCPD induced carcinogenicity at the target organ level. For that purpose, we evaluated DNA damages in selected target (kidneys and testes) and non-target (blood leukocytes, liver and bone marrow) male rat organs by the in vivo alkaline single cell gel electrophoresis (comet) assay, 3 and 24 h after 3-MCPD oral administration to Sprague-Dawley and Fisher 344 adult rats. 3-MCPD may be metabolised to a genotoxic intermediate, glycidol, whereas the predominant urinary metabolite in rats following 3-MCPD administration is beta-chlorolactic acid. Therefore, we also studied the DNA damaging effects of 3-MCPD and its metabolites, glycidol and beta-chlorolactic acid, in the in vitro comet assay on CHO cells. Our results show the absence of genotoxic potential of 3-MCPD in vivo in the target as well as in the non-target organs. Glycidol, the epoxide metabolite, induced DNA damages in CHO cells. beta-Chlorolactic acid, the main metabolite of 3-MCPD in rats, was shown to be devoid of DNA-damaging effects in vitro in mammalian cells.

Tracking single membrane targets of human autoantibodies using single nanoparticle imaging.

PubMed

Jézéquel, Julie; Dupuis, Julien P; Maingret, François; Groc, Laurent

2018-04-21

Over the past decade, an increasing number of neurological and neuropsychiatric diseases have been associated with the expression of autoantibodies directed against neuronal targets, including neurotransmitter receptors. Although cell-based assays are routinely used in clinics to detect the presence of immunoglobulins, such tests often provide heterogeneous outcomes due to their limited sensitivity, especially at low titers. Thus, there is an urging need for new methods allowing the detection of autoantibodies in seropositive patients that cannot always be clinically distinguished from seronegative ones. Here we make a case for single nanoparticle imaging approaches as a highly sensitive antibody detection assay. Through high-affinity interactions between functionalized nanoparticles and autoantibodies that recognize extracellular domains of membrane neuronal targets, single nanoparticle imaging allows a live surface staining of transmembrane proteins and gives access to their surface dynamics. We show here that this method is well-suited to detect low titers of purified immunoglobulin G (IgG) from first-episode psychotic patients and demonstrate that these IgG target glutamatergic N-Methyl-d-Aspartate receptors (NMDAR) in live hippocampal neurons. The molecular behaviors of targeted membrane receptors were indistinguishable from those of endogenous GluN1 NMDAR subunit and were virtually independent of the IgG concentration present in the sample contrary to classical cell-based assays. Single nanoparticle imaging emerges as a real-time sensitive method to detect IgG directed against neuronal surface proteins, which could be used as an additional step to rule out ambiguous seropositivity diagnoses. Copyright © 2018 Elsevier B.V. All rights reserved.

Single Nisoldipine-Sensitive Calcium Channels in Smooth Muscle Cells Isolated from Rabbit Mesenteric Artery

NASA Astrophysics Data System (ADS)

Worley, Jennings F.; Deitmer, Joachim W.; Nelson, Mark T.

1986-08-01

Single smooth muscle cells were enzymatically isolated from the rabbit mesenteric artery. At physiological levels of external Ca, these cells were relaxed and contracted on exposure to norepinephrine, caffeine, or high levels of potassium. The patch-clamp technique was used to measure unitary currents through single channels in the isolated cells. Single channels were selective for divalent cations and exhibited two conductance levels, 8 pS and 15 pS. Both types of channels were voltage-dependent, and channel activity occurred at potentials positive to -40 mV. The activity of both channel types was almost completely inhibited by 50 nM nisoldipine. These channels appear to be the pathways for voltage-dependent Ca influx in vascular smooth muscle and may be the targets of the clinically used dihydropyridines.

Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9

PubMed Central

Mandal, Pankaj K.; Ferreira, Leonardo M. R.; Collins, Ryan; Meissner, Torsten B.; Boutwell, Christian L.; Friesen, Max; Vrbanac, Vladimir; Garrison, Brian S.; Stortchevoi, Alexei; Bryder, David; Musunuru, Kiran; Brand, Harrison; Tager, Andrew M.; Allen, Todd M.; Talkowski, Michael E.; Rossi, Derrick J.; Cowan, Chad A.

2014-01-01

SUMMARY Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9 mediated genome editing in clinically relevant human somatic cells remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multi-lineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy. PMID:25517468

Ibrutinib (PCI-32765) in Chronic Lymphocytic Leukemia

PubMed Central

Jain, Nitin; O’Brien, Susan

2015-01-01

SYNOPSIS B-cell receptor (BCR) signaling is essential for chronic lymphocytic leukemia (CLL) cell survival. Many kinases in the BCR signaling pathway are currently being studied as potential therapeutic targets. These include Lyn, Syk, PI3 and Bruton tyrosine (BTK). Ibrutinib (PCI-32765) is a novel first-in-class selective inhibitor of BTK. Preclinical evidence suggests that ibrutinib inhibits CLL cell survival and proliferation. In addition, it also affects CLL cell migration and homing. Early clinical data in CLL and non-Hodgkin’s lymphoma patients is very encouraging. In relapsed-refractory patients with CLL, a 67% response rate was observed (420mg dose cohort) with single-agent ibrutinib. Long-term follow-up of these studies and other ongoing/planned studies of ibrutinib either as single-agent or in combination with monoclonal antibodies and chemoimmunotherapy is eagerly awaited. It is likely that ibrutinib and other drugs targeting the BCR pathway will become an integral component of CLL therapy. PMID:23915749

Robust high-performance nanoliter-volume single-cell multiple displacement amplification on planar substrates.

PubMed

Leung, Kaston; Klaus, Anders; Lin, Bill K; Laks, Emma; Biele, Justina; Lai, Daniel; Bashashati, Ali; Huang, Yi-Fei; Aniba, Radhouane; Moksa, Michelle; Steif, Adi; Mes-Masson, Anne-Marie; Hirst, Martin; Shah, Sohrab P; Aparicio, Samuel; Hansen, Carl L

2016-07-26

The genomes of large numbers of single cells must be sequenced to further understanding of the biological significance of genomic heterogeneity in complex systems. Whole genome amplification (WGA) of single cells is generally the first step in such studies, but is prone to nonuniformity that can compromise genomic measurement accuracy. Despite recent advances, robust performance in high-throughput single-cell WGA remains elusive. Here, we introduce droplet multiple displacement amplification (MDA), a method that uses commercially available liquid dispensing to perform high-throughput single-cell MDA in nanoliter volumes. The performance of droplet MDA is characterized using a large dataset of 129 normal diploid cells, and is shown to exceed previously reported single-cell WGA methods in amplification uniformity, genome coverage, and/or robustness. We achieve up to 80% coverage of a single-cell genome at 5× sequencing depth, and demonstrate excellent single-nucleotide variant (SNV) detection using targeted sequencing of droplet MDA product to achieve a median allelic dropout of 15%, and using whole genome sequencing to achieve false and true positive rates of 9.66 × 10(-6) and 68.8%, respectively, in a G1-phase cell. We further show that droplet MDA allows for the detection of copy number variants (CNVs) as small as 30 kb in single cells of an ovarian cancer cell line and as small as 9 Mb in two high-grade serous ovarian cancer samples using only 0.02× depth. Droplet MDA provides an accessible and scalable method for performing robust and accurate CNV and SNV measurements on large numbers of single cells.

Multiplex bioimaging of piRNA molecular pathway-regulated theragnostic effects in a single breast cancer cell using a piRNA molecular beacon.

PubMed

Lee, Youn Jung; Moon, Sung Ung; Park, Min Geun; Jung, Woon Yong; Park, Yong Keun; Song, Sung Kyu; Ryu, Je Gyu; Lee, Yong Seung; Heo, Hye Jung; Gu, Ha Na; Cho, Su Jeong; Ali, Bahy A; Al-Khedhairy, Abdulaziz A; Lee, Ilkyun; Kim, Soonhag

2016-09-01

Recently, PIWI-interacting small non-coding RNAs (piRNAs) have emerged as novel cancer biomarkers candidate because of their high expression level in various cancer types and role in the control of tumor suppressor genes. In this study, a novel breast cancer theragnostics probe based on a single system targeting the piRNA-36026 (piR-36026) molecular pathway was developed using a piR-36026 molecular beacon (MB). The piR-36026 MB successfully visualized endogenous piR-36026 biogenesis, which is highly expressed in MCF7 cells (a human breast cancer cell line), and simultaneously inhibited piR-36026-mediated cancer progression in vitro and in vivo. We discovered two tumor suppressor proteins, SERPINA1 and LRAT, that were directly regulated as endogenous piR-36026 target genes in MCF7 cells. Furthermore, multiplex bioimaging of a single MCF7 cell following treatment with piR-36026 MB clearly visualized the direct molecular interaction of piRNA-36026 with SERPINA1 or LRAT and subsequent molecular therapeutic responses including caspase-3 and PI in the nucleus. Copyright © 2016 Elsevier Ltd. All rights reserved.

Single-cell transcriptomics for microbial eukaryotes.

PubMed

Kolisko, Martin; Boscaro, Vittorio; Burki, Fabien; Lynn, Denis H; Keeling, Patrick J

2014-11-17

One of the greatest hindrances to a comprehensive understanding of microbial genomics, cell biology, ecology, and evolution is that most microbial life is not in culture. Solutions to this problem have mainly focused on whole-community surveys like metagenomics, but these analyses inevitably loose information and present particular challenges for eukaryotes, which are relatively rare and possess large, gene-sparse genomes. Single-cell analyses present an alternative solution that allows for specific species to be targeted, while retaining information on cellular identity, morphology, and partitioning of activities within microbial communities. Single-cell transcriptomics, pioneered in medical research, offers particular potential advantages for uncultivated eukaryotes, but the efficiency and biases have not been tested. Here we describe a simple and reproducible method for single-cell transcriptomics using manually isolated cells from five model ciliate species; we examine impacts of amplification bias and contamination, and compare the efficacy of gene discovery to traditional culture-based transcriptomics. Gene discovery using single-cell transcriptomes was found to be comparable to mass-culture methods, suggesting single-cell transcriptomics is an efficient entry point into genomic data from the vast majority of eukaryotic biodiversity. Copyright © 2014 Elsevier Ltd. All rights reserved.

A pretargeted nanoparticle system for tumor cell labeling

PubMed Central

Gunn, Jonathan; Park, Steven I.; Veiseh, Omid; Press, Oliver W.; Zhang, Miqin

2011-01-01

Nanoparticle-based cancer diagnostics and therapeutics can be significantly enhanced by selective tissue localization, but the strategy can be complicated by the requirement of a targeting ligand conjugated on nanoparticles, that is specific to only one or a limited few types of neoplastic cells, necessitating the development of multiple nanoparticle systems for different diseases. Here, we present a new nanoparticle system that capitalizes on a targeting pretreatment strategy, where a circulating fusion protein (FP) selectively prelabels the targeted cellular epitope, and a biotinylated iron oxide nanoparticle serves as a secondary label that binds to the FP on the target cell. This approach enables a single nanoparticle formulation to be used with any one of existing fusion proteins to bind a variety of target cells. We demonstrated this approach with two fusion proteins against two model cancer cell lines: lymphoma (Ramos) and leukemia (Jurkat), which showed 72.2% and 91.1% positive labeling, respectively. Notably, TEM analysis showed that a large nanoparticle population was endocytosed via attachment to the non-internalizing CD20 epitope. PMID:21107453

A pretargeted nanoparticle system for tumor cell labeling.

PubMed

Gunn, Jonathan; Park, Steven I; Veiseh, Omid; Press, Oliver W; Zhang, Miqin

2011-03-01

Nanoparticle-based cancer diagnostics and therapeutics can be significantly enhanced by selective tissue localization, but the strategy can be complicated by the requirement of a targeting ligand conjugated on nanoparticles, that is specific to only one or a limited few types of neoplastic cells, necessitating the development of multiple nanoparticle systems for different diseases. Here, we present a new nanoparticle system that capitalizes on a targeting pretreatment strategy, where a circulating fusion protein (FP) selectively prelabels the targeted cellular epitope, and a biotinylated iron oxide nanoparticle serves as a secondary label that binds to the FP on the target cell. This approach enables a single nanoparticle formulation to be used with any one of existing fusion proteins to bind a variety of target cells. We demonstrated this approach with two fusion proteins against two model cancer cell lines: lymphoma (Ramos) and leukemia (Jurkat), which showed 72.2% and 91.1% positive labeling, respectively. Notably, TEM analysis showed that a large nanoparticle population was endocytosed via attachment to the non-internalizing CD20 epitope.

Single-particle fusion of influenza viruses reveals complex interactions with target membranes

NASA Astrophysics Data System (ADS)

van der Borg, Guus; Braddock, Scarlett; Blijleven, Jelle S.; van Oijen, Antoine M.; Roos, Wouter H.

2018-05-01

The first step in infection of influenza A virus is contact with the host cell membrane, with which it later fuses. The composition of the target bilayer exerts a complex influence on both fusion efficiency and time. Here, an in vitro, single-particle approach is used to study this effect. Using total internal reflection fluorescence (TIRF) microscopy and a microfluidic flow cell, the hemifusion of single virions is visualized. Hemifusion efficiency and kinetics are studied while altering target bilayer cholesterol content and sialic-acid donor. Cholesterol ratios tested were 0%, 10%, 20%, and 40%. Sialic-acid donors GD1a and GYPA were used. Both cholesterol ratio and sialic-acid donors proved to have a significant effect on hemifusion efficiency. Furthermore, comparison between GD1a and GYPA conditions shows that the cholesterol dependence of the hemifusion time is severely affected by the sialic-acid donor. Only GD1a shows a clear increasing trend in hemifusion efficiency and time with increasing cholesterol concentration of the target bilayer with maximum rates for GD1A and 40% cholesterol. Overall our results show that sialic acid donor and target bilayer composition should be carefully chosen, depending on the desired hemifusion time and efficiency in the experiment.

Efficient Generation of Somatic Cell Nuclear Transfer-Competent Porcine Cells with Mutated Alleles at Multiple Target Loci by Using CRISPR/Cas9 Combined with Targeted Toxin-Based Selection System.

PubMed

Sato, Masahiro; Miyoshi, Kazuchika; Nakamura, Shingo; Ohtsuka, Masato; Sakurai, Takayuki; Watanabe, Satoshi; Kawaguchi, Hiroaki; Tanimoto, Akihide

2017-12-04

The recent advancement in genome editing such a CRISPR/Cas9 system has enabled isolation of cells with knocked multiple alleles through a one-step transfection. Somatic cell nuclear transfer (SCNT) has been frequently employed as one of the efficient tools for the production of genetically modified (GM) animals. To use GM cells as SCNT donor, efficient isolation of transfectants with mutations at multiple target loci is often required. The methods for the isolation of such GM cells largely rely on the use of drug selection-based approach using selectable genes; however, it is often difficult to isolate cells with mutations at multiple target loci. In this study, we used a novel approach for the efficient isolation of porcine cells with at least two target loci mutations by one-step introduction of CRISPR/Cas9-related components. A single guide (sg) RNA targeted to GGTA1 gene, involved in the synthesis of cell-surface α-Gal epitope (known as xenogenic antigen), is always a prerequisite. When the transfected cells were reacted with toxin-labeled BS-I-B₄ isolectin for 2 h at 37 C to eliminate α-Gal epitope-expressing cells, the surviving clones lacked α-Gal epitope expression and were highly expected to exhibit induced mutations at another target loci. Analysis of these α-Gal epitope-negative surviving cells demonstrated a 100% occurrence of genome editing at target loci. SCNT using these cells as donors resulted in the production of cloned blastocysts with the genotype similar to that of the donor cells used. Thus, this novel system will be useful for SCNT-mediated acquisition of GM cloned piglets, in which multiple target loci may be mutated.

Determination of the accuracy for targeted irradiations of cellular substructures at SNAKE

NASA Astrophysics Data System (ADS)

Siebenwirth, C.; Greubel, C.; Drexler, S. E.; Girst, S.; Reindl, J.; Walsh, D. W. M.; Dollinger, G.; Friedl, A. A.; Schmid, T. E.; Drexler, G. A.

2015-04-01

In the last 10 years the ion microbeam SNAKE, installed at the Munich 14 MV tandem accelerator, has been successfully used for radiobiological experiments by utilizing pattern irradiation without targeting single cells. Now for targeted irradiation of cellular substructures a precise irradiation device was added to the live cell irradiation setup at SNAKE. It combines a sub-micrometer single ion irradiation facility with a high resolution optical fluorescence microscope. Most systematic errors can be reduced or avoided by using the same light path in the microscope for beam spot verification as well as for and target recognition. In addition online observation of the induced cellular responses is possible. The optical microscope and the beam delivering system are controlled by an in-house developed software which integrates the open-source image analysis software, CellProfiler, for semi-automatic target recognition. In this work the targeting accuracy was determined by irradiation of a cross pattern with 55 MeV carbon ions on nucleoli in U2OS and HeLa cells stably expressing a GFP-tagged repair protein MDC1. For target recognition, nuclei were stained with Draq5 and nucleoli were stained with Syto80 or Syto83. The damage response was determined by live-cell imaging of MDC1-GFP accumulation directly after irradiation. No systematic displacement and a random distribution of about 0.7 μm (SD) in x-direction and 0.8 μm (SD) in y-direction were observed. An independent analysis after immunofluorescence staining of the DNA damage marker yH2AX yielded similar results. With this performance a target with a size similar to that of nucleoli (i.e. a diameter of about 3 μm) is hit with a probability of more than 80%, which enables the investigation of the radiation response of cellular subcompartments after targeted ion irradiation in the future.

Melatonin potentiates "inside-out" nano-thermotherapy in human breast cancer cells: a potential cancer target multimodality treatment based on melatonin-loaded nanocomposite particles.

PubMed

Xie, Wensheng; Gao, Qin; Wang, Dan; Wang, Wei; Yuan, Jie; Guo, Zhenhu; Yan, Hao; Wang, Xiumei; Sun, Xiaodan; Zhao, Lingyun

2017-01-01

With the wide recognition of oncostatic effect of melatonin, the current study proposes a potential breast cancer target multimodality treatment based on melatonin-loaded magnetic nanocomposite particles (Melatonin-MNPs). Melatonin-MNPs were fabricated by the single emulsion solvent extraction/evaporation method. Based on the facilitated transport of melatonin by the GLUT overexpressed on the cell membrane, such Melatonin-MNPs can be more favorably uptaken by MCF-7 cells compared with the melatonin-free nanocomposite particles, which indicates the cancer targeting ability of melatonin molecule. Inductive heating can be generated by exposure to the Melatonin-MNPs internalized within cancer cells under alternative magnetic field, so as to achieve the "inside-out" magnetic nano-thermotherapy. In addition to demonstrating the superior cytotoxic effect of such nano-thermotherapy over the conventional exogenous heating by metal bath, more importantly, the sustainable release of melatonin from the Melatonin-MNPs can be greatly promoted upon responsive to the magnetic heating. The multimodality treatment based on Melatonin-MNPs can lead to more significant decrease in cell viability than any single treatment, suggesting the potentiated effect of melatonin on the cytotoxic response to nano-thermotherapy. This study is the first to fabricate the precisely engineered melatonin-loaded multifunctional nanocomposite particles and demonstrate the potential in breast cancer target multimodality treatment.

A microfluidic approach to parallelized transcriptional profiling of single cells.

PubMed

Sun, Hao; Olsen, Timothy; Zhu, Jing; Tao, Jianguo; Ponnaiya, Brian; Amundson, Sally A; Brenner, David J; Lin, Qiao

2015-12-01

The ability to correlate single-cell genetic information with cellular phenotypes is of great importance to biology and medicine, as it holds the potential to gain insight into disease pathways that is unavailable from ensemble measurements. We present a microfluidic approach to parallelized, rapid, quantitative analysis of messenger RNA from single cells via RT-qPCR. The approach leverages an array of single-cell RT-qPCR analysis units formed by a set of parallel microchannels concurrently controlled by elastomeric pneumatic valves, thereby enabling parallelized handling and processing of single cells in a drastically simplified operation procedure using a relatively small number of microvalves. All steps for single-cell RT-qPCR, including cell isolation and immobilization, cell lysis, mRNA purification, reverse transcription and qPCR, are integrated on a single chip, eliminating the need for off-chip manual cell and reagent transfer and qPCR amplification as commonly used in existing approaches. Additionally, the approach incorporates optically transparent microfluidic components to allow monitoring of single-cell trapping without the need for molecular labeling that can potentially alter the targeted gene expression and utilizes a polycarbonate film as a barrier against evaporation to minimize the loss of reagents at elevated temperatures during the analysis. We demonstrate the utility of the approach by the transcriptional profiling for the induction of the cyclin-dependent kinase inhibitor 1a and the glyceraldehyde 3-phosphate dehydrogenase in single cells from the MCF-7 breast cancer cell line. Furthermore, the methyl methanesulfonate is employed to allow measurement of the expression of the genes in individual cells responding to a genotoxic stress.

Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector

PubMed Central

Kabadi, Ami M.; Ousterout, David G.; Hilton, Isaac B.; Gersbach, Charles A.

2014-01-01

Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types. PMID:25122746

Human T cells monitored by impedance spectrometry using field-effect transistor arrays: a novel tool for single-cell adhesion and migration studies.

PubMed

Law, Jessica Ka Yan; Susloparova, Anna; Vu, Xuan Thang; Zhou, Xiao; Hempel, Felix; Qu, Bin; Hoth, Markus; Ingebrandt, Sven

2015-05-15

Cytotoxic T lymphocytes (CTLs) play an important role in the immune system by recognizing and eliminating pathogen-infected and tumorigenic cells. In order to achieve their function, T cells have to migrate throughout the whole body and identify the respective targets. In conventional immunology studies, interactions between CTLs and targets are usually investigated using tedious and time-consuming immunofluorescence imaging. However, there is currently no straightforward measurement tool available to examine the interaction strengths. In the present study, adhesion strengths and migration of single human CD8(+) T cells on pre-coated field-effect transistor (FET) devices (i.e. fibronectin, anti-CD3 antibody, and anti-LFA-1 antibody) were measured using impedance spectroscopy. Adhesion strengths to different protein and antibody coatings were compared. By fitting the data to an electronically equivalent circuit model, cell-related parameters (cell membrane capacitance referring to cell morphology and seal resistance referring to adhesion strength) were obtained. This electronically-assessed adhesion strength provides a novel, fast, and important index describing the interaction efficiency. Furthermore, the size of our detection transistor gates as well as their sensitivity reaches down to single cell resolution. Real-time motions of individually migrating T cells can be traced using our FET devices. The in-house fabricated FETs used in the present study are providing a novel and very efficient insight to individual cell interactions. Copyright © 2014 Elsevier B.V. All rights reserved.

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells

NASA Astrophysics Data System (ADS)

Herce, Henry D.; Schumacher, Dominik; Schneider, Anselm F. L.; Ludwig, Anne K.; Mann, Florian A.; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M. Cristina; Hackenberger, Christian P. R.

2017-08-01

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

Unexpected heterogeneity derived from Cas9 ribonucleoprotein-introduced clonal cells at the HPRT1 locus.

PubMed

Sakuma, Tetsushi; Mochida, Keiji; Nakade, Shota; Ezure, Toru; Minagawa, Sachi; Yamamoto, Takashi

2018-04-01

Single-cell cloning is an essential technique for establishing genome-edited cell clones mediated by programmable nucleases such as CRISPR-Cas9. However, residual genome-editing activity after single-cell cloning may cause heterogeneity in the clonal cells. Previous studies showed efficient mutagenesis and rapid degradation of CRISPR-Cas9 components in cultured cells by introducing Cas9 ribonucleoproteins (RNPs). In this study, we investigated how the timing for single-cell cloning of Cas9 RNP-transfected cells affected the heterogeneity of the resultant clones. We carried out transfection of Cas9 RNPs targeting several loci in the HPRT1 gene in HCT116 cells, followed by single-cell cloning at 24, 48, 72 hr and 1 week post-transfection. After approximately 3 weeks of incubation, the clonal cells were collected and genotyped by high-resolution microchip electrophoresis and Sanger sequencing. Unexpectedly, long-term incubation before single-cell cloning resulted in highly heterogeneous clones. We used a lipofection method for transfection, and the media containing transfectable RNPs were not removed before single-cell cloning. Therefore, the active Cas9 RNPs were considered to be continuously incorporated into cells during the precloning incubation. Our findings provide a warning that lipofection of Cas9 RNPs may cause continuous introduction of gene mutations depending on the experimental procedures. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

The microRNA-200 family coordinately regulates cell adhesion and proliferation in hair morphogenesis.

PubMed

Hoefert, Jaimee E; Bjerke, Glen A; Wang, Dongmei; Yi, Rui

2018-06-04

The microRNA (miRNA)-200 (miR-200) family is highly expressed in epithelial cells and frequently lost in metastatic cancer. Despite intensive studies into their roles in cancer, their targets and functions in normal epithelial tissues remain unclear. Importantly, it remains unclear how the two subfamilies of the five-miRNA family, distinguished by a single nucleotide within the seed region, regulate their targets. By directly ligating miRNAs to their targeted mRNA regions, we identify numerous miR-200 targets involved in the regulation of focal adhesion, actin cytoskeleton, cell cycle, and Hippo/Yap signaling. The two subfamilies bind to largely distinct target sites, but many genes are coordinately regulated by both subfamilies. Using inducible and knockout mouse models, we show that the miR-200 family regulates cell adhesion and orientation in the hair germ, contributing to precise cell fate specification and hair morphogenesis. Our findings demonstrate that combinatorial targeting of many genes is critical for miRNA function and provide new insights into miR-200's functions. © 2018 Hoefert et al.

Single-cell and subcellular pharmacokinetic imaging allows insight into drug action in vivo.

PubMed

Thurber, Greg M; Yang, Katy S; Reiner, Thomas; Kohler, Rainer H; Sorger, Peter; Mitchison, Tim; Weissleder, Ralph

2013-01-01

Pharmacokinetic analysis at the organ level provides insight into how drugs distribute throughout the body, but cannot explain how drugs work at the cellular level. Here we demonstrate in vivo single-cell pharmacokinetic imaging of PARP-1 inhibitors and model drug behaviour under varying conditions. We visualize intracellular kinetics of the PARP-1 inhibitor distribution in real time, showing that PARP-1 inhibitors reach their cellular target compartment, the nucleus, within minutes in vivo both in cancer and normal cells in various cancer models. We also use these data to validate predictive finite element modelling. Our theoretical and experimental data indicate that tumour cells are exposed to sufficiently high PARP-1 inhibitor concentrations in vivo and suggest that drug inefficiency is likely related to proteomic heterogeneity or insensitivity of cancer cells to DNA-repair inhibition. This suggests that single-cell pharmacokinetic imaging and derived modelling improve our understanding of drug action at single-cell resolution in vivo.

Cell-Specific Multifunctional Processing of Heterogeneous Cell Systems in a Single Laser Pulse Treatment

PubMed Central

Lukianova-Hleb, Ekaterina Y.; Mutonga, Martin B. G.; Lapotko, Dmitri O.

2012-01-01

Current methods of cell processing for gene and cell therapies use several separate procedures for gene transfer and cell separation or elimination, because no current technology can offer simultaneous multi-functional processing of specific cell sub-sets in highly heterogeneous cell systems. Using the cell-specific generation of plasmonic nanobubbles of different sizes around cell-targeted gold nanoshells and nanospheres, we achieved simultaneous multifunctional cell-specific processing in a rapid single 70 ps laser pulse bulk treatment of heterogeneous cell suspension. This method supported the detection of cells, delivery of external molecular cargo to one type of cells and the concomitant destruction of another type of cells without damaging other cells in suspension, and real-time guidance of the two above cellular effects. PMID:23167546

Preparation and Imaging Investigation of Dual-targeted C3F8-filled PLGA Nanobubbles as a Novel Ultrasound Contrast Agent for Breast Cancer.

PubMed

Du, Jing; Li, Xiao-Yu; Hu, He; Xu, Li; Yang, Shi-Ping; Li, Feng-Hua

2018-03-01

Molecularly-targeted contrast enhanced ultrasound (US) imaging is a promising imaging strategy with large potential for improving diagnostic accuracy of conventional US imaging in breast cancer detection. Therefore, we constructed a novel dual-targeted nanosized US contrast agent (UCA) directed at both vascular endothelial growth factor receptor 2 (VEGFR2) and human epidermal growth factor receptor 2 (HER2) based on perfluoropropane (C 3 F 8 )-filled poly(lactic-co-glycolic acid) (PLGA) (NBs) for breast cancer detection. In vitro, single- or dual-targeted PLGA NBs showed high target specificities and better effects of target enhancement in VEGFR2 or HER2-positive cells. In vivo, US imaging signal in the murine breast cancer model was significantly higher (P < 0.01) for dual-targeted NBs than single-targeted and non-targeted NBs. Small animal fluorescence imaging further confirmed the special affinity of the dual-targeted nanosized contrast agent to both VEGFR2 and HER2. Immunofluorescence and immunohistochemistry staining confirmed the expressions of VEGFR2 and HER2 on tumor neovasculature and tumor cells of breast cancer. In conclusions, the feasibility of using dual-targeted PLGA NBs to enhance ultrasonic images is demonstrated in vitro and in vivo. This may be a promising approach to target biomarkers of breast cancer for two site-specific US molecular imaging.

Probing site-exclusive binding of aqueous QDs and their organelle-dependent dynamics in live cells by single molecule spectroscopy.

PubMed

Dong, Chaoqing; Chowdhury, Basudev; Irudayaraj, Joseph

2013-05-21

Understanding the biophysical and chemical interactions of nanoprobes and their fate upon entering live cells is critical for developing fundamental insights related to intracellular diagnostics, drug delivery and targeting. In this article we report herein a single molecule analysis procedure to quantitate site-specific exclusive membrane binding of N-acetyl-L-cysteine (NAC)-capped cadmium telluride (CdTe) quantum dots (QDs) in A-427 lung carcinoma cells (k(eq) = 0.075 ± 0.011 nM(-1)), its relative intracellular distribution and dynamics using fluorescence correlation spectroscopy (FCS) combined with scanning confocal fluorescence lifetime imaging (FLIM). In particular, we demonstrate that the binding efficacy of QDs to the cell membrane is directly related to their size and the targeting of QDs to specific membrane sites is exclusive. We also show that QDs are efficiently internalized by endocytosis and enclosed within the endosome and organelle-dependent diffusion dynamics can be monitored in live cells.

Synthetic PAMAM-RGD conjugates target and bind to odontoblast-like MDPC 23 cells and the predentin in tooth organ cultures.

PubMed

Hill, Elliott; Shukla, Rameshwer; Park, Steve S; Baker, James R

2007-01-01

Screening techniques now allow for the identification of small peptides that bind specifically to molecules like cells. However, despite the enthusiasm for this approach, single peptides often lack the binding affinity to target in vivo and regulate cell function. We took peptides containing the Arg-Gly Asp(RGD) motif that bind to the alpha Vbeta 3 integrin and have shown potential as therapeutics. To improve their binding affinity, we synthesized polyamidoamine (PAMAM) dendrimer-RGD conjugates that that contain 12-13 copies of the peptide. When cultured with human dermal microvessel endothelial cells (HDMEC), human vascular endothelial cells (HUVEC), or odontoblast-like MDPC-23 cells, the PAMAM dendrimer conjugate targets this receptor in a manner that is both time- and dose-dependent. Finally, this conjugate selectively targets RGD binding sites in the predentin of human tooth organ cultures. Taken together, these studies provide proof of principle that synthetic PAMAM-RGD conjugates could prove useful as carriers for the tissue-specific delivery of integrin-targeted therapeutics or imaging agents and could be used to engineer tissue regeneration.

Design of antibody-functionalized carbon nanotubes filled with radioactivable metals towards a targeted anticancer therapy

NASA Astrophysics Data System (ADS)

Spinato, Cinzia; Perez Ruiz de Garibay, Aritz; Kierkowicz, Magdalena; Pach, Elzbieta; Martincic, Markus; Klippstein, Rebecca; Bourgognon, Maxime; Wang, Julie Tzu-Wen; Ménard-Moyon, Cécilia; Al-Jamal, Khuloud T.; Ballesteros, Belén; Tobias, Gerard; Bianco, Alberto

2016-06-01

In the present work we have devised the synthesis of a novel promising carbon nanotube carrier for the targeted delivery of radioactivity, through a combination of endohedral and exohedral functionalization. Steam-purified single-walled carbon nanotubes (SWCNTs) have been initially filled with radioactive analogues (i.e. metal halides) and sealed by high temperature treatment, affording closed-ended CNTs with the filling material confined in the inner cavity. The external functionalization of these filled CNTs was then achieved by nitrene cycloaddition and followed by the derivatization with a monoclonal antibody (Cetuximab) targeting the epidermal growth factor receptor (EGFR), overexpressed by several cancer cells. The targeting efficiency of the so-obtained conjugate was evaluated by immunostaining with a secondary antibody and by incubation of the CNTs with EGFR positive cells (U87-EGFR+), followed by flow cytometry, confocal microscopy or elemental analyses. We demonstrated that our filled and functionalized CNTs can internalize more efficiently in EGFR positive cancer cells.In the present work we have devised the synthesis of a novel promising carbon nanotube carrier for the targeted delivery of radioactivity, through a combination of endohedral and exohedral functionalization. Steam-purified single-walled carbon nanotubes (SWCNTs) have been initially filled with radioactive analogues (i.e. metal halides) and sealed by high temperature treatment, affording closed-ended CNTs with the filling material confined in the inner cavity. The external functionalization of these filled CNTs was then achieved by nitrene cycloaddition and followed by the derivatization with a monoclonal antibody (Cetuximab) targeting the epidermal growth factor receptor (EGFR), overexpressed by several cancer cells. The targeting efficiency of the so-obtained conjugate was evaluated by immunostaining with a secondary antibody and by incubation of the CNTs with EGFR positive cells (U87-EGFR+), followed by flow cytometry, confocal microscopy or elemental analyses. We demonstrated that our filled and functionalized CNTs can internalize more efficiently in EGFR positive cancer cells. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07923c

One Bacterial Cell, One Complete Genome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woyke, Tanja; Tighe, Damon; Mavrommatis, Konstantinos

2010-04-26

While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated frommore » the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200?900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA). Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs), indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.« less

Folate receptor 1 (FOLR1) targeted chimeric antigen receptor (CAR) T cells for the treatment of gastric cancer

PubMed Central

Pyo, Suhkneung; Kang, Chung Hyo; Lee, Chong Ock; Lee, Heung Kyoung; Choi, Sang Un; Park, Chi Hoon

2018-01-01

Gastric cancer is a malignancy that has a high mortality rate. Although progress has been made in the treatment of gastric cancer, many patients experience cancer recurrence and metastasis. Folate receptor 1 (FOLR1) is overexpressed on the cell surface in over one-third of gastric cancer patients, but rarely is expressed in normal tissue. This makes FOLR1 a potential target for chimeric antigen receptor (CAR) T cell immunotherapy, although the function of FOLR1 has not been elucidated. CAR are engineered fusion receptor composed of an antigen recognition region and signaling domains. T cells expressing CAR have specific activation and cytotoxic effects against cancer cells containing the target antigen. In this study, we generated a CAR that targets FOLR1 composed of a single-chain variable fragment (scFv) of FOLR1 antibody and signaling domains consisting of CD28 and CD3ζ. Both FOLR1-CAR KHYG-1, a natural killer cell line, and FOLR1-CAR T cells recognized FOLR1-positive gastric cancer cells in a MHC-independent manner and induced secretion of various cytokines and caused cell death. Conclusively, this is the first study to demonstrate that CAR KHYG-1/T cells targeting FOLR1 are effective against FOLR1-positive gastric cancer cells. PMID:29874279

Convergent Transcription At Intragenic Super-Enhancers Targets AID-initiated Genomic Instability

PubMed Central

Meng, Fei-Long; Du, Zhou; Federation, Alexander; Hu, Jiazhi; Wang, Qiao; Kieffer-Kwon, Kyong-Rim; Meyers, Robin M.; Amor, Corina; Wasserman, Caitlyn R.; Neuberg, Donna; Casellas, Rafael; Nussenzweig, Michel C.; Bradner, James E.; Liu, X. Shirley; Alt, Frederick W.

2015-01-01

Summary Activation-induced cytidine deaminase (AID) initiates both somatic hypermutation (SHM) for antibody affinity maturation and DNA breakage for antibody class switch recombination (CSR) via transcription-dependent cytidine deamination of single stranded DNA targets. While largely specific for immunoglobulin genes, AID also acts on a limited set of off-targets, generating oncogenic translocations and mutations that contribute to B cell lymphoma. How AID is recruited to off-targets has been a long-standing mystery. Based on deep GRO-Seq studies of mouse and human B lineage cells activated for CSR or SHM, we report that most robust AID off-target translocations occur within highly focal regions of target genes in which sense and antisense transcription converge. Moreover, we found that such AID-targeting “convergent” transcription arises from antisense transcription that emanates from Super-Enhancers within sense transcribed gene bodies. Our findings provide an explanation for AID off-targeting to a small subset of mostly lineage-specific genes in activated B cells. PMID:25483776

Identification and Characterization of a Suite of Tumor Targeting Peptides for Non-Small Cell Lung Cancer

NASA Astrophysics Data System (ADS)

McGuire, Michael J.; Gray, Bethany Powell; Li, Shunzi; Cupka, Dorothy; Byers, Lauren Averett; Wu, Lei; Rezaie, Shaghayegh; Liu, Ying-Horng; Pattisapu, Naveen; Issac, James; Oyama, Tsukasa; Diao, Lixia; Heymach, John V.; Xie, Xian-Jin; Minna, John D.; Brown, Kathlynn C.

2014-03-01

Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071-40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands.

Identification and Characterization of a Suite of Tumor Targeting Peptides for Non-Small Cell Lung Cancer

PubMed Central

McGuire, Michael J.; Gray, Bethany Powell; Li, Shunzi; Cupka, Dorothy; Byers, Lauren Averett; Wu, Lei; Rezaie, Shaghayegh; Liu, Ying-Horng; Pattisapu, Naveen; Issac, James; Oyama, Tsukasa; Diao, Lixia; Heymach, John V.; Xie, Xian-Jin; Minna, John D.; Brown, Kathlynn C.

2014-01-01

Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071–40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands. PMID:24670678

Anti-HER2 IgY antibody-functionalized single-walled carbon nanotubes for detection and selective destruction of breast cancer cells

PubMed Central

2009-01-01

Background Nanocarrier-based antibody targeting is a promising modality in therapeutic and diagnostic oncology. Single-walled carbon nanotubes (SWNTs) exhibit two unique optical properties that can be exploited for these applications, strong Raman signal for cancer cell detection and near-infrared (NIR) absorbance for selective photothermal ablation of tumors. In the present study, we constructed a HER2 IgY-SWNT complex and demonstrated its dual functionality for both detection and selective destruction of cancer cells in an in vitro model consisting of HER2-expressing SK-BR-3 cells and HER2-negative MCF-7 cells. Methods The complex was constructed by covalently conjugating carboxylated SWNTs with anti-HER2 chicken IgY antibody, which is more specific and sensitive than mammalian IgGs. Raman signals were recorded on Raman spectrometers with a laser excitation at 785 nm. NIR irradiation was performed using a diode laser system, and cells with or without nanotube treatment were irradiated by 808 nm laser at 5 W/cm2 for 2 min. Cell viability was examined by the calcein AM/ethidium homodimer-1 (EthD-1) staining. Results Using a Raman optical microscope, we found the Raman signal collected at single-cell level from the complex-treated SK-BR-3 cells was significantly greater than that from various control cells. NIR irradiation selectively destroyed the complex-targeted breast cancer cells without harming receptor-free cells. The cell death was effectuated without the need of internalization of SWNTs by the cancer cells, a finding that has not been reported previously. Conclusion We have demonstrated that the HER2 IgY-SWNT complex specifically targeted HER2-expressing SK-BR-3 cells but not receptor-negative MCF-7 cells. The complex can be potentially used for both detection and selective photothermal ablation of receptor-positive breast cancer cells without the need of internalization by the cells. Thus, the unique intrinsic properties of SWNTs combined with high specificity and sensitivity of IgY antibodies can lead to new strategies for cancer detection and therapy. PMID:19799784

Anti-HER2 IgY antibody-functionalized single-walled carbon nanotubes for detection and selective destruction of breast cancer cells.

PubMed

Xiao, Yan; Gao, Xiugong; Taratula, Oleh; Treado, Stephen; Urbas, Aaron; Holbrook, R David; Cavicchi, Richard E; Avedisian, C Thomas; Mitra, Somenath; Savla, Ronak; Wagner, Paul D; Srivastava, Sudhir; He, Huixin

2009-10-02

Nanocarrier-based antibody targeting is a promising modality in therapeutic and diagnostic oncology. Single-walled carbon nanotubes (SWNTs) exhibit two unique optical properties that can be exploited for these applications, strong Raman signal for cancer cell detection and near-infrared (NIR) absorbance for selective photothermal ablation of tumors. In the present study, we constructed a HER2 IgY-SWNT complex and demonstrated its dual functionality for both detection and selective destruction of cancer cells in an in vitro model consisting of HER2-expressing SK-BR-3 cells and HER2-negative MCF-7 cells. The complex was constructed by covalently conjugating carboxylated SWNTs with anti-HER2 chicken IgY antibody, which is more specific and sensitive than mammalian IgGs. Raman signals were recorded on Raman spectrometers with a laser excitation at 785 nm. NIR irradiation was performed using a diode laser system, and cells with or without nanotube treatment were irradiated by 808 nm laser at 5 W/cm2 for 2 min. Cell viability was examined by the calcein AM/ethidium homodimer-1 (EthD-1) staining. Using a Raman optical microscope, we found the Raman signal collected at single-cell level from the complex-treated SK-BR-3 cells was significantly greater than that from various control cells. NIR irradiation selectively destroyed the complex-targeted breast cancer cells without harming receptor-free cells. The cell death was effectuated without the need of internalization of SWNTs by the cancer cells, a finding that has not been reported previously. We have demonstrated that the HER2 IgY-SWNT complex specifically targeted HER2-expressing SK-BR-3 cells but not receptor-negative MCF-7 cells. The complex can be potentially used for both detection and selective photothermal ablation of receptor-positive breast cancer cells without the need of internalization by the cells. Thus, the unique intrinsic properties of SWNTs combined with high specificity and sensitivity of IgY antibodies can lead to new strategies for cancer detection and therapy.

Antigenic properties of the human immunodeficiency virus envelope glycoprotein gp120 on virions bound to target cells.

PubMed

Mengistu, Meron; Ray, Krishanu; Lewis, George K; DeVico, Anthony L

2015-03-01

The HIV-1 envelope glycoprotein, gp120, undergoes multiple molecular interactions and structural rearrangements during the course of host cell attachment and viral entry, which are being increasingly defined at the atomic level using isolated proteins. In comparison, antigenic markers of these dynamic changes are essentially unknown for single HIV-1 particles bound to target cells. Such markers should indicate how neutralizing and/or non-neutralizing antibodies might interdict infection by either blocking infection or sensitizing host cells for elimination by Fc-mediated effector function. Here we address this deficit by imaging fluorescently labeled CCR5-tropic HIV-1 pseudoviruses using confocal and superresolution microscopy to track the exposure of neutralizing and non-neutralizing epitopes as they appear on single HIV-1 particles bound to target cells. Epitope exposure was followed under conditions permissive or non-permissive for viral entry to delimit changes associated with virion binding from those associated with post-attachment events. We find that a previously unexpected array of gp120 epitopes is exposed rapidly upon target cell binding. This array comprises both neutralizing and non-neutralizing epitopes, the latter being hidden on free virions yet capable of serving as potent targets for Fc-mediated effector function. Under non-permissive conditions for viral entry, both neutralizing and non-neutralizing epitope exposures were relatively static over time for the majority of bound virions. Under entry-permissive conditions, epitope exposure patterns changed over time on subsets of virions that exhibited concurrent variations in virion contents. These studies reveal that bound virions are distinguished by a broad array of both neutralizing and non-neutralizing gp120 epitopes that potentially sensitize a freshly engaged target cell for destruction by Fc-mediated effector function and/or for direct neutralization at a post-binding step. The elucidation of these epitope exposure patterns during viral entry will help clarify antibody-mediated inhibition of HIV-1 as it is measured in vitro and in vivo.

Dissection of Influenza Infection In Vivo by Single-Cell RNA Sequencing.

PubMed

Steuerman, Yael; Cohen, Merav; Peshes-Yaloz, Naama; Valadarsky, Liran; Cohn, Ofir; David, Eyal; Frishberg, Amit; Mayo, Lior; Bacharach, Eran; Amit, Ido; Gat-Viks, Irit

2018-06-01

The influenza virus is a major cause of morbidity and mortality worldwide. Yet, both the impact of intracellular viral replication and the variation in host response across different cell types remain uncharacterized. Here we used single-cell RNA sequencing to investigate the heterogeneity in the response of lung tissue cells to in vivo influenza infection. Analysis of viral and host transcriptomes in the same single cell enabled us to resolve the cellular heterogeneity of bystander (exposed but uninfected) as compared with infected cells. We reveal that all major immune and non-immune cell types manifest substantial fractions of infected cells, albeit at low viral transcriptome loads relative to epithelial cells. We show that all cell types respond primarily with a robust generic transcriptional response, and we demonstrate novel markers specific for influenza-infected as opposed to bystander cells. These findings open new avenues for targeted therapy aimed exclusively at infected cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Stand-Sit Microchip for High-Throughput, Multiplexed Analysis of Single Cancer Cells.

PubMed

Ramirez, Lisa; Herschkowitz, Jason I; Wang, Jun

2016-09-01

Cellular heterogeneity in function and response to therapeutics has been a major challenge in cancer treatment. The complex nature of tumor systems calls for the development of advanced multiplexed single-cell tools that can address the heterogeneity issue. However, to date such tools are only available in a laboratory setting and don't have the portability to meet the needs in point-of-care cancer diagnostics. Towards that application, we have developed a portable single-cell system that is comprised of a microchip and an adjustable clamp, so on-chip operation only needs pipetting and adjusting of clamping force. Up to 10 proteins can be quantitated from each cell with hundreds of single-cell assays performed in parallel from one chip operation. We validated the technology and analyzed the oncogenic signatures of cancer stem cells by quantitating both aldehyde dehydrogenase (ALDH) activities and 5 signaling proteins in single MDA-MB-231 breast cancer cells. The technology has also been used to investigate the PI3K pathway activities of brain cancer cells expressing mutant epidermal growth factor receptor (EGFR) after drug intervention targeting EGFR signaling. Our portable single-cell system will potentially have broad application in the preclinical and clinical settings for cancer diagnosis in the future.

Intestinal double-positive CD4+CD8+ T cells are highly activated memory cells with an increased capacity to produce cytokines.

PubMed

Pahar, Bapi; Lackner, Andrew A; Veazey, Ronald S

2006-03-01

Peripheral blood and intestinal CD4+CD8+ double-positive (DP) T cells have been described in several species including humans, but their function and immunophenotypic characteristics are still not clearly understood. Here we demonstrate that DP T cells are abundant in the intestinal lamina propria of normal rhesus macaques (Macaca mulatta). Moreover, DP T cells have a memory phenotype and are capable of producing different and/or higher levels of cytokines and chemokines in response to mitogen stimulation compared to CD4+ single-positive T cells. Intestinal DP T cells are also highly activated and have higher expression of CCR5, which makes them preferred targets for simian immunodeficiency virus/HIV infection. Increased levels of CD69, CD25 and HLA-DR, and lower CD62L expression were found on intestinal DP T cells populations compared to CD4+ single-positive T cells. Collectively, these findings demonstrate that intestinal and peripheral blood DP T cells are effector cells and may be important in regulating immune responses, which distinguishes them from the immature DP cells found in the thymus. Finally, these intestinal DP T cells may be important target cells for HIV infection and replication due to their activation, memory phenotype and high expression of CCR5.

Single-cell forensic short tandem repeat typing within microfluidic droplets.

PubMed

Geng, Tao; Novak, Richard; Mathies, Richard A

2014-01-07

A short tandem repeat (STR) typing method is developed for forensic identification of individual cells. In our strategy, monodisperse 1.5 nL agarose-in-oil droplets are produced with a high frequency using a microfluidic droplet generator. Statistically dilute single cells, along with primer-functionalized microbeads, are randomly compartmentalized in the droplets. Massively parallel single-cell droplet polymerase chain reaction (PCR) is performed to transfer replicas of desired STR targets from the single-cell genomic DNA onto the coencapsulated microbeads. These DNA-conjugated beads are subsequently harvested and reamplified under statistically dilute conditions for conventional capillary electrophoresis (CE) STR fragment size analysis. The 9-plex STR profiles of single cells from both pure and mixed populations of GM09947 and GM09948 human lymphoid cells show that all alleles are correctly called and allelic drop-in/drop-out is not observed. The cell mixture study exhibits a good linear relationship between the observed and input cell ratios in the range of 1:1 to 10:1. Additionally, the STR profile of GM09947 cells could be deduced even in the presence of a high concentration of cell-free contaminating 9948 genomic DNA. Our method will be valuable for the STR analysis of samples containing mixtures of cells/DNA from multiple contributors and for low-concentration samples.

Precision Genome Editing for Treating Single-gene Disorders

NASA Astrophysics Data System (ADS)

Bao, Gang

There are an estimated 6,000 human single-gene disorders, most of them have no cure. This imposes a significant burden on human health worldwide. The recent advent in developing engineered nucleases, especially CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeats and CRISPR-associated protein 9) systems provides a powerful tool for precisely modifying the human genome, thus revolutionizing the treatment of single-gene disorders. In this talk, I will present recent work in my lab on developing new tools and methods for the design and optimization of CRISPR/Cas9 systems, and the efforts in developing a clinically applicable gene correction strategy for treating sickle cell disease (SCD), which is the first single-gene disorder with molecular understanding. We optimized CRISPR/Cas9 systems targeting the beta-globin gene, and systematically evaluated their on- and off-target cleavage in different cells. We also quantified the nuclease-induced gene modification rates in CD34+ cells from SCD patients, and demonstrated that CRISPR/Cas9 based genome editing is effective in generating normal hemoglobin (HbA) and reducing sickling hemoglobin (HbS). These studies significantly facilitated our pre-clinical investigation of SCD treatment using CRISPR/Cas9 and donor templates. The opportunities and challenges in developing nuclease-based genome editing strategies for treating single-gene disorders are discussed.

Mo99 Production Plant Layout

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woloshun, Keith Albert; Dale, Gregory E.; Naranjo, Angela Carol

The NorthStar Medical Technologies 99Mo production facility configuration is envisioned to be 8 accelerator pairs irradiating 7 100Mo targets (one spare accelerator pair undergoing maintenance while the other 7 pairs are irradiating targets). The required shielding in every direction for the accelerators is initially estimated to be 10 feet of concrete. With the accelerator pairs on one (ground) level and spaced with the required shielding between adjacent pairs, the only practical path for target insertion and removal while minimizing floor space is vertical. The current scheme then requires a target vertical lift of nominally 10 feet through a shield stack.more » It is envisioned that the lift will be directly into a hot cell where an activated target can be removed from its holder and a new target attached and lowered. The hot cell is on a rail system so that a single hot cell can service all active target locations, as well as deliver the ready targets to the separations lab. On this rail system, coupled to the hot cell, will be a helium recovery and clean-up system. All helium coolant equipment is located on the upper level near to the target removal point.« less

Synaptotagmin-Like Proteins Control Formation of a Single Apical Membrane Domain in Epithelial Cells

PubMed Central

Gálvez-Santisteban, Manuel; Rodriguez-Fraticelli, Alejo E.; Bryant, David M.; Vergarajauregui, Silvia; Yasuda, Takao; Bañón-Rodríguez, Inmaculada; Bernascone, Ilenia; Datta, Anirban; Spivak, Natalie; Young, Kitty; Slim, Christiaan L.; Brakeman, Paul R.; Fukuda, Mitsunori; Mostov, Keith E.; Martín-Belmonte, Fernando

2012-01-01

SUMMARY The formation of epithelial tissues requires both the generation of apical-basal polarity and the co-ordination of this polarity between neighboring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to formation of a singular apical membrane, resulting in contribution of each cell to only a single lumen. Here, from a functional screen for genes required for 3D epithelial architecture we identify key roles for Synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PI(4,5)P2-dependent manner, where it targets Rab27-loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE Syntaxin-3. Together, Slp2-a/4-a co-ordinate the spatiotemporal organization of vectorial apical transport to ensure only a single apical surface, and thus formation of a single lumen, occurs per cell. PMID:22820376

Development and Evaluation of an Optimal Human Single-Chain Variable Fragment-Derived BCMA-Targeted CAR T Cell Vector.

PubMed

Smith, Eric L; Staehr, Mette; Masakayan, Reed; Tatake, Ishan J; Purdon, Terence J; Wang, Xiuyan; Wang, Pei; Liu, Hong; Xu, Yiyang; Garrett-Thomson, Sarah C; Almo, Steven C; Riviere, Isabelle; Liu, Cheng; Brentjens, Renier J

2018-06-06

B cell maturation antigen (BCMA) has recently been identified as an important multiple myeloma (MM)-specific target for chimeric antigen receptor (CAR) T cell therapy. In CAR T cell therapy targeting CD19 for lymphoma, host immune anti-murine CAR responses limited the efficacy of repeat dosing and possibly long-term persistence. This clinically relevant concern can be addressed by generating a CAR incorporating a human single-chain variable fragment (scFv). We screened a human B cell-derived scFv phage display library and identified a panel of BCMA-specific clones from which human CARs were engineered. Despite a narrow range of affinity for BCMA, dramatic differences in CAR T cell expansion were observed between unique scFvs in a repeat antigen stimulation assay. These results were confirmed by screening in a MM xenograft model, where only the top preforming CARs from the repeat antigen stimulation assay eradicated disease and prolonged survival. The results of this screening identified a highly effective CAR T cell therapy with properties, including rapid in vivo expansion (>10,000-fold, day 6), eradication of large tumor burden, and durable protection to tumor re-challenge. We generated a bicistronic construct including a second-generation CAR and a truncated-epithelial growth factor receptor marker. CAR T cell vectors stemming from this work are under clinical investigation. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Single-cell RNA-sequencing reveals a distinct population of proglucagon-expressing cells specific to the mouse upper small intestine.

PubMed

Glass, Leslie L; Calero-Nieto, Fernando J; Jawaid, Wajid; Larraufie, Pierre; Kay, Richard G; Göttgens, Berthold; Reimann, Frank; Gribble, Fiona M

2017-10-01

To identify sub-populations of intestinal preproglucagon-expressing (PPG) cells producing Glucagon-like Peptide-1, and their associated expression profiles of sensory receptors, thereby enabling the discovery of therapeutic strategies that target these cell populations for the treatment of diabetes and obesity. We performed single cell RNA sequencing of PPG-cells purified by flow cytometry from the upper small intestine of 3 GLU-Venus mice. Cells from 2 mice were sequenced at low depth, and from the third mouse at high depth. High quality sequencing data from 234 PPG-cells were used to identify clusters by tSNE analysis. qPCR was performed to compare the longitudinal and crypt/villus locations of cluster-specific genes. Immunofluorescence and mass spectrometry were used to confirm protein expression. PPG-cells formed 3 major clusters: a group with typical characteristics of classical L-cells, including high expression of Gcg and Pyy (comprising 51% of all PPG-cells); a cell type overlapping with Gip-expressing K-cells (14%); and a unique cluster expressing Tph1 and Pzp that was predominantly located in proximal small intestine villi and co-produced 5-HT (35%). Expression of G-protein coupled receptors differed between clusters, suggesting the cell types are differentially regulated and would be differentially targetable. Our findings support the emerging concept that many enteroendocrine cell populations are highly overlapping, with individual cells producing a range of peptides previously assigned to distinct cell types. Different receptor expression profiles across the clusters highlight potential drug targets to increase gut hormone secretion for the treatment of diabetes and obesity. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Single-cell analysis of population context advances RNAi screening at multiple levels

PubMed Central

Snijder, Berend; Sacher, Raphael; Rämö, Pauli; Liberali, Prisca; Mench, Karin; Wolfrum, Nina; Burleigh, Laura; Scott, Cameron C; Verheije, Monique H; Mercer, Jason; Moese, Stefan; Heger, Thomas; Theusner, Kristina; Jurgeit, Andreas; Lamparter, David; Balistreri, Giuseppe; Schelhaas, Mario; De Haan, Cornelis A M; Marjomäki, Varpu; Hyypiä, Timo; Rottier, Peter J M; Sodeik, Beate; Marsh, Mark; Gruenberg, Jean; Amara, Ali; Greber, Urs; Helenius, Ari; Pelkmans, Lucas

2012-01-01

Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cell's microenvironment, in image-based RNAi screens. We developed a comprehensive computational approach that employs Bayesian and multivariate methods at the single-cell level. We applied these methods to 45 RNA interference screens of various sizes, including 7 druggable genome and 2 genome-wide screens, analysing 17 different mammalian virus infections and four related cell physiological processes. Analysing cell-based screens at this depth reveals widespread RNAi-induced changes in the population context of individual cells leading to indirect RNAi effects, as well as perturbations of cell-to-cell variability regulators. We find that accounting for indirect effects improves the consistency between siRNAs targeted against the same gene, and between replicate RNAi screens performed in different cell lines, in different labs, and with different siRNA libraries. In an era where large-scale RNAi screens are increasingly performed to reach a systems-level understanding of cellular processes, we show that this is often improved by analyses that account for and incorporate the single-cell microenvironment. PMID:22531119

Realizing the potential of the Actinium-225 radionuclide generator in targeted alpha-particle therapy applications

PubMed Central

Miederer, Matthias; Scheinberg, David A.; McDevitt, Michael R.

2013-01-01

Alpha particle-emitting isotopes have been proposed as novel cytotoxic agents for augmenting targeted therapy. Properties of alpha particle radiation such as their limited range in tissue of a few cell diameters and their high linear energy transfer leading to dense radiation damage along each alpha track are promising in the treatment of cancer, especially when single cells or clusters of tumor cells are targeted. Actinium-225 (225Ac) is an alpha particle-emitting radionuclide that generates 4 net alpha particle isotopes in a short decay chain to stable 209Bi, and as such can be described as an alpha particle nanogenerator. This article reviews the literature pertaining to the research, development, and utilization of targeted 225Ac to potently and specifically affect cancer. PMID:18514364

A Tupaia paramyxovirus vector system for targeting and transgene expression.

PubMed

Engeland, Christine E; Bossow, Sascha; Hudacek, Andrew W; Hoyler, Birgit; Förster, Judith; Veinalde, Rūta; Jäger, Dirk; Cattaneo, Roberto; Ungerechts, Guy; Springfeld, Christoph

2017-09-01

Viruses from the diverse family of Paramyxoviridae include important pathogens and are applied in gene therapy and for cancer treatment. The Tupaia paramyxovirus (TPMV), isolated from the kidney of a tree shrew, does not infect human cells and neutralizing antibodies against other Paramyxoviridae do not cross-react with TPMV. Here, we present a vector system for de novo generation of infectious TPMV that allows for insertion of additional genes as well as targeting using antibody single-chain variable fragments. We show that the recombinant TPMV specifically infect cells expressing the targeted receptor and replicate in human cells. This vector system provides a valuable tool for both basic research and therapeutic applications.

Highly sensitive and quantitative detection of rare pathogens through agarose droplet microfluidic emulsion PCR at the single-cell level.

PubMed

Zhu, Zhi; Zhang, Wenhua; Leng, Xuefei; Zhang, Mingxia; Guan, Zhichao; Lu, Jiangquan; Yang, Chaoyong James

2012-10-21

Genetic alternations can serve as highly specific biomarkers to distinguish fatal bacteria or cancer cells from their normal counterparts. However, these mutations normally exist in very rare amount in the presence of a large excess of non-mutated analogs. Taking the notorious pathogen E. coli O157:H7 as the target analyte, we have developed an agarose droplet-based microfluidic ePCR method for highly sensitive, specific and quantitative detection of rare pathogens in the high background of normal bacteria. Massively parallel singleplex and multiplex PCR at the single-cell level in agarose droplets have been successfully established. Moreover, we challenged the system with rare pathogen detection and realized the sensitive and quantitative analysis of a single E. coli O157:H7 cell in the high background of 100,000 excess normal K12 cells. For the first time, we demonstrated rare pathogen detection through agarose droplet microfluidic ePCR. Such a multiplex single-cell agarose droplet amplification method enables ultra-high throughput and multi-parameter genetic analysis of large population of cells at the single-cell level to uncover the stochastic variations in biological systems.

Quantification of Functionalised Gold Nanoparticle-Targeted Knockdown of Gene Expression in HeLa Cells

PubMed Central

Jiwaji, Meesbah; Sandison, Mairi E.; Reboud, Julien; Stevenson, Ross; Daly, Rónán; Barkess, Gráinne; Faulds, Karen; Kolch, Walter; Graham, Duncan; Girolami, Mark A.; Cooper, Jonathan M.; Pitt, Andrew R.

2014-01-01

Introduction Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein. PMID:24926959

77 FR 62521 - Prospective Grant of Exclusive License: The Development of Therapeutic Agents for the Treatment...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-10-15

...- cell Lymphoma AGENCY: National Institutes of Health, Public Health Service, HHS. ACTION: Notice...) incorporating a p53 isoform antisense oligonucleotide as a single biologic therapy to treat T- cell lymphoma... inventions concern i) compositions and methods for targeted delivery of inhibitory nucleic acids to cells...

Are Th17 cells and their cytokines a therapeutic target in Guillain-Barré syndrome?

PubMed

Wu, Xiujuan; Wang, Juan; Liu, Kangding; Zhu, Jie; Zhang, Hong-Liang

2016-01-01

Guillain-Barré syndrome (GBS) is an immune-mediated inflammatory disorder of the peripheral nervous system (PNS). Experimental autoimmune neuritis (EAN) is a useful animal model for studying GBS. Currently, GBS remains a life-threatening disorder and more effective therapeutic strategies are in urgent need. Accumulating evidence has revealed that T helper (Th) 17 cells and their cytokines are pathogenic in GBS/EAN. Drugs attenuated clinical signs of GBS/EAN, in part, by decreasing Th17 cells or IL-17A. Th17 cells and their cytokines might be potential therapeutic targets. Approaches targeting Th17 cells or their cytokines are in development in treating Th17 cells-involved disorders. In this review, we summarize the up-to-date knowledge on roles of Th17 cells and their cytokines in GBS/EAN, as well potential approaches targeting Th17 cells and their cytokines as clinical applications. As Th17 cells produce different sets of pro-inflammatory cytokines and Th17-related cytokines are not exclusively produced by Th17 cells, targeting Th17 cell development may be superior to blocking a single Th17 cytokine to treat Th17 cells-involved disorders. Considering the essential role of retinoic acid-related orphan receptor γT (RORγT) and IL-23 in Th17 cell development, RORγT inhibitors or IL-23 antagonists may provide better clinical efficacy in treating GBS/EAN.

In Situ Target Engagement Studies in Adherent Cells.

PubMed

Axelsson, Hanna; Almqvist, Helena; Otrocka, Magdalena; Vallin, Michaela; Lundqvist, Sara; Hansson, Pia; Karlsson, Ulla; Lundbäck, Thomas; Seashore-Ludlow, Brinton

2018-04-20

A prerequisite for successful drugs is effective binding of the desired target protein in the complex environment of a living system. Drug-target engagement has typically been difficult to monitor in physiologically relevant models, and with current methods, especially, while maintaining spatial information. One recent technique for quantifying drug-target engagement is the cellular thermal shift assay (CETSA), in which ligand-induced protein stabilization is measured after a heat challenge. Here, we describe a CETSA protocol in live A431 cells for p38α (MAPK14), where remaining soluble protein is detected in situ, using high-content imaging in 384-well, microtiter plates. We validate this assay concept using a number of known p38α inhibitors and further demonstrate the potential of this technology for chemical probe and drug discovery purposes by performing a small pilot screen for novel p38α binders. Importantly, this protocol creates a workflow that is amenable to adherent cells in their native state and yields spatially resolved target engagement information measurable at the single-cell level.

Highlighting cancer cells with macromolecular probes

NASA Astrophysics Data System (ADS)

Tang, Sicheng; Zhang, Yang; Thapaliya, Ek Raj; Brown, Adrienne S.; Wilson, James N.; Raymo, Françisco M.

2017-02-01

Conventional fluorophore-ligand constructs for the detection of cancer cells generally produce relatively weak signals with modest contrast. The inherently low brightness accessible per biding event with the pairing of a single organic fluorophore to a single ligand as well as the contribution of unbound probes to background fluorescence are mainly responsible for these limitations. Our laboratories identified a viable structural design to improve both brightness and contrast. It is based on the integration of activatable fluorophores and targeting ligands within the same macromolecular construct. The chromophoric components are engineered to emit bright fluorescence exclusively in acidic environments. The targeting agents are designed to bind complementary receptors overexpressed on the surface of cancer cells and allow internalization of the macromolecules into acidic organelles. As a result of these properties, our macromolecular probes switch their intense emission on exclusively in the intracellular space of target cells with minimal background fluorescence from the extracellular matrix. In fact, these operating principles translate into a 170-fold enhancement in brightness, relative to equivalent but isolated chromophoric components, and a 3-fold increase in contrast, relative to model but non-activatable fluorophores. Thus, our macromolecular probes might ultimately evolve into valuable analytical tools to highlight cancer cells with optimal signal-to-noise ratios in a diversity of biomedical applications.

BRAF and MEK inhibitor therapy eliminates nestin expressing melanoma cells in human tumors.

PubMed

Doxie, Deon B; Greenplate, Allison R; Gandelman, Jocelyn S; Diggins, Kirsten E; Roe, Caroline E; Dahlman, Kimberly B; Sosman, Jeffrey A; Kelley, Mark C; Irish, Jonathan M

2018-05-19

Little is known about the in vivo impacts of targeted therapy on melanoma cell abundance and protein expression. Here, 21 antibodies were added to an established melanoma mass cytometry panel to measure 32 cellular features, distinguish malignant cells, and characterize dabrafenib and trametinib responses in BRAF V 600mut melanoma. Tumor cells were biopsied before neoadjuvant therapy and compared to cells surgically resected from the same site after 4 weeks of therapy. Approximately 50,000 cells per tumor were characterized by mass cytometry and computational tools t-SNE/viSNE, FlowSOM, and MEM. The resulting single cell view of melanoma treatment response revealed initially heterogeneous melanoma tumors were consistently cleared of Nestin expressing melanoma cells. Melanoma cells subsets that persisted to week 4 were heterogeneous but expressed SOX2 or SOX10 proteins and specifically lacked surface expression of MHC I proteins by MEM analysis. Traditional histology imaging of tissue microarrays from the same tumors confirmed mass cytometry results, including persistence of NES- SOX10+ S100β+ melanoma cells. This quantitative single cell view of melanoma treatment response revealed protein features of malignant cells that are not eliminated by targeted therapy. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Fine tuning cellular recognition: The function of the leucine rich repeat (LRR) trans-membrane protein, LRT, in muscle targeting to tendon cells.

PubMed

Gilsohn, Eli; Volk, Talila

2010-01-01

The formation of complex tissues during embryonic development is often accompanied by directed cellular migration towards a target tissue. Specific mutual recognition between the migrating cell and its target tissue leads to the arrest of the cell migratory behavior and subsequent contact formation between the two interacting cell types. Recent studies implicated a novel family of surface proteins containing a trans-membrane domain and single leucine-rich repeat (LRR) domain in inter-cellular recognition and the arrest of cell migration. Here, we describe the involvement of a novel LRR surface protein, LRT, in targeting migrating muscles towards their corresponding tendon cells in the Drosophila embryo. LRT is specifically expressed by the target tendon cells and is essential for arresting the migratory behavior of the muscle cells. Additional studies in Drosophila S2 cultured cells suggest that LRT forms a protein complex with the Roundabout (Robo) receptor, essential for guiding muscles towards their tendon partners. Genetic analysis supports a model in which LRT performs its activity non-autonomously through its interaction with the Robo receptors expressed on the muscle surfaces. These results suggest a novel mechanism of intercellular recognition through interactions between LRR family members and Robo receptors.

A compound chimeric antigen receptor strategy for targeting multiple myeloma.

PubMed

Chen, K H; Wada, M; Pinz, K G; Liu, H; Shuai, X; Chen, X; Yan, L E; Petrov, J C; Salman, H; Senzel, L; Leung, E L H; Jiang, X; Ma, Y

2018-02-01

Current clinical outcomes using chimeric-antigen receptors (CARs) against multiple myeloma show promise in the eradication of bulk disease. However, these anti-BCMA (CD269) CARs observe relapse as a common phenomenon after treatment due to the reemergence of either antigen-positive or -negative cells. Hence, the development of improvements in CAR design to target antigen loss and increase effector cell persistency represents a critical need. Here, we report on the anti-tumor activity of a CAR T-cell possessing two complete and independent CAR receptors against the multiple myeloma antigens BCMA and CS1. We determined that the resulting compound CAR (cCAR) T-cell possesses consistent, potent and directed cytotoxicity against each target antigen population. Using multiple mouse models of myeloma and mixed cell populations, we are further able to show superior in vivo survival by directed cytotoxicity against multiple populations compared to a single-expressing CAR T-cell. These findings indicate that compound targeting of BCMA and CS1 on myeloma cells can potentially be an effective strategy for augmenting the response against myeloma bulk disease and for initiation of broader coverage CAR therapy.

Bioinformatics approaches to single-cell analysis in developmental biology.

PubMed

Yalcin, Dicle; Hakguder, Zeynep M; Otu, Hasan H

2016-03-01

Individual cells within the same population show various degrees of heterogeneity, which may be better handled with single-cell analysis to address biological and clinical questions. Single-cell analysis is especially important in developmental biology as subtle spatial and temporal differences in cells have significant associations with cell fate decisions during differentiation and with the description of a particular state of a cell exhibiting an aberrant phenotype. Biotechnological advances, especially in the area of microfluidics, have led to a robust, massively parallel and multi-dimensional capturing, sorting, and lysis of single-cells and amplification of related macromolecules, which have enabled the use of imaging and omics techniques on single cells. There have been improvements in computational single-cell image analysis in developmental biology regarding feature extraction, segmentation, image enhancement and machine learning, handling limitations of optical resolution to gain new perspectives from the raw microscopy images. Omics approaches, such as transcriptomics, genomics and epigenomics, targeting gene and small RNA expression, single nucleotide and structural variations and methylation and histone modifications, rely heavily on high-throughput sequencing technologies. Although there are well-established bioinformatics methods for analysis of sequence data, there are limited bioinformatics approaches which address experimental design, sample size considerations, amplification bias, normalization, differential expression, coverage, clustering and classification issues, specifically applied at the single-cell level. In this review, we summarize biological and technological advancements, discuss challenges faced in the aforementioned data acquisition and analysis issues and present future prospects for application of single-cell analyses to developmental biology. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A new approach to the internal thermal management of cylindrical battery cells for automotive applications

NASA Astrophysics Data System (ADS)

Worwood, Daniel; Kellner, Quirin; Wojtala, Malgorzata; Widanage, W. D.; M^cGlen, Ryan; Greenwood, David; Marco, James

2017-04-01

Conventional cooling approaches that target either a singular tab or outer surface of common format cylindrical lithium-ion battery cells suffer from a high cell thermal resistance. Under an aggressive duty cycle, this resistance can result in the formation of large in-cell temperature gradients and high hot spot temperatures, which are known to accelerate ageing and further reduce performance. In this paper, a novel approach to internal thermal management of cylindrical battery cells to lower the thermal resistance for heat transport through the inside of the cell is investigated. The effectiveness of the proposed method is analysed for two common cylindrical formats when subject to highly aggressive electrical loading conditions representative of a high performance electric vehicle (EV) and hybrid electric vehicle (HEV). A mathematical model that captures the dominant thermal properties of the cylindrical cell is created and validated using experimental data. Results from the extensive simulation study indicate that the internal cooling strategy can reduce the cell thermal resistance by up to 67.8 ± 1.4% relative to single tab cooling, and can emulate the performance of a more complex pack-level double tab cooling approach whilst targeting cooling at a single tab.

Tumor-targeted IL-2 amplifies T cell-mediated immune response induced by gene therapy with single-chain IL-12

PubMed Central

Lode, Holger N.; Xiang, Rong; Duncan, Steven R.; Theofilopoulos, Argyrios N.; Gillies, Stephen D.; Reisfeld, Ralph A.

1999-01-01

Induction, maintenance, and amplification of tumor-protective immunity after cytokine gene therapy is essential for the clinical success of immunotherapeutic approaches. We investigated whether this could be achieved by single-chain IL-12 (scIL-12) gene therapy followed by tumor-targeted IL-2 using a fusion protein containing a tumor-specific recombinant anti-ganglioside GD2 antibody and IL-2 (ch14.18-IL-2) in a poorly immunogenic murine neuroblastoma model. Herein, we demonstrate the absence of liver and bone marrow metastases after a lethal challenge with NXS2 wild-type cells only in mice (five of six animals) vaccinated with scIL-12-producing NXS2 cells and given a booster injection of low-dose ch14.18-IL-2 fusion protein. This tumor-protective immunity was effective 3 months after initial vaccination, in contrast to control animals treated with a nonspecific fusion protein or an equivalent mixture of antibody and IL-2. Only vaccinated mice receiving the tumor-specific ch14.18-IL-2 fusion protein revealed a reactivation of CD8+ T cells and subsequent MHC class I-restricted tumor target cell lysis in vitro. The sequential increase in the usage of TCR chains Vβ11 and -13 in mouse CD8+ T cells after vaccination and amplification with ch14.18-IL-2 suggests that the initial polyclonal CD8+ T cell response is effectively boosted by targeted IL-2. In conclusion, we demonstrate that a successful boost of a partially protective memory T cell immune response that is induced by scIL-12 gene therapy could be generated by tumor-specific targeting of IL-2 with a ch14.18-IL-2 fusion protein. This approach could increase success rates of clinical cancer vaccine trials. PMID:10411920

Minimizing inhibition of PCR-STR typing using digital agarose droplet microfluidics.

PubMed

Geng, Tao; Mathies, Richard A

2015-01-01

The presence of PCR inhibitors in forensic and other biological samples reduces the amplification efficiency, sometimes resulting in complete PCR failure. Here we demonstrate a high-performance digital agarose droplet microfluidics technique for single-cell and single-molecule forensic short tandem repeat (STR) typing of samples contaminated with high concentrations of PCR inhibitors. In our multifaceted strategy, the mitigation of inhibitory effects is achieved by the efficient removal of inhibitors from the porous agarose microgel droplets carrying the DNA template through washing and by the significant dilution of targets and remaining inhibitors to the stochastic limit within the ultralow nL volume droplet reactors. Compared to conventional tube-based bulk PCR, our technique shows enhanced (20 ×, 10 ×, and 16 ×) tolerance of urea, tannic acid, and humic acid, respectively, in STR typing of GM09948 human lymphoid cells. STR profiling of single cells is not affected by small soluble molecules like urea and tannic acid because of their effective elimination from the agarose droplets; however, higher molecular weight humic acid still partially inhibits single-cell PCR when the concentration is higher than 200 ng/μL. Nevertheless, the full STR profile of 9948 male genomic DNA contaminated with 500 ng/μL humic acid was generated by pooling and amplifying beads carrying single-molecule 9948 DNA PCR products in a single secondary reaction. This superior performance suggests that our digital agarose droplet microfluidics technology is a promising approach for analyzing low-abundance DNA targets in the presence of inhibitors. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus.

PubMed

Wu, Yong; Gao, Tieli; Wang, Xiaolin; Hu, Youjin; Hu, Xuyun; Hu, Zhiqing; Pang, Jialun; Li, Zhuo; Xue, Jinfeng; Feng, Mai; Wu, Lingqian; Liang, Desheng

2014-03-28

Although targeted gene addition could be stimulated strikingly by a DNA double strand break (DSB) created by either zinc finger nucleases (ZFNs) or TALE nucleases (TALENs), the DSBs are really mutagenic and toxic to human cells. As a compromised solution, DNA single-strand break (SSB) or nick has been reported to mediate high efficient gene addition but with marked reduction of random mutagenesis. We previously demonstrated effective targeted gene addition at the human multicopy ribosomal DNA (rDNA) locus, a genomic safe harbor for the transgene with therapeutic potential. To improve the transgene integration efficiency by using TALENs while lowering the cytotoxicity of DSBs, we created both TALENs and TALE nickases (TALENickases) targeting this multicopy locus. A targeting vector which could integrate a GFP cassette at the rDNA locus was constructed and co-transfected with TALENs or TALENickases. Although the fraction of GFP positive cells using TALENs was greater than that using TALENickases during the first few days after transfection, it reduced to a level less than that using TALENickases after continuous culture. Our findings showed that the TALENickases were more effective than their TALEN counterparts at the multi-copy rDNA locus, though earlier studies using ZFNs and ZFNickases targeting the single-copy loci showed the reverse. Besides, TALENickases mediated the targeted integration of a 5.4 kb fragment at a frequency of up to 0.62% in HT1080 cells after drug selection, suggesting their potential application in targeted gene modification not being limited at the rDNA locus. Copyright © 2014 Elsevier Inc. All rights reserved.

Target sequencing and CRISPR/Cas editing reveal simultaneous loss of UTX and UTY in urothelial bladder cancer.

PubMed

Ahn, Jinwoo; Kim, Kwang Hyun; Park, Sanghui; Ahn, Young-Ho; Kim, Ha Young; Yoon, Hana; Lee, Ji Hyun; Bang, Duhee; Lee, Dong Hyeon

2016-09-27

UTX is a histone demethylase gene located on the X chromosome and is a frequently mutated gene in urothelial bladder cancer (UBC). UTY is a paralog of UTX located on the Y chromosome. We performed target capture sequencing on 128 genes in 40 non-metastatic UBC patients. UTX was the most frequently mutated gene (30%, 12/40). Of the genetic alterations identified, 75% were truncating mutations. UTY copy number loss was detected in 8 male patients (22.8%, 8/35). Of the 9 male patients with UTX mutations, 6 also had copy number loss (66.7%). To evaluate the functional roles of UTX and UTY in tumor progression, we designed UTX and UTY single knockout and UTX-UTY double knockout experiments using a CRISPR/Cas9 lentiviral system, and compared the proliferative capacities of two UBC cell lines in vitro. Single UTX or UTY knockout increased cell proliferation as compared to UTX-UTY wild-type cells. UTX-UTY double knockout cells exhibited greater proliferation than single knockout cells. These findings suggest both UTX and UTY function as dose-dependent suppressors of UBC development. While UTX escapes X chromosome inactivation in females, UTY may function as a male homologue of UTX, which could compensate for dosage imbalances.

Single gene-based distinction of individual microbial genomes from a mixed population of microbial cells.

PubMed

Tamminen, Manu V; Virta, Marko P J

2015-01-01

Recent progress in environmental microbiology has revealed vast populations of microbes in any given habitat that cannot be detected by conventional culturing strategies. The use of sensitive genetic detection methods such as CARD-FISH and in situ PCR have been limited by the cell wall permeabilization requirement that cannot be performed similarly on all cell types without lysing some and leaving some nonpermeabilized. Furthermore, the detection of low copy targets such as genes present in single copies in the microbial genomes, has remained problematic. We describe an emulsion-based procedure to trap individual microbial cells into picoliter-volume polyacrylamide droplets that provide a rigid support for genetic material and therefore allow complete degradation of cellular material to expose the individual genomes. The polyacrylamide droplets are subsequently converted into picoliter-scale reactors for genome amplification. The amplified genomes are labeled based on the presence of a target gene and differentiated from those that do not contain the gene by flow cytometry. Using the Escherichia coli strains XL1 and MC1061, which differ with respect to the presence (XL1), or absence (MC1061) of a single copy of a tetracycline resistance gene per genome, we demonstrate that XL1 genomes present at 0.1% of MC1061 genomes can be differentiated using this method. Using a spiked sediment microbial sample, we demonstrate that the method is applicable to highly complex environmental microbial communities as a target gene-based screen for individual microbes. The method provides a novel tool for enumerating functional cell populations in complex microbial communities. We envision that the method could be optimized for fluorescence-activated cell sorting to enrich genetic material of interest from complex environmental samples.

Targeting HSP70 and GRP78 in canine osteosarcoma cells in combination with doxorubicin chemotherapy.

PubMed

Asling, Jonathan; Morrison, Jodi; Mutsaers, Anthony J

2016-11-01

Heat shock proteins (HSPs) are molecular chaperones subdivided into several families based on their molecular weight. Due to their cytoprotective roles, these proteins may help protect cancer cells against chemotherapy-induced cell death. Investigation into the biologic activity of HSPs in a variety of cancers including primary bone tumors, such as osteosarcoma (OSA), is of great interest. Both human and canine OSA tumor samples have aberrant production of HSP70. This study assessed the response of canine OSA cells to inhibition of HSP70 and GRP78 by the ATP-mimetic VER-155008 and whether this treatment strategy could sensitize cells to doxorubicin chemotherapy. Single-agent VER-155008 treatment decreased cellular viability and clonogenic survival and increased apoptosis in canine OSA cell lines. However, combination schedules with doxorubicin after pretreatment with VER-155008 did not improve inhibition of cellular viability, apoptosis, or clonogenic survival. Treatment with VER-155008 prior to chemotherapy resulted in an upregulation of target proteins HSP70 and GRP78 in addition to the co-chaperone proteins Herp, C/EBP homologous transcription protein (CHOP), and BAG-1. The increased GRP78 was more cytoplasmic in location compared to untreated cells. Single-agent treatment also revealed a dose-dependent reduction in activated and total Akt. Based on these results, targeting GRP78 and HSP70 may have biologic activity in canine osteosarcoma. Further studies are required to determine if and how this strategy may impact the response of osteosarcoma cells to chemotherapy.

Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems.

PubMed

Yasue, Akihiro; Mitsui, Silvia Naomi; Watanabe, Takahito; Sakuma, Tetsushi; Oyadomari, Seiichi; Yamamoto, Takashi; Noji, Sumihare; Mito, Taro; Tanaka, Eiji

2014-07-16

Since the establishment of embryonic stem (ES) cell lines, the combined use of gene targeting with homologous recombination has aided in elucidating the functions of various genes. However, the ES cell technique is inefficient and time-consuming. Recently, two new gene-targeting technologies have been developed: the transcription activator-like effector nuclease (TALEN) system, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. In addition to aiding researchers in solving conventional problems, these technologies can be used to induce site-specific mutations in various species for which ES cells have not been established. Here, by targeting the Fgf10 gene through RNA microinjection in one-cell mouse embryos with the TALEN and CRISPR/Cas systems, we produced the known limb-defect phenotypes of Fgf10-deficient embryos at the F0 generation. Compared to the TALEN system, the CRISPR/Cas system induced the limb-defect phenotypes with a strikingly higher efficiency. Our results demonstrate that although both gene-targeting technologies are useful, the CRISPR/Cas system more effectively elicits single-step biallelic mutations in mice.

A system and methodology for high-content visual screening of individual intact living cells in suspension

NASA Astrophysics Data System (ADS)

Renaud, Olivier; Heintzmann, Rainer; Sáez-Cirión, Asier; Schnelle, Thomas; Mueller, Torsten; Shorte, Spencer

2007-02-01

Three dimensional imaging provides high-content information from living intact biology, and can serve as a visual screening cue. In the case of single cell imaging the current state of the art uses so-called "axial through-stacking". However, three-dimensional axial through-stacking requires that the object (i.e. a living cell) be adherently stabilized on an optically transparent surface, usually glass; evidently precluding use of cells in suspension. Aiming to overcome this limitation we present here the utility of dielectric field trapping of single cells in three-dimensional electrode cages. Our approach allows gentle and precise spatial orientation and vectored rotation of living, non-adherent cells in fluid suspension. Using various modes of widefield, and confocal microscope imaging we show how so-called "microrotation" can provide a unique and powerful method for multiple point-of-view (three-dimensional) interrogation of intact living biological micro-objects (e.g. single-cells, cell aggregates, and embryos). Further, we show how visual screening by micro-rotation imaging can be combined with micro-fluidic sorting, allowing selection of rare phenotype targets from small populations of cells in suspension, and subsequent one-step single cell cloning (with high-viability). Our methodology combining high-content 3D visual screening with one-step single cell cloning, will impact diverse paradigms, for example cytological and cytogenetic analysis on haematopoietic stem cells, blood cells including lymphocytes, and cancer cells.

Reductive nanocomplex encapsulation of cRGD-siRNA conjugates for enhanced targeting to cancer cells

PubMed Central

Zhang, Yanfen; Yang, Xiantao; Ma, Yuan; Guan, Zhu; Wu, Yun; Zhang, Lihe; Yang, Zhenjun

2017-01-01

In this study, through covalent conjugation and lipid material entrapment, a combined modification strategy was established for effective delivery of small interfering RNA (siRNA). Single strands of siRNA targeting to BRAFV600E gene (siMB3) conjugated with cRGD peptide at 3′-terminus or 5′-terminus via cleavable disulfide bond was synthesized and then annealed with corresponding strands to obtain single and bis-cRGD-siRNA conjugates. A cationic lipid material (CLD) developed by our laboratory was mixed with the conjugates to generate nanocomplexes; their uniformity and electrical property were revealed by particle size and zeta potential measurement. Compared with CLD/siBraf, CLD/cRGD-siBraf achieved higher cell uptake and more excellent tumor-targeting ability, especially 21 (sense-5′/antisense-3″-cRGD-congjugate) nanocomplex. Moreover, they can regulate multiple pathways to varying degree and reduce acidification of endosome. Compared with the gene silencing of different conjugates, single or bis-cRGD-conjugated siRNA showed little differences except 22 (5/5) which cRGD was conjugated at 5′-terminus of antisense strand and sense strand. However bis-cRGD conjugate 21 nanocomplex exhibited better specific target gene silencing at multiple time points. Furthermore, the serum stabilities of the bis-cRGD conjugates were higher than those of the single-cRGD conjugates. In conclusion, all these data suggested that CLD/bis-conjugates, especially CLD/21, can be an effective system for delivery of siRNA to target BRAFV600E gene for therapy of melanoma. PMID:29042774

Detection of single-copy functional genes in prokaryotic cells by two-pass TSA-FISH with polynucleotide probes.

PubMed

Kawakami, Shuji; Hasegawa, Takuya; Imachi, Hiroyuki; Yamaguchi, Takashi; Harada, Hideki; Ohashi, Akiyoshi; Kubota, Kengo

2012-02-01

In situ detection of functional genes with single-cell resolution is currently of interest to microbiologists. Here, we developed a two-pass tyramide signal amplification (TSA)-fluorescence in situ hybridization (FISH) protocol with PCR-derived polynucleotide probes for the detection of single-copy genes in prokaryotic cells. The mcrA gene and the apsA gene in methanogens and sulfate-reducing bacteria, respectively, were targeted. The protocol showed bright fluorescence with a good signal-to-noise ratio and achieved a high efficiency of detection (>98%). The discrimination threshold was approximately 82-89% sequence identity. Microorganisms possessing the mcrA or apsA gene in anaerobic sludge samples were successfully detected by two-pass TSA-FISH with polynucleotide probes. The developed protocol is useful for identifying single microbial cells based on functional gene sequences. Copyright © 2011 Elsevier B.V. All rights reserved.

Development of a single ion hit facility at the Pierre Sue Laboratory: a collimated microbeam to study radiological effects on targeted living cells.

PubMed

Daudin, L; Carrière, M; Gouget, B; Hoarau, J; Khodja, H

2006-01-01

A single ion hit facility is being developed at the Pierre Süe Laboratory (LPS) since 2004. This set-up will be dedicated to the study of ionising radiation effects on living cells, which will complete current research conducted on uranium chemical toxicity on renal and osteoblastic cells. The study of the response to an exposure to alpha particles will allow us to distinguish radiological and chemical toxicities of uranium, with a special emphasis on the bystander effect at low doses. Designed and installed on the LPS Nuclear microprobe, up to now dedicated to ion beam microanalysis, this set-up will enable us to deliver an exact number of light ions accelerated by a 3.75 MV electrostatic accelerator. An 'in air' vertical beam permits the irradiation of cells in conditions compatible with cell culture techniques. Furthermore, cellular monolayer will be kept in controlled conditions of temperature and atmosphere in order to diminish stress. The beam is collimated with a fused silica capillary tubing to target pre-selected cells. Motorisation of the collimator with piezo-electric actuators should enable fast irradiation without moving the sample, thus avoiding mechanical stress. An automated epifluorescence microscope, mounted on an antivibration table, allows pre- and post-irradiation cell observation. An ultra thin silicon surface barrier detector has been developed and tested to be able to shoot a cell with a single alpha particle.

MicroRNA-182 drives metastasis of primary sarcomas by targeting multiple genes

PubMed Central

Sachdeva, Mohit; Mito, Jeffrey K.; Lee, Chang-Lung; Zhang, Minsi; Li, Zhizhong; Dodd, Rebecca D.; Cason, David; Luo, Lixia; Ma, Yan; Van Mater, David; Gladdy, Rebecca; Lev, Dina C.; Cardona, Diana M.; Kirsch, David G.

2014-01-01

Metastasis causes most cancer deaths, but is incompletely understood. MicroRNAs can regulate metastasis, but it is not known whether a single miRNA can regulate metastasis in primary cancer models in vivo. We compared the expression of miRNAs in metastatic and nonmetastatic primary mouse sarcomas and found that microRNA-182 (miR-182) was markedly overexpressed in some tumors that metastasized to the lungs. By utilizing genetically engineered mice with either deletion of or overexpression of miR-182 in primary sarcomas, we discovered that deletion of miR-182 substantially decreased, while overexpression of miR-182 considerably increased, the rate of lung metastasis after amputation of the tumor-bearing limb. Additionally, deletion of miR-182 decreased circulating tumor cells (CTCs), while overexpression of miR-182 increased CTCs, suggesting that miR-182 regulates intravasation of cancer cells into the circulation. We identified 4 miR-182 targets that inhibit either the migration of tumor cells or the degradation of the extracellular matrix. Notably, restoration of any of these targets in isolation did not alter the metastatic potential of sarcoma cells injected orthotopically, but the simultaneous restoration of all 4 targets together substantially decreased the number of metastases. These results demonstrate that a single miRNA can regulate metastasis of primary tumors in vivo by coordinated regulation of multiple genes. PMID:25180607

Label-free imaging of gold nanoparticles in single live cells by photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Tian, Chao; Qian, Wei; Shao, Xia; Xie, Zhixing; Cheng, Xu; Liu, Shengchun; Cheng, Qian; Liu, Bing; Wang, Xueding

2016-03-01

Gold nanoparticles (AuNPs) have been extensively explored as a model nanostructure in nanomedicine and have been widely used to provide advanced biomedical research tools in diagnostic imaging and therapy. Due to the necessity of targeting AuNPs to individual cells, evaluation and visualization of AuNPs in the cellular level is critical to fully understand their interaction with cellular environment. Currently imaging technologies, such as fluorescence microscopy and transmission electron microscopy all have advantages and disadvantages. In this paper, we synthesized AuNPs by femtosecond pulsed laser ablation, modified their surface chemistry through sequential bioconjugation, and targeted the functionalized AuNPs with individual cancer cells. Based on their high optical absorption contrast, we developed a novel, label-free imaging method to evaluate and visualize intracellular AuNPs using photoacoustic microscopy (PAM). Preliminary study shows that the PAM imaging technique is capable of imaging cellular uptake of AuNPs in vivo at single-cell resolution, which provide an important tool for the study of AuNPs in nanomedicine.

Single cell imaging of Bruton's Tyrosine Kinase using an irreversible inhibitor

NASA Astrophysics Data System (ADS)

Turetsky, Anna; Kim, Eunha; Kohler, Rainer H.; Miller, Miles A.; Weissleder, Ralph

2014-04-01

A number of Bruton's tyrosine kinase (BTK) inhibitors are currently in development, yet it has been difficult to visualize BTK expression and pharmacological inhibition in vivo in real time. We synthesized a fluorescent, irreversible BTK binder based on the drug Ibrutinib and characterized its behavior in cells and in vivo. We show a 200 nM affinity of the imaging agent, high selectivity, and irreversible binding to its target following initial washout, resulting in surprisingly high target-to-background ratios. In vivo, the imaging agent rapidly distributed to BTK expressing tumor cells, but also to BTK-positive tumor-associated host cells.

Single-Cell RNA Sequencing of Human T Cells.

PubMed

Villani, Alexandra-Chloé; Shekhar, Karthik

2017-01-01

Understanding how populations of human T cells leverage cellular heterogeneity, plasticity, and diversity to achieve a wide range of functional flexibility, particularly during dynamic processes such as development, differentiation, and antigenic response, is a core challenge that is well suited for single-cell analysis. Hypothesis-free evaluation of cellular states and subpopulations by transcriptional profiling of single T cells can identify relationships that may be obscured by targeted approaches such as FACS sorting on cell-surface antigens, or bulk expression analysis. While this approach is relevant to all cell types, it is of particular interest in the study of T cells for which classical phenotypic criteria are now viewed as insufficient for distinguishing different T cell subtypes and transitional states, and defining the changes associated with dysfunctional T cell states in autoimmunity and tumor-related exhaustion. This unit describes a protocol to generate single-cell transcriptomic libraries of human blood CD4 + and CD8 + T cells, and also introduces the basic bioinformatic steps to process the resulting sequence data for further computational analysis. We show how cellular subpopulations can be identified from transcriptional data, and derive characteristic gene expression signatures that distinguish these states. We believe single-cell RNA-seq is a powerful technique to study the cellular heterogeneity in complex tissues, a paradigm that will be of great value for the immune system.

Imaging immune surveillance of individual natural killer cells confined in microwell arrays.

PubMed

Guldevall, Karolin; Vanherberghen, Bruno; Frisk, Thomas; Hurtig, Johan; Christakou, Athanasia E; Manneberg, Otto; Lindström, Sara; Andersson-Svahn, Helene; Wiklund, Martin; Önfelt, Björn

2010-11-12

New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level.

Micro-magnetic Structures for Biological Applications

NASA Astrophysics Data System (ADS)

Howdyshell, Marci L.

Developments in single-molecule and single-cell experiments over the past century have provided researchers with many tools to probe the responses of cells to stresses such as physical force or to the injection of foreign genes. Often these techniques target the cell membrane, although many are now advancing to probe within the cell. As these techniques are improved upon and the investigations advance toward clinical applications, it has become more critical to achieve high-throughput outcomes which in turn lead to statistically significant results. The technologies developed in this thesis are targeted at transfecting large populations of cells with controlled doses of specific exogenic material without adversely affecting cell viability. Underlying this effort is a platform of lithographically patterned ferromagnetic thin films capable of remotely manipulating and localizing magnetic microbeads attached to biological entities. A novel feature of this approach, as demonstrated here with both DNA and cells, is the opportunity for multiplexed operations on targeted biological specimens. This thesis includes two main thrusts: (1) the advancement of the trapping platforms through experimental verification of mathematical models providing the energy landscapes associated with the traps and (2) implementation of the platform as a basis for rapid and effective high-throughput microchannel and nanochannel cell electroporation devices. The electroporation devices have, in our studies, not only been demonstrated to sustain cell viability with extremely low cell mortality rates, but are also found to be effective for various types of cells. The advances over current electroporation technologies that are achieved in these efforts demonstrate the potential for detection of mRNA expression in heterogeneous cell populations and probing intracellular responses to the introduction of foreign genes into cells.

Novel approaches in function-driven single-cell genomics.

PubMed

Doud, Devin F R; Woyke, Tanja

2017-07-01

Deeper sequencing and improved bioinformatics in conjunction with single-cell and metagenomic approaches continue to illuminate undercharacterized environmental microbial communities. This has propelled the 'who is there, and what might they be doing' paradigm to the uncultivated and has already radically changed the topology of the tree of life and provided key insights into the microbial contribution to biogeochemistry. While characterization of 'who' based on marker genes can describe a large fraction of the community, answering 'what are they doing' remains the elusive pinnacle for microbiology. Function-driven single-cell genomics provides a solution by using a function-based screen to subsample complex microbial communities in a targeted manner for the isolation and genome sequencing of single cells. This enables single-cell sequencing to be focused on cells with specific phenotypic or metabolic characteristics of interest. Recovered genomes are conclusively implicated for both encoding and exhibiting the feature of interest, improving downstream annotation and revealing activity levels within that environment. This emerging approach has already improved our understanding of microbial community functioning and facilitated the experimental analysis of uncharacterized gene product space. Here we provide a comprehensive review of strategies that have been applied for function-driven single-cell genomics and the future directions we envision. © FEMS 2017.

Novel approaches in function-driven single-cell genomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Doud, Devin F. R.; Woyke, Tanja

Deeper sequencing and improved bioinformatics in conjunction with single-cell and metagenomic approaches continue to illuminate undercharacterized environmental microbial communities. This has propelled the 'who is there, and what might they be doing' paradigm to the uncultivated and has already radically changed the topology of the tree of life and provided key insights into the microbial contribution to biogeochemistry. While characterization of 'who' based on marker genes can describe a large fraction of the community, answering 'what are they doing' remains the elusive pinnacle for microbiology. Function-driven single-cell genomics provides a solution by using a function-based screen to subsample complex microbialmore » communities in a targeted manner for the isolation and genome sequencing of single cells. This enables single-cell sequencing to be focused on cells with specific phenotypic or metabolic characteristics of interest. Recovered genomes are conclusively implicated for both encoding and exhibiting the feature of interest, improving downstream annotation and revealing activity levels within that environment. This emerging approach has already improved our understanding of microbial community functioning and facilitated the experimental analysis of uncharacterized gene product space. Here we provide a comprehensive review of strategies that have been applied for function-driven single-cell genomics and the future directions we envision.« less

Novel approaches in function-driven single-cell genomics

DOE PAGES

Doud, Devin F. R.; Woyke, Tanja

2017-06-07

Deeper sequencing and improved bioinformatics in conjunction with single-cell and metagenomic approaches continue to illuminate undercharacterized environmental microbial communities. This has propelled the 'who is there, and what might they be doing' paradigm to the uncultivated and has already radically changed the topology of the tree of life and provided key insights into the microbial contribution to biogeochemistry. While characterization of 'who' based on marker genes can describe a large fraction of the community, answering 'what are they doing' remains the elusive pinnacle for microbiology. Function-driven single-cell genomics provides a solution by using a function-based screen to subsample complex microbialmore » communities in a targeted manner for the isolation and genome sequencing of single cells. This enables single-cell sequencing to be focused on cells with specific phenotypic or metabolic characteristics of interest. Recovered genomes are conclusively implicated for both encoding and exhibiting the feature of interest, improving downstream annotation and revealing activity levels within that environment. This emerging approach has already improved our understanding of microbial community functioning and facilitated the experimental analysis of uncharacterized gene product space. Here we provide a comprehensive review of strategies that have been applied for function-driven single-cell genomics and the future directions we envision.« less

Complementary Approaches to Existing Target Based Drug Discovery for Identifying Novel Drug Targets.

PubMed

Vasaikar, Suhas; Bhatia, Pooja; Bhatia, Partap G; Chu Yaiw, Koon

2016-11-21

In the past decade, it was observed that the relationship between the emerging New Molecular Entities and the quantum of R&D investment has not been favorable. There might be numerous reasons but few studies stress the introduction of target based drug discovery approach as one of the factors. Although a number of drugs have been developed with an emphasis on a single protein target, yet identification of valid target is complex. The approach focuses on an in vitro single target, which overlooks the complexity of cell and makes process of validation drug targets uncertain. Thus, it is imperative to search for alternatives rather than looking at success stories of target-based drug discovery. It would be beneficial if the drugs were developed to target multiple components. New approaches like reverse engineering and translational research need to take into account both system and target-based approach. This review evaluates the strengths and limitations of known drug discovery approaches and proposes alternative approaches for increasing efficiency against treatment.

Application of porous metal enrichment probe sampling to single cell analysis using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS).

PubMed

Fu, Qiang; Tang, Jun; Cui, Meng; Xing, Junpeng; Liu, Zhiqiang; Liu, Shuying

2016-01-01

There is an increasing need for analyzing metabolism in a single cell, which is important to understand the nature of cellular heterogeneity, disease, growth and specialization, etc. However, single cell analysis is often challenging for the traces of samples. In the present study, porous metal enrichment probe sampling combined with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) has been applied for in situ analysis of live onion epidemic cell. Porous probe, treated by corroding copper wire with HCl, was directly inserted into a single cell to get cell solution. A self-made linear actuator was enough to control the penetration of probe into the target cell accurately. Then samples on the tip of probe were eluted and detected by a commercial MALDI-TOF-MS directly. The formation of porous microstructure on the probe surface increased the adsorptive capacity of cell solution. The sensitivity of porous probe sampling was 6 times higher than uncorroded probes generally. This method provides a sensitive and convenient way for the sampling and detection of single cell solution. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Efficient priming of CD4 T cells by Langerin-expressing dendritic cells targeted with porcine epidemic diarrhea virus spike protein domains in pigs.

PubMed

Subramaniam, Sakthivel; Cao, Dianjun; Tian, Debin; Cao, Qian M; Overend, Christopher; Yugo, Danielle M; Matzinger, Shannon R; Rogers, Adam J; Heffron, C Lynn; Catanzaro, Nicholas; Kenney, Scott P; Opriessnig, Tanja; Huang, Yao-Wei; Labarque, Geoffrey; Wu, Stephen Q; Meng, Xiang-Jin

2017-01-02

Porcine epidemic diarrhea virus (PEDV) first emerged in the United States in 2013 causing high mortality and morbidity in neonatal piglets with immense economic losses to the swine industry. PEDV is an alpha-coronavirus replicating primarily in porcine intestinal cells. PEDV vaccines are available in Asia and Europe, and conditionally-licensed vaccines recently became available in the United States but the efficacies of these vaccines in eliminating PEDV from swine populations are questionable. In this study, the immunogenicity of a subunit vaccine based on the spike protein of PEDV, which was directly targeted to porcine dendritic cells (DCs) expressing Langerin, was assessed. The PEDV S antigen was delivered to the dendritic cells through a single-chain antibody specific to Langerin and the targeted cells were stimulated with cholera toxin adjuvant. This approach, known as "dendritic cell targeting," greatly improved PEDV S antigen-specific T cell interferon-γ responses in the CD4 pos CD8 pos T cell compartment in pigs as early as 7days upon transdermal administration. When the vaccine protein was targeted to Langerin pos DCs systemically through intramuscular vaccination, it induced higher serum IgG and IgA responses in pigs, though these responses require a booster dose, and the magnitude of T cell responses were lower as compared to transdermal vaccination. We conclude that PEDV spike protein domains targeting Langerin-expressing dendritic cells significantly increased CD4 T cell immune responses in pigs. The results indicate that the immunogenicity of protein subunit vaccines can be greatly enhanced by direct targeting of the vaccine antigens to desirable dendritic cell subsets in pigs. Copyright © 2016 Elsevier B.V. All rights reserved.

Nanochannel Electroporation as a Platform for Living Cell Interrogation in Acute Myeloid Leukemia.

PubMed

Zhao, Xi; Huang, Xiaomeng; Wang, Xinmei; Wu, Yun; Eisfeld, Ann-Kathrin; Schwind, Sebastian; Gallego-Perez, Daniel; Boukany, Pouyan E; Marcucci, Guido I; Lee, Ly James

2015-12-01

A living cell interrogation platform based on nanochannel electroporation is demonstrated with analysis of RNAs in single cells. This minimally invasive process is based on individual cells and allows both multi-target analysis and stimulus-response analysis by sequential deliveries. The unique platform possesses a great potential to the comprehensive and lysis-free nucleic acid analysis on rare or hard-to-transfect cells.

Single-band upconversion nanoprobes for multiplexed simultaneous in situ molecular mapping of cancer biomarkers.

PubMed

Zhou, Lei; Wang, Rui; Yao, Chi; Li, Xiaomin; Wang, Chengli; Zhang, Xiaoyan; Xu, Congjian; Zeng, Aijun; Zhao, Dongyuan; Zhang, Fan

2015-04-24

The identification of potential diagnostic markers and target molecules among the plethora of tumour oncoproteins for cancer diagnosis requires facile technology that is capable of quantitatively analysing multiple biomarkers in tumour cells and tissues. Diagnostic and prognostic classifications of human tumours are currently based on the western blotting and single-colour immunohistochemical methods that are not suitable for multiplexed detection. Herein, we report a general and novel method to prepare single-band upconversion nanoparticles with different colours. The expression levels of three biomarkers in breast cancer cells were determined using single-band upconversion nanoparticles, western blotting and immunohistochemical technologies with excellent correlation. Significantly, the application of antibody-conjugated single-band upconversion nanoparticle molecular profiling technology can achieve the multiplexed simultaneous in situ biodetection of biomarkers in breast cancer cells and tissue specimens and produce more accurate results for the simultaneous quantification of proteins present at low levels compared with classical immunohistochemical technology.

Single quantum dot tracking reveals the impact of nanoparticle surface on intracellular state.

PubMed

Zahid, Mohammad U; Ma, Liang; Lim, Sung Jun; Smith, Andrew M

2018-05-08

Inefficient delivery of macromolecules and nanoparticles to intracellular targets is a major bottleneck in drug delivery, genetic engineering, and molecular imaging. Here we apply live-cell single-quantum-dot imaging and tracking to analyze and classify nanoparticle states after intracellular delivery. By merging trajectory diffusion parameters with brightness measurements, multidimensional analysis reveals distinct and heterogeneous populations that are indistinguishable using single parameters alone. We derive new quantitative metrics of particle loading, cluster distribution, and vesicular release in single cells, and evaluate intracellular nanoparticles with diverse surfaces following osmotic delivery. Surface properties have a major impact on cell uptake, but little impact on the absolute cytoplasmic numbers. A key outcome is that stable zwitterionic surfaces yield uniform cytosolic behavior, ideal for imaging agents. We anticipate that this combination of quantum dots and single-particle tracking can be widely applied to design and optimize next-generation imaging probes, nanoparticle therapeutics, and biologics.

Negative Enrichment and Isolation of Circulating Tumor Cells for Whole Genome Amplification.

PubMed

Kanwar, Nisha; Done, Susan J

2017-01-01

Circulating tumor cells (CTCs) are a rare population of cells found in the peripheral blood of patients with many types of cancer such as breast, prostate, colon, and lung cancers. Higher numbers of these cells in blood are associated with a poorer prognosis of patients. Genomic profiling of CTCs would help characterize markers specific for the identification of these cells in blood, and also define genomic alterations that give these cells a metastatic advantage over other cells in the primary tumor. Here, we describe an immunomagnetic method to enrich CTCs from the blood of patients with breast cancer, followed by single-cell laser capture microdissection to isolate single CTCs. Whole genome amplification of isolated CTCs allows for many downstream applications to be performed to aide in their characterization, such as whole genome or exome sequencing, Single Nucleotide Polymorphism (SNP) and copy number analysis, and targeted sequencing or quantitative Polymerase Chain Reaction (qPCR) for genomic analyses.

CRISPR-Cas9 nuclear dynamics and target recognition in living cells

PubMed Central

Ma, Hanhui; Tu, Li-Chun; Zhang, Shaojie; Grunwald, David

2016-01-01

The bacterial CRISPR-Cas9 system has been repurposed for genome engineering, transcription modulation, and chromosome imaging in eukaryotic cells. However, the nuclear dynamics of clustered regularly interspaced short palindromic repeats (CRISPR)–associated protein 9 (Cas9) guide RNAs and target interrogation are not well defined in living cells. Here, we deployed a dual-color CRISPR system to directly measure the stability of both Cas9 and guide RNA. We found that Cas9 is essential for guide RNA stability and that the nuclear Cas9–guide RNA complex levels limit the targeting efficiency. Fluorescence recovery after photobleaching measurements revealed that single mismatches in the guide RNA seed sequence reduce the target residence time from >3 h to as low as <2 min in a nucleotide identity- and position-dependent manner. We further show that the duration of target residence correlates with cleavage activity. These results reveal that CRISPR discriminates between genuine versus mismatched targets for genome editing via radical alterations in residence time. PMID:27551060

Quantum Dot Platform for Single-Cell Molecular Profiling

NASA Astrophysics Data System (ADS)

Zrazhevskiy, Pavel S.

In-depth understanding of the nature of cell physiology and ability to diagnose and control the progression of pathological processes heavily rely on untangling the complexity of intracellular molecular mechanisms and pathways. Therefore, comprehensive molecular profiling of individual cells within the context of their natural tissue or cell culture microenvironment is essential. In principle, this goal can be achieved by tagging each molecular target with a unique reporter probe and detecting its localization with high sensitivity at sub-cellular resolution, primarily via microscopy-based imaging. Yet, neither widely used conventional methods nor more advanced nanoparticle-based techniques have been able to address this task up to date. High multiplexing potential of fluorescent probes is heavily restrained by the inability to uniquely match probes with corresponding molecular targets. This issue is especially relevant for quantum dot probes---while simultaneous spectral imaging of up to 10 different probes is possible, only few can be used concurrently for staining with existing methods. To fully utilize multiplexing potential of quantum dots, it is necessary to design a new staining platform featuring unique assignment of each target to a corresponding quantum dot probe. This dissertation presents two complementary versatile approaches towards achieving comprehensive single-cell molecular profiling and describes engineering of quantum dot probes specifically tailored for each staining method. Analysis of expanded molecular profiles is achieved through augmenting parallel multiplexing capacity with performing several staining cycles on the same specimen in sequential manner. In contrast to other methods utilizing quantum dots or other nanoparticles, which often involve sophisticated probe synthesis, the platform technology presented here takes advantage of simple covalent bioconjugation and non-covalent self-assembly mechanisms for straightforward probe preparation and specimen labeling, requiring no advanced technical skills and being directly applicable for a wide range of molecular profiling studies. Utilization of quantum dot platform for single-cell molecular profiling promises to greatly benefit both biomedical research and clinical diagnostics by providing a tool for addressing phenotypic heterogeneity within large cell populations, opening access to studying low-abundance events often masked or completely erased by batch processing, and elucidating biomarker signatures of diseases critical for accurate diagnostics and targeted therapy.

Mechanisms of diminished natural killer cell activity in pregnant women and neonates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baley, J.E.; Schacter, B.Z.

1985-05-01

Because alterations in natural killer (NK) activity in the perinatal period may be important in the maintenance of a healthy pregnancy, the mechanisms by which these alterations are mediated in neonates and in pregnant and postpartum women was examined. NK activity, as measured in a 4-hr /sup 51/Cr-release assay and compared with adult controls, is significantly diminished in all three trimesters of pregnancy and in immediately postpartum women. In postpartum women, NK activity appears to be higher than in pregnant women, although this does not reach statistical significance. Pregnant and postpartum women have normal numbers of large granular lymphocytes andmore » normal target cell binding in an agarose single cell assay but decreased lysis of the bound target cells. NK activity of mononuclear cells from postpartum women, in addition, demonstrate a shift in distribution to higher levels of resistance to gamma-irradiation. Further, sera from postpartum women cause a similar shift to increased radioresistance in mononuclear cells from adult controls. Because radioresistance is a property of interleukin 2-stimulated NK, the shift to radioresistance may represent lymphokine-mediated stimulation occurring during parturition. In contrast, cord blood cells have a more profound decrease in NK activity as determined by /sup 51/Cr-release assay and decreases in both binding and lysis of bound target cells in the single cell assay. The resistance of NK activity in cord cells to gamma-irradiation is also increased, as seen in postpartum women. Cord blood serum, however, did not alter radioresistance or inhibit NK activity. The results suggest that the observed diminished NK activity in pregnant women and neonates arise by different mechanisms: an absence of mature NK cells in the neonate and an alteration of the NK cell in pregnancy leading to decreased killing.« less

Targeted depletion of a MDSC subset unmasks pancreatic ductal adenocarcinoma to adaptive immunity

PubMed Central

Stromnes, Ingunn M.; Brockenbrough, Scott; Izeradjene, Kamel; Carlson, Markus A.; Cuevas, Carlos; Simmons, Randi M.; Greenberg, Philip D.; Hingorani, Sunil R.

2015-01-01

Objective Pancreatic ductal adenocarcinoma (PDA) is characterized by a robust desmoplasia, including the notable accumulation of immunosuppressive cells that shield neoplastic cells from immune detection. Immune evasion may be further enhanced if the malignant cells fail to express high levels of antigens that are sufficiently immunogenic to engender an effector T cell response. In this report, we investigate the predominant subsets of immunosuppressive cancer-conditioned myeloid cells that chronicle and shape pancreas cancer progression. We show that selective depletion of one subset of myeloid-derived suppressor cells (MDSC) in an autochthonous, genetically engineered mouse model (GEMM) of PDA unmasks the ability of the adaptive immune response to engage and target tumor epithelial cells. Methods A combination of in vivo and in vitro studies were performed employing a GEMM that faithfully recapitulates the cardinal features of human PDA. The predominant cancer-conditioned myeloid cell subpopulation was specifically targeted in vivo and the biological outcomes determined. Results PDA orchestrates the induction of distinct subsets of cancer-associated myeloid cells through the production of factors known to influence myelopoeisis. These immature myeloid cells inhibit the proliferation and induce apoptosis of activated T cells. Targeted depletion of granulocytic MDSC (Gr-MDSC) in autochthonous PDA increases the intratumoral accumulation of activated CD8 T cells and apoptosis of tumor epithelial cells, and also remodels the tumor stroma. Conclusions Neoplastic ductal cells of the pancreas induce distinct myeloid cell subsets that promote tumor cell survival and accumulation. Targeted depletion of a single myeloid subset, the Gr-MDSC, can unmask an endogenous T cell response, revealing an unexpected latent immunity and invoking targeting of Gr-MDSC as a potential strategy to exploit for treating this highly lethal disease. PMID:24555999

Monitoring of Dual CRISPR/Cas9-Mediated Steroidogenic Acute Regulatory Protein Gene Deletion and Cholesterol Accumulation Using High-Resolution Fluorescence In Situ Hybridization in a Single Cell

PubMed Central

Lee, Jinwoo; Jefcoate, Colin

2017-01-01

Recent advances in fluorescence microscopy, coupled with CRISPR/Cas9 gene editing technology, provide opportunities for understanding gene regulation at the single-cell level. The application of direct imaging shown here provides an in situ side-by-side comparison of CRISPR/Cas9-edited cells and adjacent unedited cells. We apply this methodology to the steroidogenic acute regulatory protein (StAR) gene in Y-1 adrenal cells and MA-10 testis cells. StAR is a gatekeeper protein that controls the access of cholesterol from the cytoplasm to the inner mitochondria. The loss of this mitochondrial cholesterol transfer mediator rapidly increases lipid droplets in cells, as seen in StAR−/− mice. Here, we describe a dual CRISPR/Cas9 strategy marked by GFP/mCherry expression that deletes StAR activity within 12 h. We used single-molecule fluorescence in situ hybridization (sm-FISH) imaging to directly monitor the time course of gene editing in single cells. We achieved StAR gene deletion at high efficiency dual gRNA targeting to the proximal promoter and exon 2. Seventy percent of transfected cells showed a slow DNA deletion as measured by PCR, and loss of Br-cAMP stimulated transcription. This DNA deletion was seen by sm-FISH in both loci of individual cells relative to non-target Cyp11a1 and StAR exon 7. sm-FISH also distinguishes two effects on stimulated StAR expression without this deletion. Br-cAMP stimulation of primary and spliced StAR RNA at the gene loci were removed within 4 h in this dual CRISPR/Cas9 strategy before any effect on cytoplasmic mRNA and protein occurred. StAR mRNA disappeared between 12 and 24 h in parallel with this deletion, while cholesterol ester droplets increased fourfold. These alternative changes match distinct StAR expression processes. This dual gRNA and sm-FISH approach to CRISPR/Cas9 editing facilitates rapid testing of editing strategies and immediate assessment of single-cell adaptation responses without the perturbation of clonal expansion procedures. PMID:29118738

Hairpin-Hairpin Molecular Beacon Interactions for Detection of Survivin mRNA in Malignant SW480 Cells.

PubMed

Ratajczak, Katarzyna; Krazinski, Bartlomiej E; Kowalczyk, Anna E; Dworakowska, Beata; Jakiela, Slawomir; Stobiecka, Magdalena

2018-05-07

Cancer biomarkers offer unique prospects for the development of cancer diagnostics and therapy. One of such biomarkers, protein survivin (Sur), exhibits strong antiapoptotic and proliferation-enhancing properties and is heavily expressed in multiple cancers. Thus, it can be utilized to provide new modalities for modulating the cell-growth rate, essential for effective cancer treatment. Herein, we have focused on the development of a new survivin-based cancer detection platform for colorectal cancer cells SW480 using a turn-on fluorescence oligonucleotide molecular beacon (MB) probe, encoded to recognize Sur messenger RNA (mRNA). Contrary to the expectations, we have found that both the complementary target oligonucleotide strands as well as the single- and double-mismatch targets, instead of exhibiting the anticipated simple random conformations, preferentially formed secondary structure motifs by folding into small-loop hairpin structures. Such a conformation may interfere with, or even undermine, the biorecognition process. To gain better understanding of the interactions involved, we have replaced the classical Tyagi-Kramer model of interactions between a straight target oligonucleotide strand and a hairpin MB with a new model to account for the hairpin-hairpin interactions as the biorecognition principle. A detailed mechanism of these interactions has been proposed. Furthermore, in experimental work, we have demonstrated an efficient transfection of malignant SW480 cells with SurMB probes containing a fluorophore Joe (SurMB-Joe) using liposomal nanocarriers. The green emission from SurMB-Joe in transfected cancer cells, due to the hybridization of the SurMB-Joe loop with Sur mRNA hairpin target, corroborates Sur overexpression. On the other hand, healthy human-colon epithelial cells CCD 841 CoN show only negligible expression of survivin mRNA. These experiments provide the proof-of-concept for distinguishing between the cancer and normal cells by the proposed hairpin-hairpin interaction method. The single nucleotide polymorphism sensitivity and a low detection limit of 26 nM (S/N = 3σ) for complementary targets have been achieved.

Competing targets of microRNA-608 affect anxiety and hypertension

PubMed Central

Hanin, Geula; Shenhar-Tsarfaty, Shani; Yayon, Nadav; Hoe, Yau Yin; Bennett, Estelle R.; Sklan, Ella H.; Rao, Dabeeru. C.; Rankinen, Tuomo; Bouchard, Claude; Geifman-Shochat, Susana; Shifman, Sagiv; Greenberg, David S.; Soreq, Hermona

2014-01-01

MicroRNAs (miRNAs) can repress multiple targets, but how a single de-balanced interaction affects others remained unclear. We found that changing a single miRNA–target interaction can simultaneously affect multiple other miRNA–target interactions and modify physiological phenotype. We show that miR-608 targets acetylcholinesterase (AChE) and demonstrate weakened miR-608 interaction with the rs17228616 AChE allele having a single-nucleotide polymorphism (SNP) in the 3′-untranslated region (3′UTR). In cultured cells, this weakened interaction potentiated miR-608-mediated suppression of other targets, including CDC42 and interleukin-6 (IL6). Postmortem human cortices homozygote for the minor rs17228616 allele showed AChE elevation and CDC42/IL6 decreases compared with major allele homozygotes. Additionally, minor allele heterozygote and homozygote subjects showed reduced cortisol and elevated blood pressure, predicting risk of anxiety and hypertension. Parallel suppression of the conserved brain CDC42 activity by intracerebroventricular ML141 injection caused acute anxiety in mice. We demonstrate that SNPs in miRNA-binding regions could cause expanded downstream effects changing important biological pathways. PMID:24722204

Local delivery of interleukin-12 using T cells targeting VEGF receptor-2 eradicates multiple vascularized tumors in mice.

PubMed

Chinnasamy, Dhanalakshmi; Yu, Zhiya; Kerkar, Sid P; Zhang, Ling; Morgan, Richard A; Restifo, Nicholas P; Rosenberg, Steven A

2012-03-15

We investigated the feasibility of delivering the proinflammatory cytokine interleukin (IL)-12 into tumor using T cells genetically engineered to express a chimeric antigen receptor (CAR) against the VEGF receptor-2 (VEGFR-2). Two different strains of mice bearing five different established subcutaneous tumors were treated with syngeneic T cells cotransduced with an anti-VEGFR-2 CAR and a constitutively expressed single-chain murine IL-12 or an inducible IL-12 gene after host lymphodepletion. Tumor regression, survival of mice, and persistence of the transferred cells were evaluated. Adoptive transfer of syngeneic T cells cotransduced with an anti-VEGFR-2 CAR and a constitutively expressing single-chain IL-12 resulted in the regression of five different established tumors of different histologies without the need for IL-2 administration. T cells transduced with either anti-VEGFR-2 CAR or single-chain IL-12 alone did not alter the tumor growth indicating that both of them had to be expressed in the same cell to mediate tumor regression. Anti-VEGFR-2 CAR and IL-12-cotransduced T cells infiltrated the tumors, expanded, and persisted for prolonged periods. The antitumor effect did not require the presence of host T and B cells but was dependent on host IL-12R-expressing cells. The anti-VEGFR-2 CAR changed the immunosuppressive tumor environment by altering/reducing both the systemic and the intratumoral CD11b(+)Gr1(+) myeloid suppressor cell subsets that expressed VEGFR-2. These results suggest that targeted delivery of IL-12 into the tumor environment with T cells redirected against VEGFR-2 is a promising approach for treating patients with a variety of solid tumor types.

Define the Twist ATX LPAR1 Signaling Axis in Promoting Obesity Associated Triple Negative Breast Cancer

DTIC Science & Technology

2017-05-01

developed CRISPR technology to examine if Twist enhances ATX and LPAR1 expression. Specifically, we performed lentiviral transduction of Twist...targeting gRNA into breast cancer cells MDA-MB-578 and SUM-1315, and selected single cell colony with Twist knockout. We chose CRISPR -gRNA over the...shRNA system which was originally proposed, as CRISPR provides higher specificity and fewer off-target effects. To verify knockout of Twist, we first

The flavonoid quercetin transiently inhibits the activity of taxol and nocodazole through interference with the cell cycle

PubMed Central

Samuel, Temesgen; Fadlalla, Khalda; Turner, Timothy; Yehualaeshet, Teshome E.

2010-01-01

Quercetin is a flavonoid with anticancer properties. In this study, we examined the effects of quercetin on cell cycle, viability and proliferation of cancer cells, either singly or in combination with the microtubule-targeting drugs taxol and nocodazole. Although quercetin induced cell death in a dose dependent manner, 12.5-50μM quercetin inhibited the activity of both taxol and nocodazole to induce G2/M arrest in various cell lines. Quercetin also partially restored drug-induced loss in viability of treated cells for up to 72 hours. This antagonism of microtubule-targeting drugs was accompanied by a delay in cell cycle progression and inhibition of the buildup of cyclin-B1 at the microtubule organizing center of treated cells. However, quercetin did not inhibit the microtubule targeting of taxol or nocodazole. Despite the short-term protection of cells by quercetin, colony formation and clonogenicity of HCT116 cells were still suppressed by quercetin or quercetin-taxol combination. The status of cell adherence to growth matrix was critical in determining the sensitivity of HCT116 cells to quercetin. We conclude that while long-term exposure of cancer cells to quercetin may prevent cell proliferation and survival, the interference of quercetin with cell cycle progression diminishes the efficacy of microtubule-targeting drugs to arrest cells at G2/M. PMID:21058190

Cell-targetable DNA nanocapsules for spatiotemporal release of caged bioactive small molecules

NASA Astrophysics Data System (ADS)

Veetil, Aneesh T.; Chakraborty, Kasturi; Xiao, Kangni; Minter, Myles R.; Sisodia, Sangram S.; Krishnan, Yamuna

2017-12-01

Achieving triggered release of small molecules with spatial and temporal precision at designated cells within an organism remains a challenge. By combining a cell-targetable, icosahedral DNA-nanocapsule loaded with photoresponsive polymers, we show cytosolic delivery of small molecules with the spatial resolution of single endosomes in specific cells in Caenorhabditis elegans. Our technology can report on the extent of small molecules released after photoactivation as well as pinpoint the location at which uncaging of the molecules occurred. We apply this technology to release dehydroepiandrosterone (DHEA), a neurosteroid that promotes neurogenesis and neuron survival, and determined the timescale of neuronal activation by DHEA, using light-induced release of DHEA from targeted DNA nanocapsules. Importantly, sequestration inside the DNA capsule prevents photocaged DHEA from activating neurons prematurely. Our methodology can in principle be generalized to diverse neurostimulatory molecules.

Targeting the cell cycle and the PI3K pathway: a possible universal strategy to reactivate innate tumor suppressor programmes in cancer cells.

PubMed

David-Pfeuty, Thérèse; Legraverend, Michel; Ludwig, Odile; Grierson, David S

2010-04-01

Corruption of the Rb and p53 pathways occurs in virtually all human cancers. This could be because it lends oncogene-bearing cells a surfeit of Cdk activity and growth, enabling them to elaborate strategies to evade tumor-suppressive mechanisms and divide inappropriately. Targeting both Cdk activities and the PI3K pathway might be therefore a potentially universal means to palliate their deficiency in cancer cells. We showed that the killing efficacy of roscovitine and 16 other purines and potentiation of roscovitine-induced apoptosis by the PI3K inhibitor, LY294002, decreased with increasing corruption of the Rb and p53 pathways. Further, we showed that purines differing by a single substitution, which exerted little lethal effect on distant cell types in rich medium, could display widely-differing cytotoxicity profiles toward the same cell types in poor medium. Thus, closely-related compounds targeting similar Cdks may interact with different targets that could compete for their interaction with therapeutically-relevant Cdk targets. In the perspective of clinical development in association with the PI3K pathway inhibitors, it might thus be advisable to select tumor cell type-specific Cdk inhibitors on the basis of their toxicity in cell-culture-based assays performed at a limiting serum concentration sufficient to suppress their interaction with undesirable crossreacting targets whose range and concentration would depend on the cell genotype.

An integrated passive micromixer-magnetic separation-capillary electrophoresis microdevice for rapid and multiplex pathogen detection at the single-cell level.

PubMed

Jung, Jae Hwan; Kim, Gha-Young; Seo, Tae Seok

2011-10-21

Here we report an integrated microdevice consisting of an efficient passive mixer, a magnetic separation chamber, and a capillary electrophoretic microchannel in which DNA barcode assay, target pathogen separation, and barcode DNA capillary electrophoretic analysis were performed sequentially within 30 min for multiplex pathogen detection at the single-cell level. The intestine-shaped serpentine 3D micromixer provides a high mixing rate to generate magnetic particle-pathogenic bacteria-DNA barcode labelled AuNP complexes quantitatively. After magnetic separation and purification of those complexes, the barcode DNA strands were released and analyzed by the microfluidic capillary electrophoresis within 5 min. The size of the barcode DNA strand was controlled depending on the target bacteria (Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella typhimurium), and the different elution time of the barcode DNA peak in the electropherogram allows us to recognize the target pathogen with ease in the monoplex as well as in the multiplex analysis. In addition, the quantity of the DNA barcode strand (∼10(4)) per AuNP is enough to be observed in the laser-induced confocal fluorescence detector, thereby making single-cell analysis possible. This novel integrated microdevice enables us to perform rapid, sensitive, and multiplex pathogen detection with sample-in-answer-out capability to be applied for biosafety testing, environmental screening, and clinical trials.

Focal calcium monitoring with targeted nanosensors at the cytosolic side of endoplasmic reticulum

NASA Astrophysics Data System (ADS)

Hou, Yanyan; Arai, Satoshi; Takei, Yoshiaki; Murata, Atsushi; Takeoka, Shinji; Suzuki, Madoka

2016-01-01

Ca2+ distribution is spatially and temporally non-uniform inside cells due to cellular compartmentalization. However, Ca2+ sensing with small organic dyes, such as fura-2 and fluo-4, has been practically applied at a single cell level where the averaged signal from freely diffusing dye molecules is acquired. In this study, we aimed to target azide-functionalized fura-2 (N3-fura-2) to a specific site of subcellular compartments to realize focal Ca2+ sensing. Using scAVD (single-chain avidin)-biotin interaction and a copper-free click reaction system, we linked N3-fura-2 to specifically-targeted scAVD protein fused with a red fluorescent protein mCherry, so that Ca2+ sensors conjugated with four N3-fura-2 dyes with dibenzocyclooctyne (DBCO)-PEG4-biotin as a linker were generated at subcellular compartments in living cells. In cytoplasm, N3-fura-2 showed a prolonged retention period after binding to scAVD. Furthermore, the reacted N3-fura-2 was retained inside cells even after free dyes were washed out by methanol fixation. When scAVD was overexpressed on endoplasmic reticulum (ER) membranes, N3-fura-2 was accumulated on ER membranes. Upon histamine stimulation, which increases cytosolic Ca2+ concentration, ER-localized N3-fura-2 successfully sensed the Ca2+ level changes at the cytosolic side of ER membrane. Our study demonstrated specific targeting of N3-fura-2 to subcellular compartments and the ability of sensing focal Ca2+ level changes with the specifically targeted Ca2+ sensors.

Gene therapy using genetically modified lymphocytes targeting VEGFR-2 inhibits the growth of vascularized syngenic tumors in mice.

PubMed

Chinnasamy, Dhanalakshmi; Yu, Zhiya; Theoret, Marc R; Zhao, Yangbing; Shrimali, Rajeev K; Morgan, Richard A; Feldman, Steven A; Restifo, Nicholas P; Rosenberg, Steven A

2010-11-01

Immunotherapies based on adoptive cell transfer are highly effective in the treatment of metastatic melanoma, but the use of this approach in other cancer histologies has been hampered by the identification of appropriate target molecules. Immunologic approaches targeting tumor vasculature provide a means for the therapy of multiple solid tumor types. We developed a method to target tumor vasculature, using genetically redirected syngeneic or autologous T cells. Mouse and human T cells were engineered to express a chimeric antigen receptor (CAR) targeted against VEGFR-2, which is overexpressed in tumor vasculature and is responsible for VEGF-mediated tumor progression and metastasis. Mouse and human T cells expressing the relevant VEGFR-2 CARs mediated specific immune responses against VEGFR-2 protein as well as VEGFR-2-expressing cells in vitro. A single dose of VEGFR-2 CAR-engineered mouse T cells plus exogenous IL-2 significantly inhibited the growth of 5 different types of established, vascularized syngeneic tumors in 2 different strains of mice and prolonged the survival of mice. T cells transduced with VEGFR-2 CAR showed durable and increased tumor infiltration, correlating with their antitumor effect. This approach provides a potential method for the gene therapy of a variety of human cancers.

Construction of METHFR shRNA/5-fluorouracil co-loaded folate-targeted chitosan polymeric nanoparticles and its anti-carcinoma effect on gastric cells growth

NASA Astrophysics Data System (ADS)

Xin, Lin; Fan, Ji-Chang; Le, Yi-Guan; Zeng, Fei; Cheng, Hua; Hu, Xiao-yun; Cao, Jia-Qing

2016-05-01

PEGylated and folate-targeted chitosan polymeric nanoparticles (FPNs) for the treatment of gastric carcinoma were prepared successfully. OQC-anchored folate conjugates were synthesized and used in assembling FPNs nano-system for enhancing intracellular uptake against folate receptor overexpressing cancer cells. The results indicated that folate-targeted chitosan polymeric nanoparticles (CPNs) can reverse drug-resistant SGC-7901 cells of 5-fluorouracil (5-FU) compared with non-targeted CPNs. Increased therapeutic efficiency of 5-FU/METHFR shRNA co-loaded PNs were also tested in SGC-7901 cells and compaed with 5-FU or METHFR shRNA in solution, which was associated with increased cell inhibition function for single drug group and synergistic effects of 5-FU and METHFR shRNA at 2.0 µg/mL FPNs concentration. In addition, the cell accumulation levels of 5-FU in SGC-7901 cells was time dependent for these nanoparticles. FPNs (effective diameter: 83.2 ± 1.1 nm; polydispersity index: 0.193) could significantly boost cellular accumulation of 5-FU and overcome the drug efflux mechanism of MDR than 5-FU-loaded NPNs and 5-FU in solution. In conclusion, ligand-targeted PNs can be used as a potentially effective drug delivery system.

Large-Scale Chemical Similarity Networks for Target Profiling of Compounds Identified in Cell-Based Chemical Screens

PubMed Central

Lo, Yu-Chen; Senese, Silvia; Li, Chien-Ming; Hu, Qiyang; Huang, Yong; Damoiseaux, Robert; Torres, Jorge Z.

2015-01-01

Target identification is one of the most critical steps following cell-based phenotypic chemical screens aimed at identifying compounds with potential uses in cell biology and for developing novel disease therapies. Current in silico target identification methods, including chemical similarity database searches, are limited to single or sequential ligand analysis that have limited capabilities for accurate deconvolution of a large number of compounds with diverse chemical structures. Here, we present CSNAP (Chemical Similarity Network Analysis Pulldown), a new computational target identification method that utilizes chemical similarity networks for large-scale chemotype (consensus chemical pattern) recognition and drug target profiling. Our benchmark study showed that CSNAP can achieve an overall higher accuracy (>80%) of target prediction with respect to representative chemotypes in large (>200) compound sets, in comparison to the SEA approach (60–70%). Additionally, CSNAP is capable of integrating with biological knowledge-based databases (Uniprot, GO) and high-throughput biology platforms (proteomic, genetic, etc) for system-wise drug target validation. To demonstrate the utility of the CSNAP approach, we combined CSNAP's target prediction with experimental ligand evaluation to identify the major mitotic targets of hit compounds from a cell-based chemical screen and we highlight novel compounds targeting microtubules, an important cancer therapeutic target. The CSNAP method is freely available and can be accessed from the CSNAP web server (http://services.mbi.ucla.edu/CSNAP/). PMID:25826798

Microfluidic devices for the controlled manipulation of small volumes

DOEpatents

Ramsey, Michael J; Jacobson, Stephen C

2012-09-18

A method for conducting a broad range of biochemical analyses or manipulations on a series of nano- to subnanoliter reaction volumes and an apparatus for carrying out the same are disclosed. The invention is implemented on a fluidic microchip to provide high serial throughput. In particular, the disclosed device is a microfabricated channel device that can manipulate nanoliter or subnanoliter reaction volumes in a controlled manner to produce results at rates of 1 to 10 Hz per channel. The reaction volumes are manipulated in serial fashion analogous to a digital shift register. The invention has application to such problems as screening molecular or cellular targets using single beads from split-synthesis combinatorial libraries, screening single cells for RNA or protein expression, genetic diagnostic screening at the single cell level, or performing single cell signal transduction studies.

Microfluidic devices for the controlled manipulation of small volumes

DOEpatents

Ramsey, J Michael [Knoxville, TN; Jacobson, Stephen C [Knoxville, TN

2007-07-03

A method for conducting a broad range of biochemical analyses or manipulations on a series of nano- to subnanoliter reaction volumes and an apparatus for carrying out the same are disclosed. The invention is implemented on a fluidic microchip to provide high serial throughput. In particular, the disclosed device is a microfabricated channel device that can manipulate nanoliter or subnanoliter reaction volumes in a controlled manner to produce results at rates of 1 to 10 Hz per channel. The reaction volumes are manipulated in serial fashion analogous to a digital shift register. The invention has application to such problems as screening molecular or cellular targets using single beads from split-synthesis combinatorial libraries, screening single cells for RNA or protein expression, genetic diagnostic screening at the single cell level, or performing single cell signal transduction studies.

Design of multifunctional nanoparticles for combined in-vivo imaging and advanced drug delivery

NASA Astrophysics Data System (ADS)

Leary, James F.

2018-02-01

Design of multifunctional nanoparticles for multimodal in-vivo imaging and advanced targeting to diseased single cells for massive parallel processing nanomedicine approaches requires careful overall design and a multilayered approach. Initial core materials can include non-toxic metals which not only serve as an x-ray contrast agent for CAT scan imaging, but can contain T1 or T2 contrast agents for MRI imaging. One choice is superparamagnetic iron oxide NPs which also allow for convenient magnetic manipulation during manufacturing but also for re-positioning inside the body and for single cell hyperthermia therapies. To permit real-time fluorescence-guided surgery, fluorescence molecules can be included. Advanced targeting can be achieved by attaching antibodies, peptides, aptamers, or other targeting molecules to the nanoparticle in a multilayered approach producing "programmable nanoparticles" whereby the "programming" means controlling a sequence of multi-step targeting methods. Addition of membrane permeating peptides can facilitate uptake by the cell. Addition of "stealth" molecules (e.g. PEG or chitosan) to the outer surfaces of the nanoparticles can permit greatly enhanced circulation times in-vivo which in turn lead to lower amounts of drug exposure to the patient which can reduce undesirable side effects. Nanoparticles with incomplete layers can be removed by affinity purification methods to minimize mistargeting events in-vivo. Nanoscale imaging of these manufactured, multifunctional nanoparticles can be achieved either directly through superresolution microscopy or indirectly through single nanoparticle zeta-sizing or x-ray correlation microscopy. Since these multifunctional nanoparticles are best analyzed by technologies permitting analysis in aqueous environments, superresolution microscopy is, in most cases, the preferred method.

Laser activated nanothermolysis of leukemia cells monitored by photothermal microscopy

NASA Astrophysics Data System (ADS)

Lapotko, Dmitri; Lukianova, Ekaterina; Shnip, Alexander; Zheltov, George; Potapnev, Michail; Savitsky, Valeriy; Klimovich, Olga; Oraevsky, Alexander

2005-04-01

We are developing new diagnostic and therapeutic technologies for leukemia based on selective targeting of leukemia cells with gold nanoparticles and thermomechanical destruction of the tumor cells with laser-induced microbubbles. Clusters of spherical gold nanoparticles that have strong optical absorption of laser pulses at 532 nm served as nucleation sites of vapor microbubbles. The nanoparticles were targeted selectively to leukemia cells using leukemia-specific surface receptors and a set of two monoclonal antibodies. Application of a primary myeloid-specific antibody to tumor cells followed by targeting the cells with 30-nm nanoparticles conjugated with a secondary antibody (IgG) resulted in formation of nanoparticulate clusters due to aggregation of IgGs. Formation of clusters resulted in substantial decrease of the damage threshold for target cells. The results encourage development of Laser Activated Nanothermolysis as a Cell Elimination Therapy (LANCET) for leukemia. The proposed technology can be applied separately or in combination with chemotherapy for killing leukemia cells without damage to other blood cells. Potential applications include initial reduction of concentration of leukemia cells in blood prior to chemotherapy and treatment of residual tumor cells after the chemotherapy. Laser-induced bubbles in individual cells and cell damage were monitored by analyzing profile of photothermal response signals over the entire cell after irradiation with a single 10-ns long laser pulse. Photothermal microscopy was utilized for imaging formation of microbubbles around nanoparticulate clusters.

Confocal Raman microspectroscopic study of folate receptor-targeted delivery of 6-mercaptopurine-embedded gold nanoparticles in a single cell.

PubMed

Park, Jin; Jeon, Won Il; Lee, So Yeong; Ock, Kwang-Su; Seo, Ji Hye; Park, Jinho; Ganbold, Erdene-Ochir; Cho, Keunchang; Song, Nam Woong; Joo, Sang-Woo

2012-05-01

We investigate the cellular uptake behaviors and efficacy of folate-coated gold nanoparticles (AuNPs) for the targeted drug delivery system in human cancer cells. Folate-conjugated AuNPs embedded with a purine analogue cancer drug of 6-mercaptopurine (6MP) were assembled via a 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) coupling reaction between the amino group of 4-aminobenzenethiol (ABT) and the carboxyl group of folic acid. The assembly of folate and 6MP on AuNPs has been examined by absorption spectroscopy, transmission electron microscopy (TEM), and confocal Raman spectroscopy. The internalization of the conjugated AuNPs inside the folate receptor-positive HeLa and KB cells was checked by TEM and dark-field microscopy (DFM) combined with label-free confocal spectroscopy over the depth variable z at a micrometer resolution. DFM live cell imaging of folate-conjugated AuNPs in HeLa cells indicated that the targeted AuNPs appeared to attach on the cell surfaces and enter into the cell with an hour. The cell viability was also compared to estimate the efficacy of folate-conjugated AuNP delivery systems. Folate receptor-targeted AuNP systems appeared to decrease cancer cell viability both in vitro and in vivo more than did the use of the 6MP-coated AuNPs drug without any targeting systems. Copyright © 2012 Wiley Periodicals, Inc.

Photothermal and photoacoustic processes of laser activated nano-thermolysis of cells

NASA Astrophysics Data System (ADS)

Lapotko, Dmitri; Lukianova, Ekaterina; Mitskevich, Pavel; Smolnikova, Victoria; Potapnev, Michail; Konopleva, Marina; Andreeff, Michael; Oraevsky, Alexander

2007-02-01

Laser Activated Nano-Thermolysis was recently proposed for selective damage of individual target (cancer) cells by pulsed laser induced microbubbles around superheated clusters of optically absorbing nanoparticles (NP). One of the clinical applications of this technology is the elimination of residual tumor cells from human blood and bone marrow. Clinical standards for the safety and efficacy of such procedure require the development and verification of highly selective and controllable mechanisms of cell killing. Our previous experiments showed that laser-induced microbubble is the main damaging factor in the case cell irradiation by short laser pulses above the threshold. Our current aim was to study the cell damage mechanisms and analyze selectivity and efficacy of cell damage as a function of NP parameters, NP-cell interaction conditions, and conditions of bubble generation around NP and NP clusters in cells. Generation of laser-induced bubbles around gold NP with diameters 10-250 nm was studied in Acute Myeloblast Leukemia (AML) cultures, normal stem and model K562 human cells. Short laser pulses (10 ns, 532 nm) were applied to those cells in vitro and the processes in cells were investigated with photothermal, fluorescent and atomic force microscopies and also with fluorescence flow cytometry. We have found that the best selectivity of cell damage is achieved by (1) forming large clusters of optically absorbing NP in target cells and (2) irradiating the cells with single laser pulses with the lowest fluence that can generate microbubble only around large clusters but not around single NP. Laser microbubbles with the lifetime from 20 ns to 2000 ns generated in individual cells caused damage and lysis of the cellular membrane and consequently cell death. Laser microbubbles did not damage normal cells around the damaged target (tumor) cell. Laser irradiation with equal fluence did not cause any damage of cells without accumulated NP clusters.

Pretargeting with bispecific fusion proteins facilitates delivery of nanoparticles to tumor cells with distinct surface antigens.

PubMed

Yang, Qi; Parker, Christina L; Lin, Yukang; Press, Oliver W; Park, Steven I; Lai, Samuel K

2017-06-10

Tumor heterogeneity, which describes the genetically and phenotypically distinct subpopulations of tumor cells present within the same tumor or patient, presents a major challenge to targeted delivery of diagnostic and/or therapeutic agents. An ideal targeting strategy should deliver a given nanocarrier to the full diversity of cancer cells, which is difficult to achieve with conventional ligand-conjugated nanoparticles. We evaluated pretargeting (i.e., multistep targeting) as a strategy to facilitate nanoparticle delivery to multiple target cells by measuring the uptake of biotinylated nanoparticles by lymphoma cells with distinct surface antigens pretreated with different bispecific streptavidin-scFv fusion proteins. Fusion proteins targeting CD20 or tumor-associated glycoprotein 72 (TAG-72) mediated the specific in vitro uptake of 100nm biotin-functionalized nanoparticles by Raji and Jurkat lymphoma cells (CD20-positive and TAG-72-positive cells, respectively). Greater uptake was observed for pretargeted nanoparticles with increasing amounts of surface biotin, with 6- to 18-fold higher uptake vs. non-biotinylated nanoparticle and fusion protein controls. Fully biotin-modified particles remained resistant to cultured macrophage cell uptake, although they were still quickly cleared from systemic circulation in vivo (t 1/2 <1h). For single Raji tumor-bearing mice, pretargeting with CD20-specific FP significantly increased nanoparticle tumor targeting. In mice bearing both Raji and Jurkat tumors, pretargeting with both fusion proteins markedly increased nanoparticle targeting to both tumor types, compared to animals dosed with nanoparticles alone. These in vitro and in vivo observations support further evaluations of pretargeting fusion protein cocktails as a strategy to enhance nanoparticle delivery to a diverse array of molecularly distinct target cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Outer Membrane Targeting, Ultrastructure, and Single Molecule Localization of the Enteropathogenic Escherichia coli Type IV Pilus Secretin BfpB

PubMed Central

Lieberman, Joshua A.; Frost, Nicholas A.; Hoppert, Michael; Fernandes, Paula J.; Vogt, Stefanie L.; Raivio, Tracy L.; Blanpied, Thomas A.

2012-01-01

Type IV pili (T4P) are filamentous surface appendages required for tissue adherence, motility, aggregation, and transformation in a wide array of bacteria and archaea. The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is a prototypical T4P and confirmed virulence factor. T4P fibers are assembled by a complex biogenesis machine that extrudes pili through an outer membrane (OM) pore formed by the secretin protein. Secretins constitute a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4P, type II secretion, type III secretion, and phage assembly. Here, we determine that the lipoprotein transport pathway is not required for targeting the BfpB secretin protein of the EPEC T4P to the OM and describe the ultrastructure of the single particle averaged structures of the assembled complex by transmission electron microscopy. Furthermore, we use photoactivated localization microscopy to determine the distribution of single BfpB molecules fused to photoactivated mCherry. In contrast to findings in other T4P systems, we found that BFP components predominantly have an uneven distribution through the cell envelope and are only found at one or both poles in a minority of cells. In addition, we report that concurrent mutation of both the T4bP secretin and the retraction ATPase can result in viable cells and found that these cells display paradoxically low levels of cell envelope stress response activity. These results imply that secretins can direct their own targeting, have complex distributions and provide feedback information on the state of pilus biogenesis. PMID:22247509

Transportation of single cell and microbubbles by phase-shift introduced to standing leaky surface acoustic waves

PubMed Central

Meng, Long; Cai, Feiyan; Zhang, Zidong; Niu, Lili; Jin, Qiaofeng; Yan, Fei; Wu, Junru; Wang, Zhanhui; Zheng, Hairong

2011-01-01

A microfluidic device was developed to precisely transport a single cell or multiple microbubbles by introducing phase-shifts to a standing leaky surface acoustic wave (SLSAW). The device consists of a polydimethyl-siloxane (PDMS) microchannel and two phase-tunable interdigital transducers (IDTs) for the generation of the relative phase for the pair of surface acoustic waves (SAW) propagating along the opposite directions forming a standing wave. When the SAW contacts the fluid medium inside the microchannel, some of SAW energy is coupled to the fluid and the SAW becomes the leaky surface wave. By modulating the relative phase between two IDTs, the positions of pressure nodes of the SLSAW in the microchannel change linearly resulting in the transportation of a single cell or microbubbles. The results also reveal that there is a good linear relationship between the relative phase and the displacement of a single cell or microbubbles. Furthermore, the single cell and the microbubbles can be transported over a predetermined distance continuously until they reach the targeted locations. This technique has its distinct advantages, such as precise position-manipulation, simple to implement, miniature size, and noninvasive character, which may provide an effective method for the position-manipulation of a single cell and microbubbles in many biological and biomedical applications. PMID:22662056

Gold nanoparticle-mediated laser stimulation causes a complex stress signal in neuronal cells

NASA Astrophysics Data System (ADS)

Johannsmeier, Sonja; Heeger, Patrick; Terakawa, Mitsuhiro; Kalies, Stefan; Heisterkamp, Alexander; Ripken, Tammo; Heinemann, Dag

2017-07-01

Gold nanoparticle mediated laser stimulation of neuronal cells allows for cell activation on a single-cell level. It could therefore be considered an alternative to classical electric neurostimulation. The physiological impact of this new approach has not been intensively studied so far. Here, we investigate the targeted cell's reaction to a laser stimulus based on its calcium response. A complex cellular reaction involving multiple sources has been revealed.

Thermo-sensitive liposomes loaded with doxorubicin and lysine modified single-walled carbon nanotubes as tumor-targeting drug delivery system.

PubMed

Zhu, Xiali; Xie, Yingxia; Zhang, Yingjie; Huang, Heqing; Huang, Shengnan; Hou, Lin; Zhang, Huijuan; Li, Zhi; Shi, Jinjin; Zhang, Zhenzhong

2014-11-01

This report focuses on the thermo-sensitive liposomes loaded with doxorubicin and lysine-modified single-walled carbon nanotube drug delivery system, which was designed to enhance the anti-tumor effect and reduce the side effects of doxorubicin. Doxorubicin-lysine/single-walled carbon nanotube-thermo-sensitive liposomes was prepared by reverse-phase evaporation method, the mean particle size was 232.0 ± 5.6 nm, and drug entrapment efficiency was 86.5 ± 3.7%. The drug release test showed that doxorubicin released more quickly at 42℃ than at 37℃. Compared with free doxorubicin, doxorubicin-lysine/single-walled carbon nanotube-thermo-sensitive liposomes could efficiently cross the cell membranes and afford higher anti-tumor efficacy on the human hepatic carcinoma cell line (SMMC-7721) cells in vitro. For in vivo experiments, the relative tumor volumes of the sarcomaia 180-bearing mice in thermo-sensitive liposomes group and doxorubicin group were significantly smaller than those of N.S. group. Meanwhile, the combination of near-infrared laser irradiation at 808 nm significantly enhanced the tumor growth inhibition both on SMMC-7721 cells and the sarcomaia 180-bearing mice. The quality of life such as body weight, mental state, food and water intake of sarcomaia 180 tumor-bearing mice treated with doxorubicin-lysine/single-walled carbon nanotube-thermo-sensitive liposomes were much higher than those treated with doxorubicin. In conclusion, doxorubicin-lysine/single-walled carbon nanotube-thermo-sensitive liposomes combined with near-infrared laser irradiation at 808 nm may potentially provide viable clinical strategies for targeting delivery of anti-cancer drugs. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Feasibility of mesenchymal stem cell culture expansion for a phase I clinical trial in multiple sclerosis.

PubMed

Planchon, Sarah M; Lingas, Karen T; Reese Koç, Jane; Hooper, Brittney M; Maitra, Basabi; Fox, Robert M; Imrey, Peter B; Drake, Kylie M; Aldred, Micheala A; Lazarus, Hillard M; Cohen, Jeffrey A

2018-01-01

Multiple sclerosis is an inflammatory, neurodegenerative disease of the central nervous system for which therapeutic mesenchymal stem cell transplantation is under study. Published experience of culture-expanding multiple sclerosis patients' mesenchymal stem cells for clinical trials is limited. To determine the feasibility of culture-expanding multiple sclerosis patients' mesenchymal stem cells for clinical use. In a phase I trial, autologous, bone marrow-derived mesenchymal stem cells were isolated from 25 trial participants with multiple sclerosis and eight matched controls, and culture-expanded to a target single dose of 1-2 × 10 6 cells/kg. Viability, cell product identity and sterility were assessed prior to infusion. Cytogenetic stability was assessed by single nucleotide polymorphism analysis of mesenchymal stem cells from 18 multiple sclerosis patients and five controls. One patient failed screening. Mesenchymal stem cell culture expansion was successful for 24 of 25 multiple sclerosis patients and six of eight controls. The target dose was achieved in 16-62 days, requiring two to three cell passages. Growth rate and culture success did not correlate with demographic or multiple sclerosis disease characteristics. Cytogenetic studies identified changes on one chromosome of one control (4.3%) after extended time in culture. Culture expansion of mesenchymal stem cells from multiple sclerosis patients as donors is feasible. However, culture time should be minimized for cell products designated for therapeutic administration.

Experimental Proof for the Role of Nonlinear Photoionization in Plasmonic Phototherapy.

PubMed

Minai, Limor; Zeidan, Adel; Yeheskely-Hayon, Daniella; Yudovich, Shimon; Kviatkovsky, Inna; Yelin, Dvir

2016-07-13

Targeting individual cells within a heterogeneous tissue is a key challenge in cancer therapy, encouraging new approaches for cancer treatment that complement the shortcomings of conventional therapies. The highly localized interactions triggered by focused laser beams promise great potential for targeting single cells or small cell clusters; however, most laser-tissue interactions often involve macroscopic processes that may harm healthy nearby tissue and reduce specificity. Specific targeting of living cells using femtosecond pulses and nanoparticles has been demonstrated promising for various potential therapeutic applications including drug delivery via optoporation, drug release, and selective cell death. Here, using an intense resonant femtosecond pulse and cell-specific gold nanorods, we show that at certain irradiation parameters cell death is triggered by nonlinear plasmonic photoionization and not by thermally driven processes. The experimental results are supported by a physical model for the pulse-particle-medium interactions. A good correlation is found between the calculated total number and energy of the generated free electrons and the observed cell death, suggesting that femtosecond photoionization plays the dominant role in cell death.

Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

DOE PAGES

Schumann, Kathrin; Lin, Steven; Boyer, Eric; ...

2015-07-27

T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4 + T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs).more » Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ~40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 ( PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ~20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.« less

Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schumann, Kathrin; Lin, Steven; Boyer, Eric

T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4 + T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs).more » Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ~40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 ( PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ~20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.« less

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue

PubMed Central

Gerdes, Michael J.; Sevinsky, Christopher J.; Sood, Anup; Adak, Sudeshna; Bello, Musodiq O.; Bordwell, Alexander; Can, Ali; Corwin, Alex; Dinn, Sean; Filkins, Robert J.; Hollman, Denise; Kamath, Vidya; Kaanumalle, Sireesha; Kenny, Kevin; Larsen, Melinda; Lazare, Michael; Lowes, Christina; McCulloch, Colin C.; McDonough, Elizabeth; Pang, Zhengyu; Rittscher, Jens; Santamaria-Pang, Alberto; Sarachan, Brion D.; Seel, Maximilian L.; Seppo, Antti; Shaikh, Kashan; Sui, Yunxia; Zhang, Jingyu; Ginty, Fiona

2013-01-01

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics. PMID:23818604

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue.

PubMed

Gerdes, Michael J; Sevinsky, Christopher J; Sood, Anup; Adak, Sudeshna; Bello, Musodiq O; Bordwell, Alexander; Can, Ali; Corwin, Alex; Dinn, Sean; Filkins, Robert J; Hollman, Denise; Kamath, Vidya; Kaanumalle, Sireesha; Kenny, Kevin; Larsen, Melinda; Lazare, Michael; Li, Qing; Lowes, Christina; McCulloch, Colin C; McDonough, Elizabeth; Montalto, Michael C; Pang, Zhengyu; Rittscher, Jens; Santamaria-Pang, Alberto; Sarachan, Brion D; Seel, Maximilian L; Seppo, Antti; Shaikh, Kashan; Sui, Yunxia; Zhang, Jingyu; Ginty, Fiona

2013-07-16

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.

Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus.

PubMed

Merkle, Florian T; Neuhausser, Werner M; Santos, David; Valen, Eivind; Gagnon, James A; Maas, Kristi; Sandoe, Jackson; Schier, Alexander F; Eggan, Kevin

2015-05-12

The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs), but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knockin at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel) mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knockin hPSC reporter cell lines free of unwanted mutations at the targeted locus. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

DNA targeting specificity of RNA-guided Cas9 nucleases.

PubMed

Hsu, Patrick D; Scott, David A; Weinstein, Joshua A; Ran, F Ann; Konermann, Silvana; Agarwala, Vineeta; Li, Yinqing; Fine, Eli J; Wu, Xuebing; Shalem, Ophir; Cradick, Thomas J; Marraffini, Luciano A; Bao, Gang; Zhang, Feng

2013-09-01

The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.

To Nick or Not to Nick: Comparison of I-SceI Single- and Double-Strand Break-Induced Recombination in Yeast and Human Cells

PubMed Central

Katz, Samantha S.; Gimble, Frederick S.; Storici, Francesca

2014-01-01

Genetic modification of a chromosomal locus to replace an existing dysfunctional allele with a corrected sequence can be accomplished through targeted gene correction using the cell's homologous recombination (HR) machinery. Gene targeting is stimulated by generation of a DNA double-strand break (DSB) at or near the site of correction, but repair of the break via non-homologous end-joining without using the homologous template can lead to deleterious genomic changes such as in/del mutations, or chromosomal rearrangements. By contrast, generation of a DNA single-strand break (SSB), or nick, can stimulate gene correction without the problems of DSB repair because the uncut DNA strand acts as a template to permit healing without alteration of genetic material. Here, we examine the ability of a nicking variant of the I-SceI endonuclease (K223I I-SceI) to stimulate gene targeting in yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK-293) cells. K223I I-SceI is proficient in both yeast and human cells and promotes gene correction up to 12-fold. We show that K223I I-SceI-driven recombination follows a different mechanism than wild-type I-SceI-driven recombination, thus indicating that the initial DNA break that stimulates recombination is not a low-level DSB but a nick. We also demonstrate that K223I I-SceI efficiently elevates gene targeting at loci distant from the break site in yeast cells. These findings establish the capability of the I-SceI nickase to enhance recombination in yeast and human cells, strengthening the notion that nicking enzymes could be effective tools in gene correction strategies for applications in molecular biology, biotechnology, and gene therapy. PMID:24558436

Simultaneous Drug Targeting of the Promoter MYC G-Quadruplex and BCL2 i-Motif in Diffuse Large B-Cell Lymphoma Delays Tumor Growth.

PubMed

Kendrick, Samantha; Muranyi, Andrea; Gokhale, Vijay; Hurley, Laurence H; Rimsza, Lisa M

2017-08-10

Secondary DNA structures are uniquely poised as therapeutic targets due to their molecular switch function in turning gene expression on or off and scaffold-like properties for protein and small molecule interaction. Strategies to alter gene transcription through these structures thus far involve targeting single DNA conformations. Here we investigate the feasibility of simultaneously targeting different secondary DNA structures to modulate two key oncogenes, cellular-myelocytomatosis (MYC) and B-cell lymphoma gene-2 (BCL2), in diffuse large B-cell lymphoma (DLBCL). Cotreatment with previously identified ellipticine and pregnanol derivatives that recognize the MYC G-quadruplex and BCL2 i-motif promoter DNA structures lowered mRNA levels and subsequently enhanced sensitivity to a standard chemotherapy drug, cyclophosphamide, in DLBCL cell lines. In vivo repression of MYC and BCL2 in combination with cyclophosphamide also significantly slowed tumor growth in DLBCL xenograft mice. Our findings demonstrate concurrent targeting of different DNA secondary structures offers an effective, precise, medicine-based approach to directly impede transcription and overcome aberrant pathways in aggressive malignancies.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Mi; University of Chinese Academy of Sciences, Beijing 100049; Liu, Lianqing, E-mail: lqliu@sia.cn

Highlights: •Nanoscale cellular ultra-structures of macrophages were observed. •The binding affinities of FcγRs were measured directly on macrophages. •The nanoscale distributions of FcγRs were mapped on macrophages. -- Abstract: Fc gamma receptors (FcγR), widely expressed on effector cells (e.g., NK cells, macrophages), play an important role in clinical cancer immunotherapy. The binding of FcγRs to the Fc portions of antibodies that are attached to the target cells can activate the antibody-dependent cell-mediated cytotoxicity (ADCC) killing mechanism which leads to the lysis of target cells. In this work, we used atomic force microscopy (AFM) to observe the cellular ultra-structures and measuremore » the biophysical properties (affinity and distribution) of FcγRs on single macrophages in aqueous environments. AFM imaging was used to obtain the topographies of macrophages, revealing the nanoscale cellular fine structures. For molecular interaction recognition, antibody molecules were attached onto AFM tips via a heterobifunctional polyethylene glycol (PEG) crosslinker. With AFM single-molecule force spectroscopy, the binding affinities of FcγRs were quantitatively measured on single macrophages. Adhesion force mapping method was used to localize the FcγRs, revealing the nanoscale distribution of FcγRs on local areas of macrophages. The experimental results can improve our understanding of FcγRs on macrophages; the established approach will facilitate further research on physiological activities involved in antibody-based immunotherapy.« less

Single Targeted Exon Mutation Creates a True Congenic Mouse for Competitive Hematopoietic Stem Cell Transplantation: The C57BL/6-CD45.1(STEM) Mouse.

PubMed

Mercier, Francois E; Sykes, David B; Scadden, David T

2016-06-14

Defining the molecular regulators of hematopoietic stem and progenitor cells (HSPCs) requires in vivo functional analyses. Competitive bone marrow transplants (BMTs) compare control and test HSPCs to demonstrate the functional role of a genetic change or chemical perturbation. Competitive BMT is enabled by antibodies that specifically recognize hematopoietic cells from congenic mouse strains due to variants of the cell surface protein CD45, designated CD45.1 and CD45.2. The current congenic competitor strain, B6.SJL-Ptprc(a) Pepc(b)/BoyJ (CD45.1), has a substantial inherent disadvantage in competition against the C57BL/6 (CD45.2) strain, confounding experimental interpretation. Despite backcrossing, the congenic interval over which the B6.SJL-Ptprc(a) Pepc(b)/BoyJ strain differs is almost 40 Mb encoding ∼300 genes. Here, we demonstrate that a single amino acid change determines the CD45.1 epitope. Further, we report on the single targeted exon mutant (STEM) mouse strain, CD45.1(STEM), which is functionally equivalent to CD45.2 cells in competitive BMT. This strain will permit the precise definition of functional roles for candidate genes using in vivo HSPC assays. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy.

PubMed

Igarashi, Yuka; Uchiyama, Toru; Minegishi, Tomoko; Takahashi, Sirirat; Watanabe, Nobuyuki; Kawai, Toshinao; Yamada, Masafumi; Ariga, Tadashi; Onodera, Masafumi

2017-09-15

Clinical improvement in stem cell gene therapy (SCGT) for primary immunodeficiencies depends on the engraftment levels of genetically corrected cells, and tracing the transgene in each hematopoietic lineage is therefore extremely important in evaluating the efficacy of SCGT. We established a single cell-based droplet digital PCR (sc-ddPCR) method consisting of the encapsulation of a single cell into each droplet, followed by emulsion PCR with primers and probes specific for the transgene. A fluorescent signal in a droplet indicates the presence of a single cell carrying the target gene in its genome, and this system can clearly determine the ratio of transgene-positive cells in the entire population at the genomic level. Using sc-ddPCR, we analyzed the engraftment of vector-transduced cells in two patients with severe combined immunodeficiency (SCID) who were treated with SCGT. Sufficient engraftment of the transduced cells was limited to the T cell lineage in peripheral blood (PB), and a small percentage of CD34 + cells exhibited vector integration in bone marrow, indicating that the transgene-positive cells in PB might have differentiated from a small population of stem cells or lineage-restricted precursor cells. sc-ddPCR is a simplified and powerful tool for the detailed assessment of transgene-positive cell distribution in patients treated with SCGT.

Targeted delivery of fluorogenic peptide aptamers into live microalgae by femtosecond laser photoporation at single-cell resolution.

PubMed

Maeno, Takanori; Uzawa, Takanori; Kono, Izumi; Okano, Kazunori; Iino, Takanori; Fukita, Keisuke; Oshikawa, Yuki; Ogawa, Taro; Iwata, Osamu; Ito, Takuro; Suzuki, Kengo; Goda, Keisuke; Hosokawa, Yoichiroh

2018-05-29

Microalgae-based metabolic engineering has been proven effective for producing valuable substances such as food supplements, pharmaceutical drugs, biodegradable plastics, and biofuels in the past decade. The ability to accurately visualize and quantify intracellular metabolites in live microalgae is essential for efficient metabolic engineering, but remains a major challenge due to the lack of characterization methods. Here we demonstrate it by synthesizing fluorogenic peptide aptamers with specific binding affinity to a target metabolite and delivering them into live microalgae by femtosecond laser photoporation at single-cell resolution. As a proof-of-principle demonstration of our method, we use it to characterize Euglena gracilis, a photosynthetic unicellular motile microalgal species, which is capable of producing paramylon (a carbohydrate granule similar to starch). Specifically, we synthesize a peptide aptamer containing a paramylon-binding fluorescent probe, 7-nitrobenzofurazan, and introduce it into E. gracilis cells one-by-one by suppressing their mobility with mannitol and transiently perforating them with femtosecond laser pulses at 800 nm for photoporation. To demonstrate the method's practical utility in metabolic engineering, we perform spatially and temporally resolved fluorescence microscopy of single live photoporated E. gracilis cells under different culture conditions. Our method holds great promise for highly efficient microalgae-based metabolic engineering.

Selection of a novel CD19 aptamer for targeted delivery of doxorubicin to lymphoma cells.

PubMed

Hu, Yan; Li, Xiaoou; An, Yacong; Duan, Jinhong; Yang, Xian-Da

2018-06-01

CD19 is overexpressed in most human B cell malignancies and considered an important tumor marker for diagnosis and treatment. Aptamers are oligonucleotides that may potentially serve as tumor-homing ligand for targeted cancer therapy with excellent affinity and specificity. In this study, we selected a novel CD19 aptamer (LC1) that was a 59-nucleotide single strand DNA. The aptamer could bind to recombinant CD19 protein with a K d of 85.4 nM, and had minimal cross reactivity to bovine serum albumin (BSA) or ovalbumin (OVA). Moreover, the aptamer was found capable of binding with the CD19-positive lymphoma cells (Ramos and Raji), but not the CD19-negative cell lines (Jurkat and NB4). An aptamer-doxorubicin complex (Apt-Dox) was also formulated, and selectively delivered doxorubicin to CD19-positive lymphoma cells in vitro . The results indicate that aptamer LC1 can recognize CD19-positive tumor cells and may potentially function as a CD19-targeting ligand.

Targeting Notch signalling pathway of cancer stem cells.

PubMed

Venkatesh, Vandana; Nataraj, Raghu; Thangaraj, Gopenath S; Karthikeyan, Murugesan; Gnanasekaran, Ashok; Kaginelli, Shanmukhappa B; Kuppanna, Gobianand; Kallappa, Chandrashekrappa Gowdru; Basalingappa, Kanthesh M

2018-01-01

Cancer stem cells (CSCs) have been defined as cells within tumor that possess the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor. CSCs have been increasingly identified in blood cancer, prostate, ovarian, lung, melanoma, pancreatic, colon, brain and many more malignancies. CSCs have slow growth rate and are resistant to chemotherapy and radiotherapy that lead to the failure of traditional current therapy. Eradicating the CSCs and recurrence, is promising aspect for the cure of cancer. The CSCs like any other stem cells activate the signal transduction pathways that involve the development and tissue homeostasis, which include Notch signaling pathway. The new treatment targets these pathway that control stem-cell replication, survival and differentiation that are under development. Notch inhibitors either single or in combination with chemotherapy drugs have been developed to treat cancer and its recurrence. This approach of targeting signaling pathway of CSCs represents a promising future direction for the therapeutic strategy to cure cancer.

At the Bench: Chimeric antigen receptor (CAR) T cell therapy for the treatment of B cell malignancies.

PubMed

Daniyan, Anthony F O; Brentjens, Renier J

2016-12-01

The chimeric antigen receptor (CAR) represents the epitome of cellular engineering and is one of the best examples of rational biologic design of a synthetic molecule. The CAR is a single polypeptide with modular domains, consisting of an antibody-derived targeting moiety, fused in line with T cell-derived signaling domains, allowing for T cell activation upon ligand binding. T cells expressing a CAR are able to eradicate selectively antigen-expressing tumor cells in a MHC-independent fashion. CD19, a tumor-associated antigen (TAA) present on normal B cells, as well as most B cell-derived malignancies, was an early target of this technology. Through years of experimental refinement and preclinical optimization, autologously derived CD19-targeting CAR T cells have been successfully, clinically deployed, resulting in dramatic and durable antitumor responses but not without therapy-associated toxicity. As CD19-targeted CAR T cells continue to show clinical success, work at the bench continues to be undertaken to increase further the efficacy of this therapy, while simultaneously minimizing the risk for treatment-related morbidities. In this review, we cover the history and evolution of CAR technology and its adaptation to targeting CD19. Furthermore, we discuss the future of CAR T cell therapy and the need to ask, as well as answer, critical questions as this treatment modality is being translated to the clinic. © Society for Leukocyte Biology.

BRCAA1 antibody- and Her2 antibody-conjugated amphiphilic polymer engineered CdSe/ZnS quantum dots for targeted imaging of gastric cancer

NASA Astrophysics Data System (ADS)

Li, Chao; Ji, Yang; Wang, Can; Liang, Shujing; Pan, Fei; Zhang, Chunlei; Chen, Feng; Fu, Hualin; Wang, Kan; Cui, Daxiang

2014-05-01

Successful development of safe and highly effective nanoprobes for targeted imaging of in vivo early gastric cancer is a great challenge. Herein, we choose the CdSe/ZnS (core-shell) quantum dots (QDs) as prototypical materials, synthesized one kind of a new amphiphilic polymer including dentate-like alkyl chains and multiple carboxyl groups, and then used the prepared amphiphilic polymer to modify QDs. The resultant amphiphilic polymer engineered QDs (PQDs) were conjugated with BRCAA1 and Her2 monoclonal antibody, and prepared BRCAA1 antibody- and Her2 antibody-conjugated QDs were used for in vitro MGC803 cell labeling and in vivo targeted imaging of gastric cancer cells. Results showed that the PQDs exhibited good water solubility, strong photoluminescence (PL) intensity, and good biocompatibility. BRCAA1 antibody- and Her2 antibody-conjugated QD nanoprobes successfully realized targeted imaging of in vivo gastric cancer MGC803 cells. In conclusion, BRCAA1 antibody- and Her2 antibody-conjugated PQDs have great potential in applications such as single cell labeling and in vivo tracking, and targeted imaging and therapeutic effects' evaluation of in vivo early gastric cancer cells in the near future.

Response-dependent dynamics of cell-specific inhibition in cortical networks in vivo

PubMed Central

El-Boustani, Sami; Sur, Mriganka

2014-01-01

In the visual cortex, inhibitory neurons alter the computations performed by target cells via combination of two fundamental operations, division and subtraction. The origins of these operations have been variously ascribed to differences in neuron classes, synapse location or receptor conductances. Here, by utilizing specific visual stimuli and single optogenetic probe pulses, we show that the function of parvalbumin-expressing and somatostatin-expressing neurons in mice in vivo is governed by the overlap of response timing between these neurons and their targets. In particular, somatostatin-expressing neurons respond at longer latencies to small visual stimuli compared with their target neurons and provide subtractive inhibition. With large visual stimuli, however, they respond at short latencies coincident with their target cells and switch to provide divisive inhibition. These results indicate that inhibition mediated by these neurons is a dynamic property of cortical circuits rather than an immutable property of neuronal classes. PMID:25504329

Fluorescence- and magnetic-activated cell sorting strategies to separate spermatozoa involving plural contributors from biological mixtures for human identification

PubMed Central

Xu, Yan; Xie, Jianhui; Chen, Ronghua; Cao, Yu; Ping, Yuan; Xu, Qingwen; Hu, Wei; Wu, Dan; Gu, Lihua; Zhou, Huaigu; Chen, Xin; Zhao, Ziqin; Zhong, Jiang; Li, Rui

2016-01-01

No effective method has been developed to distinguish sperm cells originating from different men in multi-suspect sexual assault cases. Here we combined MACS and FACS to isolate single donor sperm cells from forensic mixture samples including female vaginal epithelial cells and sperm cells from multiple contributors. Sperms from vaginal swab were isolated by MACS using FITC-conjugated A kinase anchor protein 3 (AKAP3) antibody; target individual sperm cells involving two or three donors were separated by FACS using FITC-labeled blood group A/B antigen antibody. This procedure was further tested in two mock multi-suspect sexual assault samples and one practical casework sample. Our results showed that complete single donor STR profiles could be successfully obtained from sperm/epithelial cell and sperm mixtures from two contributors. For unbalanced sperm/epithelial cells and sperm cells mixtures, sensitivity results revealed that target cells could be detected at as low as 1:32 and 1:8 mixed ratios, respectively. Although highly relies on cell number and blood types or secretor status of the individuals, this procedure would still be useful tools for forensic DNA analysis of multi-suspect sexual assault cases by the combined use of FACS and MACS based on sperm-specific AKAP3 antigen and human blood type antigen. PMID:27857155

A chimeric antigen receptor for TRAIL-receptor 1 induces apoptosis in various types of tumor cells.

PubMed

Kobayashi, Eiji; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Hamana, Hiroshi; Nakagawa, Hidetoshi; Jin, Aishun; Lin, Zhezhu; Muraguchi, Atsushi

2014-10-31

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its associated receptors (TRAIL-R/TR) are attractive targets for cancer therapy because TRAIL induces apoptosis in tumor cells through TR while having little cytotoxicity on normal cells. Therefore, many agonistic monoclonal antibodies (mAbs) specific for TR have been produced, and these induce apoptosis in multiple tumor cell types. However, some TR-expressing tumor cells are resistant to TR-specific mAb-induced apoptosis. In this study, we constructed a chimeric antigen receptor (CAR) of a TRAIL-receptor 1 (TR1)-specific single chain variable fragment (scFv) antibody (TR1-scFv-CAR) and expressed it on a Jurkat T cell line, the KHYG-1 NK cell line, and human peripheral blood lymphocytes (PBLs). We found that the TR1-scFv-CAR-expressing Jurkat cells killed target cells via TR1-mediated apoptosis, whereas TR1-scFv-CAR-expressing KHYG-1 cells and PBLs killed target cells not only via TR1-mediated apoptosis but also via CAR signal-induced cytolysis, resulting in cytotoxicity on a broader range if target cells than with TR1-scFv-CAR-expressing Jurkat cells. The results suggest that TR1-scFv-CAR could be a new candidate for cancer gene therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

Targeted Vascular Drug Delivery in Cerebral Cancer.

PubMed

Humle, Nanna; Johnsen, Kasper Bendix; Arendt, Gitte Abildgaard; Nielsen, Rikke Paludan; Moos, Torben; Thomsen, Louiza Bohn

2016-01-01

This review presents the present-day literature on the anatomy and physiological mechanisms of the blood-brain barrier and the problematic of cerebral drug delivery in relation to malignant brain tumors. First step in treatment of malignant brain tumors is resection, but there is a high risk of single remnant infiltrative tumor cells in the outer zone of the brain tumor. These infiltrative single-cells will be supplied by capillaries with an intact BBB as opposed to the partly leaky BBB found in the tumor tissue before resection. Even though BBB penetrance of a chemotherapeutic agent is considered irrelevant though the limited success rate for chemotherapeutic treatability of GBM tumors indicate otherwise. Therefore drug delivery strategies to cerebral cancer after resection should be tailored to being able to both penetrate the intact BBB and target the cancer cells. In this review the intact bloodbrain barrier and cerebral cancer with main focus on glioblastoma multiforme (GBM) is introduced. The GBM induced formation of a blood-tumor barrier and the consequences hereof is described and discussed with emphasis on the impact these changes of the BBB has on drug delivery to GBM. The most commonly used drug carriers for drug delivery to GBM is described and the current drug delivery strategies for glioblastoma multiforme including possible routes through the BBB and epitopes, which can be targeted on the GBM cells is outlined. Overall, this review aims to address targeted drug delivery in GBM treatment when taking the differing permeability of the BBB into consideration.

Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells

PubMed Central

Rossotti, Martín; Tabares, Sofía; Alfaya, Lucía; Leizagoyen, Carmen; Moron, Gabriel; González-Sapienza, Gualberto

2015-01-01

BACKGROUND Owing to their minimal size, high production yield, versatility and robustness, the recombinant variable domain (nanobody) of camelid single chain antibodies are valued affinity reagents for research, diagnostic, and therapeutic applications. While their preparation against purified antigens is straightforward, the generation of nanobodies to difficult targets such as multi-pass or complex membrane cell receptors remains challenging. Here we devised a platform for high throughput identification of nanobodies to cell receptor based on the use of a biotin handle. METHODS Using a biotin-acceptor peptide tag, the in vivo biotinylation of nanobodies in 96 well culture blocks was optimized allowing their parallel analysis by flow cytometry and ELISA, and their direct used for pull-down/MS target identification. RESULTS The potential of this strategy was demonstrated by the selection and characterization of panels of nanobodies to Mac-1 (CD11b/CD18), MHC II and the mouse Ly-5 leukocyte common antigen (CD45) receptors, from a VHH library obtained from a llama immunized with mouse bone marrow derived dendritic cells. By on and off switching of the addition of biotin, the method also allowed the epitope binning of the selected Nbs directly on cells. CONCLUSIONS This strategy streamline the selection of potent nanobodies to complex antigens, and the selected nanobodies constitute ready-to-use biotinylated reagents. GENERAL SIGNIFICANCE This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets. PMID:25819371

Hapten-derivatized nanoparticle targeting and imaging of gene expression by multimodality imaging systems.

PubMed

Cheng, C-M; Chu, P-Y; Chuang, K-H; Roffler, S R; Kao, C-H; Tseng, W-L; Shiea, J; Chang, W-D; Su, Y-C; Chen, B-M; Wang, Y-M; Cheng, T-L

2009-01-01

Non-invasive gene monitoring is important for most gene therapy applications to ensure selective gene transfer to specific cells or tissues. We developed a non-invasive imaging system to assess the location and persistence of gene expression by anchoring an anti-dansyl (DNS) single-chain antibody (DNS receptor) on the cell surface to trap DNS-derivatized imaging probes. DNS hapten was covalently attached to cross-linked iron oxide (CLIO) to form a 39+/-0.5 nm DNS-CLIO nanoparticle imaging probe. DNS-CLIO specifically bound to DNS receptors but not to a control single-chain antibody receptor. DNS-CLIO (100 microM Fe) was non-toxic to both B16/DNS (DNS receptor positive) and B16/phOx (control receptor positive) cells. Magnetic resonance (MR) imaging could detect as few as 10% B16/DNS cells in a mixture in vitro. Importantly, DNS-CLIO specifically bound to a B16/DNS tumor, which markedly reduced signal intensity. Similar results were also shown with DNS quantum dots, which specifically targeted CT26/DNS cells but not control CT26/phOx cells both in vitro and in vivo. These results demonstrate that DNS nanoparticles can systemically monitor the expression of DNS receptor in vivo by feasible imaging systems. This targeting strategy may provide a valuable tool to estimate the efficacy and specificity of different gene delivery systems and optimize gene therapy protocols in the clinic.

Genetic modification of mesenchymal stem cells to express a single-chain antibody against EGFRvIII on the cell surface.

PubMed

Balyasnikova, Irina V; Franco-Gou, Rosa; Mathis, J Michael; Lesniak, Maciej S

2010-06-01

Human adult mesenchymal stem cells (hMSCs) are under active investigation as cellular carriers for gene therapy. hMSCs possess natural tropism toward tumours; however, the targeting of hMSCs to specific cell populations within tumours is unexplored. In the case of glioblastoma multiforme (GBM), at least half of the tumours express EGFRvIII on the cell surface, an ideal target for antibody-mediated gene/drug delivery. In this study, we investigated the feasibility of genetically modifying hMSCs to express a single-chain antibody (scFv) to EGFRvIII on their surfaces. Nucleofection was used to transfect hMSCs with cDNA encoding scFv EGFRvIII fused with PDGFR or human B7-1 transmembrane domains. The expression of scFv EGFRvIII on the cell surface was assessed by FACS. A stable population of scFv EGFRvIII-expressing hMSCs was selected, based on antibiotic resistance, and enriched using FACS. We found that nucleofection allows the efficient expression of scFv EGFRvIII on the cell surface of hMSCs. hMSCs transfected with the construct encoding scFv EGFRvIII as a fusion with PDGFRtm showed scFv EGFRvIII expression in up to 86% of cells. Most importantly, human MSCs expressing scFv against EGFRvIII demonstrated enhanced binding to U87-EGFRvIII cells in vitro and significantly increased retention in human U87-EGFRvIII-expressing tumours in vivo. In summary, we provide the first conclusive evidence of genetic modification of hMSCs with a single-chain antibody against an antigen expressed on the surface of tumour cells, thereby opening up a new venue for enhanced delivery of gene therapy applications in the context of malignant brain cancer. Copyright 2009 John Wiley & Sons, Ltd.

Targeted Multiplex Imaging Mass Spectrometry in Transmission Geometry for Subcellular Spatial Resolution

PubMed Central

Lavenant, Gwendoline Thiery; Zavalin, Andrey I.; Caprioli, Richard M.

2013-01-01

Targeted multiplex Imaging Mass Spectrometry utilizes several different antigen-specific primary antibodies, each directly labeled with a unique photocleavable mass tag, to detect multiple antigens in a single tissue section. Each photocleavable mass tag bound to an antibody has a unique molecular weight and can be readily ionized by laser desorption ionization mass spectrometry. This manuscript describes a mass spectrometry method that allows imaging of targeted single cells within tissue using transmission geometry laser desorption ionization mass spectrometry. Transmission geometry focuses the laser beam on the back side of the tissue placed on a glass slide, providing a 2 μm diameter laser spot irradiating the biological specimen. This matrix-free method enables simultaneous localization at the sub-cellular level of multiple antigens using specific tagged antibodies. We have used this technology to visualize the co-expression of synaptophysin and two major hormones peptides, insulin and somatostatin, in duplex assays in beta and delta cells contained in a human pancreatic islet. PMID:23397138

Targeted Multiplex Imaging Mass Spectrometry in Transmission Geometry for Subcellular Spatial Resolution

NASA Astrophysics Data System (ADS)

Thiery-Lavenant, Gwendoline; Zavalin, Andre I.; Caprioli, Richard M.

2013-04-01

Targeted multiplex imaging mass spectrometry utilizes several different antigen-specific primary antibodies, each directly labeled with a unique photocleavable mass tag, to detect multiple antigens in a single tissue section. Each photocleavable mass tag bound to an antibody has a unique molecular weight and can be readily ionized by laser desorption ionization mass spectrometry. This article describes a mass spectrometry method that allows imaging of targeted single cells within tissue using transmission geometry laser desorption ionization mass spectrometry. Transmission geometry focuses the laser beam on the back side of the tissue placed on a glass slide, providing a 2 μm diameter laser spot irradiating the biological specimen. This matrix-free method enables simultaneous localization at the sub-cellular level of multiple antigens using specific tagged antibodies. We have used this technology to visualize the co-expression of synaptophysin and two major hormones peptides, insulin and somatostatin, in duplex assays in beta and delta cells contained in a human pancreatic islet.

Cryptosporidium as a testbed for single cell genome characterization of unicellular eukaryotes.

PubMed

Troell, Karin; Hallström, Björn; Divne, Anna-Maria; Alsmark, Cecilia; Arrighi, Romanico; Huss, Mikael; Beser, Jessica; Bertilsson, Stefan

2016-06-23

Infectious disease involving multiple genetically distinct populations of pathogens is frequently concurrent, but difficult to detect or describe with current routine methodology. Cryptosporidium sp. is a widespread gastrointestinal protozoan of global significance in both animals and humans. It cannot be easily maintained in culture and infections of multiple strains have been reported. To explore the potential use of single cell genomics methodology for revealing genome-level variation in clinical samples from Cryptosporidium-infected hosts, we sorted individual oocysts for subsequent genome amplification and full-genome sequencing. Cells were identified with fluorescent antibodies with an 80 % success rate for the entire single cell genomics workflow, demonstrating that the methodology can be applied directly to purified fecal samples. Ten amplified genomes from sorted single cells were selected for genome sequencing and compared both to the original population and a reference genome in order to evaluate the accuracy and performance of the method. Single cell genome coverage was on average 81 % even with a moderate sequencing effort and by combining the 10 single cell genomes, the full genome was accounted for. By a comparison to the original sample, biological variation could be distinguished and separated from noise introduced in the amplification. As a proof of principle, we have demonstrated the power of applying single cell genomics to dissect infectious disease caused by closely related parasite species or subtypes. The workflow can easily be expanded and adapted to target other protozoans, and potential applications include mapping genome-encoded traits, virulence, pathogenicity, host specificity and resistance at the level of cells as truly meaningful biological units.

Quantitative analysis of circadian single cell oscillations in response to temperature

PubMed Central

Kramer, Achim; Herzel, Hanspeter

2018-01-01

Body temperature rhythms synchronize circadian oscillations in different tissues, depending on the degree of cellular coupling: the responsiveness to temperature is higher when single circadian oscillators are uncoupled. So far, the role of coupling in temperature responsiveness has only been studied in organotypic tissue slices of the central circadian pacemaker, because it has been assumed that peripheral target organs behave like uncoupled multicellular oscillators. Since recent studies indicate that some peripheral tissues may exhibit cellular coupling as well, we asked whether peripheral network dynamics also influence temperature responsiveness. Using a novel technique for long-term, high-resolution bioluminescence imaging of primary cultured cells, exposed to repeated temperature cycles, we were able to quantitatively measure period, phase, and amplitude of central (suprachiasmatic nuclei neuron dispersals) and peripheral (mouse ear fibroblasts) single cell oscillations in response to temperature. Employing temperature cycles of different lengths, and different cell densities, we found that some circadian characteristics appear cell-autonomous, e.g. period responses, while others seem to depend on the quality/degree of cellular communication, e.g. phase relationships, robustness of the oscillation, and amplitude. Overall, our findings indicate a strong dependence on the cell’s ability for intercellular communication, which is not only true for neuronal pacemakers, but, importantly, also for cells in peripheral tissues. Hence, they stress the importance of comparative studies that evaluate the degree of coupling in a given tissue, before it may be used effectively as a target for meaningful circadian manipulation. PMID:29293562

Phenotypic drug profiling in droplet microfluidics for better targeting of drug-resistant tumors.

PubMed

Sarkar, S; Cohen, N; Sabhachandani, P; Konry, T

2015-12-07

Acquired drug resistance is a key factor in the failure of chemotherapy. Due to intratumoral heterogeneity, cancer cells depict variations in intracellular drug uptake and efflux at the single cell level, which may not be detectable in bulk assays. In this study we present a droplet microfluidics-based approach to assess the dynamics of drug uptake, efflux and cytotoxicity in drug-sensitive and drug-resistant breast cancer cells. An integrated droplet generation and docking microarray was utilized to encapsulate single cells as well as homotypic cell aggregates. Drug-sensitive cells showed greater death in the presence or absence of Doxorubicin (Dox) compared to the drug-resistant cells. We observed heterogeneous Dox uptake in individual drug-sensitive cells while the drug-resistant cells showed uniformly low uptake and retention. Dox-resistant cells were classified into distinct subsets based on their efflux properties. Cells that showed longer retention of extracellular reagents also demonstrated maximal death. We further observed homotypic fusion of both cell types in droplets, which resulted in increased cell survival in the presence of high doses of Dox. Our results establish the applicability of this microfluidic platform for quantitative drug screening in single cells and multicellular interactions.

Greater than the sum of its parts: single-nucleus sequencing identifies convergent evolution of independent EGFR mutants in GBM.

PubMed

Gini, Beatrice; Mischel, Paul S

2014-08-01

Single-cell sequencing approaches are needed to characterize the genomic diversity of complex tumors, shedding light on their evolutionary paths and potentially suggesting more effective therapies. In this issue of Cancer Discovery, Francis and colleagues develop a novel integrative approach to identify distinct tumor subpopulations based on joint detection of clonal and subclonal events from bulk tumor and single-nucleus whole-genome sequencing, allowing them to infer a subclonal architecture. Surprisingly, the authors identify convergent evolution of multiple, mutually exclusive, independent EGFR gain-of-function variants in a single tumor. This study demonstrates the value of integrative single-cell genomics and highlights the biologic primacy of EGFR as an actionable target in glioblastoma. ©2014 American Association for Cancer Research.

Single-cell codetection of metabolic activity, intracellular functional proteins, and genetic mutations from rare circulating tumor cells.

PubMed

Zhang, Yu; Tang, Yin; Sun, Shuai; Wang, Zhihua; Wu, Wenjun; Zhao, Xiaodong; Czajkowsky, Daniel M; Li, Yan; Tian, Jianhui; Xu, Ling; Wei, Wei; Deng, Yuliang; Shi, Qihui

2015-10-06

The high glucose uptake and activation of oncogenic signaling pathways in cancer cells has long made these features, together with the mutational spectrum, prime diagnostic targets of circulating tumor cells (CTCs). Further, an ability to characterize these properties at a single cell resolution is widely believed to be essential, as the known extensive heterogeneity in CTCs can obscure important correlations in data obtained from cell population-based methods. However, to date, it has not been possible to quantitatively measure metabolic, proteomic, and genetic data from a single CTC. Here we report a microchip-based approach that allows for the codetection of glucose uptake, intracellular functional proteins, and genetic mutations at the single-cell level from rare tumor cells. The microchip contains thousands of nanoliter grooves (nanowells) that isolate individual CTCs and allow for the assessment of their glucose uptake via imaging of a fluorescent glucose analog, quantification of a panel of intracellular signaling proteins using a miniaturized antibody barcode microarray, and retrieval of the individual cell nuclei for subsequent off-chip genome amplification and sequencing. This approach integrates molecular-scale information on the metabolic, proteomic, and genetic status of single cells and permits the inference of associations between genetic signatures, energy consumption, and phosphoproteins oncogenic signaling activities in CTCs isolated from blood samples of patients. Importantly, this microchip chip-based approach achieves this multidimensional molecular analysis with minimal cell loss (<20%), which is the bottleneck of the rare cell analysis.

A novel albumin nanocomplex containing both small interfering RNA and gold nanorods for synergetic anticancer therapy

NASA Astrophysics Data System (ADS)

Choi, Jin-Ha; Hwang, Hai-Jin; Shin, Seung Won; Choi, Jeong-Woo; Um, Soong Ho; Oh, Byung-Keun

2015-05-01

Therapeutic nanocomplexes have been extensively developed for the effective treatment of aggressive cancers because of their outstanding versatility, easy manipulation, and low cytotoxicity. In this study, we describe the synthesis of a novel bovine serum albumin (BSA)-based nanocomplex harboring both Bcl-2-specific small interfering RNA (siRNA) and gold (Au) nanorods (siRNA and rods encapsulated in BSA; SREB) with the aim of developing a targeted breast cancer therapeutic. The SREB complexes contained 2 × 105 siRNA molecules and eight Au nanorods per BSA complex and were successively functionalized with polyethylene glycol (PEG) and anti-ErbB-2 antibodies to facilitate active targeting. The synergetic therapeutic activity originating from the two components effectively induced cell death (~80% reduction in viability compared with control cells) in target breast cancer cells after a single dose of laser irradiation. Intracellular SREB nanocomplex decomposition by proteolytic enzymes resulted in simultaneous RNA interference and thermal ablation, thus leading to apoptosis in the targeted cancer cells. Moreover, these therapeutic effects were sustained for approximately 72 hours. The intrinsic biocompatibility, multifunctionality, and potent in vitro anticancer properties of these SREB nanocomplexes indicate that they have great therapeutic potential for in vivo targeted cancer therapy, in addition to other areas of nanomedicine.Therapeutic nanocomplexes have been extensively developed for the effective treatment of aggressive cancers because of their outstanding versatility, easy manipulation, and low cytotoxicity. In this study, we describe the synthesis of a novel bovine serum albumin (BSA)-based nanocomplex harboring both Bcl-2-specific small interfering RNA (siRNA) and gold (Au) nanorods (siRNA and rods encapsulated in BSA; SREB) with the aim of developing a targeted breast cancer therapeutic. The SREB complexes contained 2 × 105 siRNA molecules and eight Au nanorods per BSA complex and were successively functionalized with polyethylene glycol (PEG) and anti-ErbB-2 antibodies to facilitate active targeting. The synergetic therapeutic activity originating from the two components effectively induced cell death (~80% reduction in viability compared with control cells) in target breast cancer cells after a single dose of laser irradiation. Intracellular SREB nanocomplex decomposition by proteolytic enzymes resulted in simultaneous RNA interference and thermal ablation, thus leading to apoptosis in the targeted cancer cells. Moreover, these therapeutic effects were sustained for approximately 72 hours. The intrinsic biocompatibility, multifunctionality, and potent in vitro anticancer properties of these SREB nanocomplexes indicate that they have great therapeutic potential for in vivo targeted cancer therapy, in addition to other areas of nanomedicine. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr00211g

IT-25DEVELOPMENTALLY REGULATED ANTIGENS FOR IMMUNOLOGIC TARGETING OF MEDULLOBLASTOMA SUBTYPES

PubMed Central

Pham, Christina; Flores, Catherine; Pei, Yanxin; Wechsler-Reya, Robert; Mitchell, Duane

2014-01-01

INTRODUCTION: Medulloblastoma (MB) remains incurable in one third of patients despite aggressive multi-modality standard therapies. Immunotherapy presents a promising alternative by specifically targeting cancer cells. To date, there have been no successful immunologic applications targeting MB. Emerging evidence from integrated genomic studies has suggested MB variants arise from deregulation of pathways affecting proliferation of progenitor cell populations within the developing cerebellum. Using total embryonic RNA as a source of tumor rejection antigens is attractive because it can be delivered as a single vaccine, target both known and unknown fetal proteins, and can be refined to preferentially treat distinct MB subtypes. METHODS: We have created two transplantable, syngeneic animal MB models recapitulating human SHH and Group 3 variants to investigate the immunologic targeting of different MB subtypes. We generated T cells specific to the developing mouse cerebellum (P5) and tested their reactivity to target cells pulsed with total RNA from two MB subtypes and the normal brain. Immune responses were evaluated by measuring cytokine secretion following re-stimulation of activated T cells with both normal and tumor cell targets. In vivo antitumor efficacy was also tested in survival studies of intracranial tumor-bearing animals. RESULTS: We generated T cells specific to the developing cerebellum in vitro, confirming the immunogenicity of developmentally regulated antigens. Additionally, we have shown that developmental antigen-specific T cells produce high levels of Th1-type cytokines in response to tumor cells of two immunologically distinct subtypes of MB. Interestingly, developmental antigen specific T cells do not show cross reactivity with the normal brain or subsequent stages of the developing brain after P5. Targeting developmental antigens also conferred a significant survival benefit in a treatment model of Group 3 tumor bearing animals. CONCLUSIONS: Developmental antigens can safely target multiple MB subtypes with equal effectiveness compared to previously established total tumor strategies.

Targeted sorting of single virus-infected cells of the coccolithophore Emiliania huxleyi.

PubMed

Martínez Martínez, Joaquín; Poulton, Nicole J; Stepanauskas, Ramunas; Sieracki, Michael E; Wilson, William H

2011-01-01

Discriminating infected from healthy cells is the first step to understanding the mechanisms and ecological implications of viral infection. We have developed a method for detecting, sorting, and performing molecular analysis of individual, infected cells of the important microalga Emiliania huxleyi, based on known physiological responses to viral infection. Of three fluorescent dyes tested, FM 1-43 (for detecting membrane blebbing) gave the most unequivocal and earliest separation of cells. Furthermore, we were able to amplify the genomes of single infected cells using Multiple Displacement Amplification. This novel method to reliably discriminate infected from healthy cells in cultures will allow researchers to answer numerous questions regarding the mechanisms and implications of viral infection of E. huxleyi. The method may be transferable to other virus-host systems.

Diffusion dynamics of the Keap1–Cullin3 interaction in single live cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baird, Liam; Dinkova-Kostova, Albena T., E-mail: a.dinkovakostova@dundee.ac.uk; Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD

2013-03-29

Highlights: ► We developed a quantitative FRAP-based system to study the Keap1–Cul3 interaction. ► We show that Keap1–EGFP and mCherry–Cul3 interact in single live cells. ► We used inducers which target distinct cysteine sensors of Keap1 and differ 4000-fold in potency. ► Inducers cause Nrf2 stabilization, nuclear translocation, and target gene expression. ► Inducers of four different types do not dissociate the Keap1–EGFP:mCherry–Cul3 complex. -- Abstract: Transcription factor NF-E2 p45-related factor 2 (Nrf2) regulates the expression of a network of genes encoding drug-detoxification, anti-inflammatory, and metabolic enzymes, as well as proteins involved in the regulation of cellular redox homeostasis. Undermore » basal conditions, Kelch-like ECH associated protein 1 (Keap1) targets Nrf2 for ubiquitination and proteasomal degradation via association with Cullin3 (Cul3)-based Rbx1 E3 ubiquitin ligase. Various small molecules (inducers) activate Nrf2 leading to upregulation of cytoprotective gene expression. Inducers chemically modify specific cysteine residues of Keap1 which ultimately loses its ability to target Nrf2 for degradation. Dissociation of the Keap1–Cul3 complex by inducers is one possible mechanism, but evidence in single live cells is lacking. To investigate the diffusion dynamics of the Keap1–Cul3 interaction and the effect of inducers, we developed a quantitative fluorescence recovery after photobleaching (FRAP)-based system using Keap1–EGFP and mCherry–Cul3 fusion proteins. We show that Keap1–EGFP and mCherry–Cul3 interact in single live cells. Exposure for 1 h to small-molecule inducers of 4 different types, the oleanane triterpenoid CDDO, the isothiocyanate sulforaphane, the sulfoxythiocarbamate STCA, and the oxidant hydrogen peroxide which target distinct cysteine sensors within Keap1 with potencies which differ by nearly 4000-fold, does not dissociate the Keap1–Cul3 complex. As inducers cause conformational changes in Keap1, we conclude that changes in conformation rather than dissociation from Cul3 inactivate the repressor function of Keap1 leading to Nrf2 stabilization.« less

Targeted reconstruction of T cell receptor sequence from single cell RNA-seq links CDR3 length to T cell differentiation state

PubMed Central

Yates, Kathleen B.; Bi, Kevin; Darko, Samuel; Godec, Jernej; Gerdemann, Ulrike; Swadling, Leo; Douek, Daniel C.; Klenerman, Paul; Barnes, Eleanor J.; Sharpe, Arlene H.

2017-01-01

Abstract The T cell compartment must contain diversity in both T cell receptor (TCR) repertoire and cell state to provide effective immunity against pathogens. However, it remains unclear how differences in the TCR contribute to heterogeneity in T cell state. Single cell RNA-sequencing (scRNA-seq) can allow simultaneous measurement of TCR sequence and global transcriptional profile from single cells. However, current methods for TCR inference from scRNA-seq are limited in their sensitivity and require long sequencing reads, thus increasing the cost and decreasing the number of cells that can be feasibly analyzed. Here we present TRAPeS, a publicly available tool that can efficiently extract TCR sequence information from short-read scRNA-seq libraries. We apply it to investigate heterogeneity in the CD8+ T cell response in humans and mice, and show that it is accurate and more sensitive than existing approaches. Coupling TRAPeS with transcriptome analysis of CD8+ T cells specific for a single epitope from Yellow Fever Virus (YFV), we show that the recently described ‘naive-like’ memory population have significantly longer CDR3 regions and greater divergence from germline sequence than do effector-memory phenotype cells. This suggests that TCR usage is associated with the differentiation state of the CD8+ T cell response to YFV. PMID:28934479

Zinc-finger protein-targeted gene regulation: Genomewide single-gene specificity

PubMed Central

Tan, Siyuan; Guschin, Dmitry; Davalos, Albert; Lee, Ya-Li; Snowden, Andrew W.; Jouvenot, Yann; Zhang, H. Steven; Howes, Katherine; McNamara, Andrew R.; Lai, Albert; Ullman, Chris; Reynolds, Lindsey; Moore, Michael; Isalan, Mark; Berg, Lutz-Peter; Campos, Bradley; Qi, Hong; Spratt, S. Kaye; Case, Casey C.; Pabo, Carl O.; Campisi, Judith; Gregory, Philip D.

2003-01-01

Zinc-finger protein transcription factors (ZFP TFs) can be designed to control the expression of any desired target gene, and thus provide potential therapeutic tools for the study and treatment of disease. Here we report that a ZFP TF can repress target gene expression with single-gene specificity within the human genome. A ZFP TF repressor that binds an 18-bp recognition sequence within the promoter of the endogenous CHK2 gene gives a >10-fold reduction in CHK2 mRNA and protein. This level of repression was sufficient to generate a functional phenotype, as demonstrated by the loss of DNA damage-induced CHK2-dependent p53 phosphorylation. We determined the specificity of repression by using DNA microarrays and found that the ZFP TF repressed a single gene (CHK2) within the monitored genome in two different cell types. These data demonstrate the utility of ZFP TFs as precise tools for target validation, and highlight their potential as clinical therapeutics. PMID:14514889

Recombinant protein production from stable mammalian cell lines and pools.

PubMed

Hacker, David L; Balasubramanian, Sowmya

2016-06-01

We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. Copyright © 2016 Elsevier Ltd. All rights reserved.

Classes and continua of hippocampal CA1 inhibitory neurons revealed by single-cell transcriptomics.

PubMed

Harris, Kenneth D; Hochgerner, Hannah; Skene, Nathan G; Magno, Lorenza; Katona, Linda; Bengtsson Gonzales, Carolina; Somogyi, Peter; Kessaris, Nicoletta; Linnarsson, Sten; Hjerling-Leffler, Jens

2018-06-18

Understanding any brain circuit will require a categorization of its constituent neurons. In hippocampal area CA1, at least 23 classes of GABAergic neuron have been proposed to date. However, this list may be incomplete; additionally, it is unclear whether discrete classes are sufficient to describe the diversity of cortical inhibitory neurons or whether continuous modes of variability are also required. We studied the transcriptomes of 3,663 CA1 inhibitory cells, revealing 10 major GABAergic groups that divided into 49 fine-scale clusters. All previously described and several novel cell classes were identified, with three previously described classes unexpectedly found to be identical. A division into discrete classes, however, was not sufficient to describe the diversity of these cells, as continuous variation also occurred between and within classes. Latent factor analysis revealed that a single continuous variable could predict the expression levels of several genes, which correlated similarly with it across multiple cell types. Analysis of the genes correlating with this variable suggested it reflects a range from metabolically highly active faster-spiking cells that proximally target pyramidal cells to slower-spiking cells targeting distal dendrites or interneurons. These results elucidate the complexity of inhibitory neurons in one of the simplest cortical structures and show that characterizing these cells requires continuous modes of variation as well as discrete cell classes.

A novel anti-alpha-fetoprotein single-chain variable fragment displays anti-tumor effects in HepG2 cells as a single agent or in combination with paclitaxel.

PubMed

Ji, Xiaonan; Shen, Yanli; Sun, Hao; Gao, Xiangdong

2016-08-01

Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival time. The function of alpha-fetoprotein (AFP) as a regulatory factor in the growth of HCC cells has been well defined. The aim of this study was to investigate the use of a novel AFP-specific single-chain variable fragment that blocked AFP and inhibited HCC cell growth. The results indicated that the anti-AFP single-chain variable fragment (scFv) induced growth inhibition of AFP-expressing HCC cell lines in vitro through induction of G1 cell cycle arrest and apoptosis. The mechanism of apoptosis probably involved with blocking AFP internalization and regulation of the PTEN/PI3K/Akt signaling network. Moreover, the anti-AFP-scFv also effectively sensitized the HepG2 cells to paclitaxel (PTX) at a lower concentration. The combination effect of PTX and anti-AFP-scFv displayed a synergistic effect on HepG2 cells both in vitro and in vivo. Our results demonstrated that targeting AFP by specific antibodies has potential immunotherapeutic efficacy in human HCC.

Self-Assembly of Gold Nanoparticles Shows Microenvironment-Mediated Dynamic Switching and Enhanced Brain Tumor Targeting

PubMed Central

Feng, Qishuai; Shen, Yajing; Fu, Yingjie; Muroski, Megan E.; Zhang, Peng; Wang, Qiaoyue; Xu, Chang; Lesniak, Maciej S.; Li, Gang; Cheng, Yu

2017-01-01

Inorganic nanoparticles with unique physical properties have been explored as nanomedicines for brain tumor treatment. However, the clinical applications of the inorganic formulations are often hindered by the biological barriers and failure to be bioeliminated. The size of the nanoparticle is an essential design parameter which plays a significant role to affect the tumor targeting and biodistribution. Here, we report a feasible approach for the assembly of gold nanoparticles into ~80 nm nanospheres as a drug delivery platform for enhanced retention in brain tumors with the ability to be dynamically switched into the single formulation for excretion. These nanoassemblies can target epidermal growth factor receptors on cancer cells and are responsive to tumor microenvironmental characteristics, including high vascular permeability and acidic and redox conditions. Anticancer drug release was controlled by a pH-responsive mechanism. Intracellular L-glutathione (GSH) triggered the complete breakdown of nanoassemblies to single gold nanoparticles. Furthermore, in vivo studies have shown that nanospheres display enhanced tumor-targeting efficiency and therapeutic effects relative to single-nanoparticle formulations. Hence, gold nanoassemblies present an effective targeting strategy for brain tumor treatment. PMID:28638474

Self-Assembly of Gold Nanoparticles Shows Microenvironment-Mediated Dynamic Switching and Enhanced Brain Tumor Targeting.

PubMed

Feng, Qishuai; Shen, Yajing; Fu, Yingjie; Muroski, Megan E; Zhang, Peng; Wang, Qiaoyue; Xu, Chang; Lesniak, Maciej S; Li, Gang; Cheng, Yu

2017-01-01

Inorganic nanoparticles with unique physical properties have been explored as nanomedicines for brain tumor treatment. However, the clinical applications of the inorganic formulations are often hindered by the biological barriers and failure to be bioeliminated. The size of the nanoparticle is an essential design parameter which plays a significant role to affect the tumor targeting and biodistribution. Here, we report a feasible approach for the assembly of gold nanoparticles into ~80 nm nanospheres as a drug delivery platform for enhanced retention in brain tumors with the ability to be dynamically switched into the single formulation for excretion. These nanoassemblies can target epidermal growth factor receptors on cancer cells and are responsive to tumor microenvironmental characteristics, including high vascular permeability and acidic and redox conditions. Anticancer drug release was controlled by a pH-responsive mechanism. Intracellular L-glutathione (GSH) triggered the complete breakdown of nanoassemblies to single gold nanoparticles. Furthermore, in vivo studies have shown that nanospheres display enhanced tumor-targeting efficiency and therapeutic effects relative to single-nanoparticle formulations. Hence, gold nanoassemblies present an effective targeting strategy for brain tumor treatment.

EphB4 as a therapeutic target in mesothelioma

PubMed Central

2013-01-01

Background Malignant pleural mesothelioma (MPM) often develops decades following exposure to asbestos. Current best therapy produces a response in only half of patients, and the median survival with this therapy remains under a year. A search for novel targets and therapeutics is underway, and recently identified targets include VEGF, Notch, and EphB4-Ephrin-B2. Each of these targets has dual activity, promoting tumor cell growth as well as tumor angiogenesis. Methods We investigated EphB4 expression in 39 human mesothelioma tissues by immunohistochemistry. Xenograft tumors established with human mesothelioma cells were treated with an EphB4 inhibitor (monomeric soluble EphB4 fused to human serum albumin, or sEphB4-HSA). The combinatorial effect of sEphB4-HSA and biologic agent was also studied. Results EphB4 was overexpressed in 72% of mesothelioma tissues evaluated, with 85% of epithelioid and 38% of sarcomatoid subtypes demonstrating overexpression. The EphB4 inhibitor sEphB4-HSA was highly active as a single agent to inhibit tumor growth, accompanied by tumor cell apoptosis and inhibition of PI3K and Src signaling. Combination of sEphB4-HSA and the anti-VEGF antibody (Bevacizumab) was superior to each agent alone and led to complete tumor regression. Conclusion EphB4 is a potential therapeutic target in mesothelioma. Clinical investigation of sEphB4-HSA as a single agent and in combination with VEGF inhibitors is warranted. PMID:23721559

Phenformin enhances the therapeutic benefit of BRAFV600E inhibition in melanoma

PubMed Central

Yuan, Ping; Ito, Koichi; Perez-Lorenzo, Rolando; Del Guzzo, Christina; Lee, Jung Hyun; Shen, Che-Hung; Bosenberg, Marcus W.; McMahon, Martin; Cantley, Lewis C.; Zheng, Bin

2013-01-01

Biguanides, such as the diabetes therapeutics metformin and phenformin, have demonstrated antitumor activity both in vitro and in vivo. The energy-sensing AMP-activated protein kinase (AMPK) is known to be a major cellular target of biguanides. Based on our discovery of cross-talk between the AMPK and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) signaling pathways, we investigated the antitumor effects of combining phenformin with a BRAF inhibitor PLX4720 on the proliferation of BRAF-mutated melanoma cells in vitro and on BRAF-driven tumor growth in vivo. Cotreatment of BRAF-mutated melanoma cell lines with phenformin and PLX4720 resulted in synergistic inhibition of cell viability, compared with the effects of the single agent alone. Moreover, treatment with phenformin significantly delayed the development of resistance to PLX4720 in cultured melanoma cells. Biochemical analyses showed that phenformin and PLX4720 exerted cooperative effects on inhibiting mTOR signaling and inducing apoptosis. Noticeably, phenformin selectively targeted subpopulations of cells expressing JARID1B, a marker for slow cycling melanoma cells, whereas PLX4720 selectively targeted JARID1B-negative cells. Finally, in contrast to their use as single agents, the combination of phenformin and PLX4720 induced tumor regression in both nude mice bearing melanoma xenografts and in a genetically engineered BRAFV600E/PTENnull-driven mouse model of melanoma. These results strongly suggest that significant therapeutic advantage may be achieved by combining AMPK activators such as phenformin with BRAF inhbitors for the treatment of melanoma. PMID:24145418

Phenformin enhances the therapeutic benefit of BRAF(V600E) inhibition in melanoma.

PubMed

Yuan, Ping; Ito, Koichi; Perez-Lorenzo, Rolando; Del Guzzo, Christina; Lee, Jung Hyun; Shen, Che-Hung; Bosenberg, Marcus W; McMahon, Martin; Cantley, Lewis C; Zheng, Bin

2013-11-05

Biguanides, such as the diabetes therapeutics metformin and phenformin, have demonstrated antitumor activity both in vitro and in vivo. The energy-sensing AMP-activated protein kinase (AMPK) is known to be a major cellular target of biguanides. Based on our discovery of cross-talk between the AMPK and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) signaling pathways, we investigated the antitumor effects of combining phenformin with a BRAF inhibitor PLX4720 on the proliferation of BRAF-mutated melanoma cells in vitro and on BRAF-driven tumor growth in vivo. Cotreatment of BRAF-mutated melanoma cell lines with phenformin and PLX4720 resulted in synergistic inhibition of cell viability, compared with the effects of the single agent alone. Moreover, treatment with phenformin significantly delayed the development of resistance to PLX4720 in cultured melanoma cells. Biochemical analyses showed that phenformin and PLX4720 exerted cooperative effects on inhibiting mTOR signaling and inducing apoptosis. Noticeably, phenformin selectively targeted subpopulations of cells expressing JARID1B, a marker for slow cycling melanoma cells, whereas PLX4720 selectively targeted JARID1B-negative cells. Finally, in contrast to their use as single agents, the combination of phenformin and PLX4720 induced tumor regression in both nude mice bearing melanoma xenografts and in a genetically engineered BRAF(V600E)/PTEN(null)-driven mouse model of melanoma. These results strongly suggest that significant therapeutic advantage may be achieved by combining AMPK activators such as phenformin with BRAF inhbitors for the treatment of melanoma.

Combined MUC1-specific nanobody-tagged PEG-polyethylenimine polyplex targeting and transcriptional targeting of tBid transgene for directed killing of MUC1 over-expressing tumour cells.

PubMed

Sadeqzadeh, Elham; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud; Rasaee, Mohammad J; Parhamifar, Ladan; Moghimi, S Moein

2011-11-30

We provide evidence for combining a single domain antibody (nanobody)-based targeting approach with transcriptional targeting as a safe way to deliver lethal transgenes to MUC1 over-expressing cancer cells. From a nanobody immune library, we have isolated an anti-DF3/Mucin1 (MUC1) nanobody with high specificity for the MUC1 antigen, which is an aberrantly glycosylated glycoprotein over-expressed in tumours of epithelial origin. The anti-MUC1 nanobody was covalently linked to the distal end of poly(ethylene glycol)(3500) (PEG(3500)) in PEG(3500)-25kDa polyethylenimine (PEI) conjugates and the resultant macromolecular entity successfully condensed plasmids coding a transcriptionally targeted truncated-Bid (tBid) killer gene under the control of the cancer-specific MUC1 promoter. The engineered polyplexes exhibited favourable physicochemical characteristics for transfection and dramatically elevated the level of Bid/tBid expression in both MUC1 over-expressing caspase 3-deficient (MCF7 cells) and caspase 3-positive (T47D and SKBR3) tumour cell lines and, concomitantly, induced considerable cell death. Neither transgene expression nor cell death occurred when the MUC1 promoter was replaced with the CNS-specific synapsin I promoter. Since PEGylated PEI was only responsible for DNA compaction and played no significant role in direct transfection and cell killing, our attempts overcome previously reported PEI-mediated apoptotic and necrotic cell death, which is advantageous for future in vivo transcriptional targeting as this will minimize (or eliminate) non-targeted cell damage. Copyright © 2011 Elsevier B.V. All rights reserved.

Dynamic Single-Use Bioreactors Used in Modern Liter- and m(3)- Scale Biotechnological Processes: Engineering Characteristics and Scaling Up.

PubMed

Löffelholz, Christian; Kaiser, Stephan C; Kraume, Matthias; Eibl, Regine; Eibl, Dieter

2014-01-01

During the past 10 years, single-use bioreactors have been well accepted in modern biopharmaceutical production processes targeting high-value products. Up to now, such processes have mainly been small- or medium-scale mammalian cell culture-based seed inoculum, vaccine or antibody productions. However, recently first attempts have been made to modify existing single-use bioreactors for the cultivation of plant cells and tissue cultures, and microorganisms. This has even led to the development of new single-use bioreactor types. Moreover, due to safety issues it has become clear that single-use bioreactors are the "must have" for expanding human stem cells delivering cell therapeutics, the biopharmaceuticals of the next generation. So it comes as no surprise that numerous different dynamic single-use bioreactor types, which are suitable for a wide range of applications, already dominate the market today. Bioreactor working principles, main applications, and bioengineering data are presented in this review, based on a current overview of greater than milliliter-scale, commercially available, dynamic single-use bioreactors. The focus is on stirred versions, which are omnipresent in R&D and manufacturing, and in particular Sartorius Stedim's BIOSTAT family. Finally, we examine development trends for single-use bioreactors, after discussing proven approaches for fast scaling-up processes.

DNA strand-displacement-induced fluorescence enhancement for highly sensitive and selective assay of multiple microRNA in cancer cells.

PubMed

Wu, Ping; Tu, Yunqiu; Qian, Yingdan; Zhang, Hui; Cai, Chenxin

2014-01-28

We report a new strategy for evaluating multiple miRNA expressions in cancer cells based on DNA strand-displacement-induced fluorescence enhancement. This assay has the ability to discriminate the target from even single-base mismatched sequences or other miRNAs.

Re-programming tumour cell metabolism to treat cancer: no lone target for lonidamine.

PubMed

Bhutia, Yangzom D; Babu, Ellappan; Ganapathy, Vadivel

2016-06-01

Tumour cell metabolism is very different from normal cell metabolism; cancer cells re-programme the metabolic pathways that occur in normal cells in such a manner that it optimizes their proliferation, growth and survival. Although this metabolic re-programming obviously operates to the advantage of the tumour, it also offers unique opportunities for effective cancer therapy. Molecules that target the tumour cell-specific metabolic pathways have potential as novel anti-cancer drugs. Lonidamine belongs to this group of molecules and is already in use in some countries for cancer treatment. It has been known for a long time that lonidamine interferes with energy production in tumour cells by inhibiting hexokinase II (HKII), a glycolytic enzyme. However, subsequent studies have uncovered additional pharmacological targets for the drug, which include the electron transport chain and the mitochondrial permeability transition pore, thus expanding the pharmacological effects of the drug on tumour cell metabolism. A study by Nancolas et al. in a recent issue of the Biochemical Journal identifies two additional new targets for lonidamine: the pyruvate transporter in the mitochondria and the H(+)-coupled monocarboxylate transporters in the plasma membrane (PM). It is thus becoming increasingly apparent that the anti-cancer effects of lonidamine do not occur through a single target; the drug works at multiple sites. Irrespective of the molecular targets, what lonidamine does in the end is to undo what the tumour cells have done in terms of re-programming cellular metabolism and mitochondrial function. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

High-performance single cell genetic analysis using microfluidic emulsion generator arrays.

PubMed

Zeng, Yong; Novak, Richard; Shuga, Joe; Smith, Martyn T; Mathies, Richard A

2010-04-15

High-throughput genetic and phenotypic analysis at the single cell level is critical to advance our understanding of the molecular mechanisms underlying cellular function and dysfunction. Here we describe a high-performance single cell genetic analysis (SCGA) technique that combines high-throughput microfluidic emulsion generation with single cell multiplex polymerase chain reaction (PCR). Microfabricated emulsion generator array (MEGA) devices containing 4, 32, and 96 channels are developed to confer a flexible capability of generating up to 3.4 x 10(6) nanoliter-volume droplets per hour. Hybrid glass-polydimethylsiloxane diaphragm micropumps integrated into the MEGA chips afford uniform droplet formation, controlled generation frequency, and effective transportation and encapsulation of primer functionalized microbeads and cells. A multiplex single cell PCR method is developed to detect and quantify both wild type and mutant/pathogenic cells. In this method, microbeads functionalized with multiple forward primers targeting specific genes from different cell types are used for solid-phase PCR in droplets. Following PCR, the droplets are lysed and the beads are pooled and rapidly analyzed by multicolor flow cytometry. Using Escherichia coli bacterial cells as a model, we show that this technique enables digital detection of pathogenic E. coli O157 cells in a high background of normal K12 cells, with a detection limit on the order of 1/10(5). This result demonstrates that multiplex SCGA is a promising tool for high-throughput quantitative digital analysis of genetic variation in complex populations.

High-Performance Single Cell Genetic Analysis Using Microfluidic Emulsion Generator Arrays

PubMed Central

Zeng, Yong; Novak, Richard; Shuga, Joe; Smith, Martyn T.; Mathies, Richard A.

2010-01-01

High-throughput genetic and phenotypic analysis at the single cell level is critical to advance our understanding of the molecular mechanisms underlying cellular function and dysfunction. Here we describe a high-performance single cell genetic analysis (SCGA) technique that combines high-throughput microfluidic emulsion generation with single cell multiplex PCR. Microfabricated emulsion generator array (MEGA) devices containing 4, 32 and 96 channels are developed to confer a flexible capability of generating up to 3.4 × 106 nanoliter-volume droplets per hour. Hybrid glass-polydimethylsiloxane diaphragm micropumps integrated into the MEGA chips afford uniform droplet formation, controlled generation frequency, and effective transportation and encapsulation of primer functionalized microbeads and cells. A multiplex single cell PCR method is developed to detect and quantify both wild type and mutant/pathogenic cells. In this method, microbeads functionalized with multiple forward primers targeting specific genes from different cell types are used for solid-phase PCR in droplets. Following PCR, the droplets are lysed, the beads are pooled and rapidly analyzed by multi-color flow cytometry. Using E. coli bacterial cells as a model, we show that this technique enables digital detection of pathogenic E. coli O157 cells in a high background of normal K12 cells, with a detection limit on the order of 1:105. This result demonstrates that multiplex SCGA is a promising tool for high-throughput quantitative digital analysis of genetic variation in complex populations. PMID:20192178

Toxicity of an α-Pore-forming Toxin Depends on the Assembly Mechanism on the Target Membrane as Revealed by Single Molecule Imaging*

PubMed Central

Subburaj, Yamunadevi; Ros, Uris; Hermann, Eduard; Tong, Rudi; García-Sáez, Ana J.

2015-01-01

α-Pore-forming toxins (α-PFTs) are ubiquitous defense tools that kill cells by opening pores in the target cell membrane. Despite their relevance in host/pathogen interactions, very little is known about the pore stoichiometry and assembly pathway leading to membrane permeabilization. Equinatoxin II (EqtII) is a model α-PFT from sea anemone that oligomerizes and forms pores in sphingomyelin-containing membranes. Here, we determined the spatiotemporal organization of EqtII in living cells by single molecule imaging. Surprisingly, we found that on the cell surface EqtII did not organize into a unique oligomeric form. Instead, it existed as a mixture of oligomeric species mostly including monomers, dimers, tetramers, and hexamers. Mathematical modeling based on our data supported a new model in which toxin clustering happened in seconds and proceeded via condensation of EqtII dimer units formed upon monomer association. Furthermore, altering the pathway of EqtII assembly strongly affected its toxic activity, which highlights the relevance of the assembly mechanism on toxicity. PMID:25525270

Viral interference with DNA repair by targeting of the single-stranded DNA binding protein RPA.

PubMed

Banerjee, Pubali; DeJesus, Rowena; Gjoerup, Ole; Schaffhausen, Brian S

2013-10-01

Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.

Synthesis and radiosensitization properties of hydrogen peroxide and sodium hyaluronate complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosli, Nur Ratasha Alia Md.; Mohamed, Faizal; Heng, Cheong Kai

2014-09-03

Cancer cells which are large in size are resistant towards radiation therapy due to the presence of large amount of anti-oxidative enzymes and hypoxic cancer cells. Thus radiosensitizer agents have been developed to enhance the therapeutic effect of radiotherapy by increasing the sensitivity of these cancer cells towards radiation. This study is conducted to investigate the radiosensitization properties of radiosensitizer complex containing hydrogen peroxide and sodium hyaluronate. Combination with sodium hyaluronate may decrease reactivity of hydrogen peroxide but maintain the oxygen concentration needed for radiosensitizing effect. HepG2 cancer cells are cultured as the mean of test subject. Cancer cell samplesmore » which are targeted and not targeted with these radiosensitizers are irradiated with 2Gy single fractionated dose. Results obtained shows that the cancer cells which are not targeted with radiosensitizers has a cell viability of 98.80±0.37% after a time interval of 48 hours and has even repopulated over 100% after a 72 hour time interval. This shows that the cancer cells are resistant towards radiation. However, when the cancer cells are targeted with radiosensitizers prior to irradiation, there is a reduction of cell viability by 25.50±10.81% and 10.30±5.10% at time intervals of 48 and 72 hours respectively. This indicates that through the use of these radiosensitizers, cancer cells are more sensitive towards radiation.« less

Synthesis and radiosensitization properties of hydrogen peroxide and sodium hyaluronate complex

NASA Astrophysics Data System (ADS)

Rosli, Nur Ratasha Alia Md.; Mohamed, Faizal; Heng, Cheong Kai; Rahman, Irman Abdul; Ahmad, Ainee Fatimah; Mohamad, Hur Munawar Kabir

2014-09-01

Cancer cells which are large in size are resistant towards radiation therapy due to the presence of large amount of anti-oxidative enzymes and hypoxic cancer cells. Thus radiosensitizer agents have been developed to enhance the therapeutic effect of radiotherapy by increasing the sensitivity of these cancer cells towards radiation. This study is conducted to investigate the radiosensitization properties of radiosensitizer complex containing hydrogen peroxide and sodium hyaluronate. Combination with sodium hyaluronate may decrease reactivity of hydrogen peroxide but maintain the oxygen concentration needed for radiosensitizing effect. HepG2 cancer cells are cultured as the mean of test subject. Cancer cell samples which are targeted and not targeted with these radiosensitizers are irradiated with 2Gy single fractionated dose. Results obtained shows that the cancer cells which are not targeted with radiosensitizers has a cell viability of 98.80±0.37% after a time interval of 48 hours and has even repopulated over 100% after a 72 hour time interval. This shows that the cancer cells are resistant towards radiation. However, when the cancer cells are targeted with radiosensitizers prior to irradiation, there is a reduction of cell viability by 25.50±10.81% and 10.30±5.10% at time intervals of 48 and 72 hours respectively. This indicates that through the use of these radiosensitizers, cancer cells are more sensitive towards radiation.

Targeting of follicle stimulating hormone peptide-conjugated dendrimers to ovarian cancer cells

NASA Astrophysics Data System (ADS)

Modi, Dimple A.; Sunoqrot, Suhair; Bugno, Jason; Lantvit, Daniel D.; Hong, Seungpyo; Burdette, Joanna E.

2014-02-01

Ovarian cancer is the most lethal gynecological malignancy. Current treatment modalities include a combination of surgery and chemotherapy, which often lead to loss of fertility in premenopausal women and a myriad of systemic side effects. To address these issues, we have designed poly(amidoamine) (PAMAM) dendrimers to selectively target the follicle stimulating hormone receptor (FSHR), which is overexpressed by tumorigenic ovarian cancer cells but not by immature primordial follicles and other non-tumorigenic cells. Fluorescein-labeled generation 5 (G5) PAMAM dendrimers were conjugated with the binding peptide domain of FSH (FSH33) that has a high affinity to FSHR. The targeted dendrimers exhibited high receptor selectivity to FSHR-expressing OVCAR-3 cells, resulting in significant uptake and downregulation of an anti-apoptotic protein survivin, while showing minimal interactions with SKOV-3 cells that do not express FSHR. The selectivity of the FSH33-targeted dendrimers was further validated in 3D organ cultures of normal mouse ovaries. Immunostaining of the conjugates revealed their selective binding and uptake by ovarian surface epithelium (OSE) cells that express FSHR, while sparing the immature primordial follicles. In addition, an in vivo study monitoring tissue accumulation following a single intraperitoneal (i.p.) injection of the conjugates showed significantly higher accumulation of FSH33-targeted dendrimers in the ovary and oviduct compared to the non-targeted conjugates. These proof-of-concept findings highlight the potential of these FSH33-targeted dendrimers to serve as a delivery platform for anti-ovarian cancer drugs, while reducing their systemic side effects by preventing nonspecific uptake by the primordial follicles.Ovarian cancer is the most lethal gynecological malignancy. Current treatment modalities include a combination of surgery and chemotherapy, which often lead to loss of fertility in premenopausal women and a myriad of systemic side effects. To address these issues, we have designed poly(amidoamine) (PAMAM) dendrimers to selectively target the follicle stimulating hormone receptor (FSHR), which is overexpressed by tumorigenic ovarian cancer cells but not by immature primordial follicles and other non-tumorigenic cells. Fluorescein-labeled generation 5 (G5) PAMAM dendrimers were conjugated with the binding peptide domain of FSH (FSH33) that has a high affinity to FSHR. The targeted dendrimers exhibited high receptor selectivity to FSHR-expressing OVCAR-3 cells, resulting in significant uptake and downregulation of an anti-apoptotic protein survivin, while showing minimal interactions with SKOV-3 cells that do not express FSHR. The selectivity of the FSH33-targeted dendrimers was further validated in 3D organ cultures of normal mouse ovaries. Immunostaining of the conjugates revealed their selective binding and uptake by ovarian surface epithelium (OSE) cells that express FSHR, while sparing the immature primordial follicles. In addition, an in vivo study monitoring tissue accumulation following a single intraperitoneal (i.p.) injection of the conjugates showed significantly higher accumulation of FSH33-targeted dendrimers in the ovary and oviduct compared to the non-targeted conjugates. These proof-of-concept findings highlight the potential of these FSH33-targeted dendrimers to serve as a delivery platform for anti-ovarian cancer drugs, while reducing their systemic side effects by preventing nonspecific uptake by the primordial follicles. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr05042d

Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

PubMed

Prada, Ilaria; Meldolesi, Jacopo

2016-08-09

Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.

A dual chain chimeric antigen receptor (CAR) in the native antibody format for targeting immune cells towards cancer cells without the need of an scFv.

PubMed

Faitschuk, E; Nagy, V; Hombach, A A; Abken, H

2016-10-01

Adoptive cell therapy with chimeric antigen receptor (CAR)-modified T cells showed remarkable therapeutic efficacy in the treatment of leukaemia/lymphoma. However, the application to a variety of cancer entities is often constricted by the non-availability of a single chain antibody (scFv), which is usually the targeting domain in a CAR, while antibodies in the natural format are often available. To overcome the limitation, we designed a CAR that uses an antibody in its natural configuration for binding. Such CAR consists of two chains, the immunoglobulin light and heavy chain with their constant regions, whereby the heavy chain is anchored to the membrane and linked to an intracellular signalling domain for T-cell activation. The two chains form a stable heterodimer, a so-called dual chain CAR (dcCAR), and bind with high affinity and in a specific manner to their cognate antigen. By specific binding, the dcCAR activates engineered T cells for the release of pro-inflammatory cytokines and for target cell lysis. We provide evidence by three examples that the dcCAR format is universally applicable and thereby broadens the CAR cell therapy towards a larger variety of targets for which an scFv antibody is not available.

Design and Validation of CRISPR/Cas9 Systems for Targeted Gene Modification in Induced Pluripotent Stem Cells.

PubMed

Lee, Ciaran M; Zhu, Haibao; Davis, Timothy H; Deshmukh, Harshahardhan; Bao, Gang

2017-01-01

The CRISPR/Cas9 system is a powerful tool for precision genome editing. The ability to accurately modify genomic DNA in situ with single nucleotide precision opens up new possibilities for not only basic research but also biotechnology applications and clinical translation. In this chapter, we outline the procedures for design, screening, and validation of CRISPR/Cas9 systems for targeted modification of coding sequences in the human genome and how to perform genome editing in induced pluripotent stem cells with high efficiency and specificity.

Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells.

PubMed

Fraietta, Joseph A; Nobles, Christopher L; Sammons, Morgan A; Lundh, Stefan; Carty, Shannon A; Reich, Tyler J; Cogdill, Alexandria P; Morrissette, Jennifer J D; DeNizio, Jamie E; Reddy, Shantan; Hwang, Young; Gohil, Mercy; Kulikovskaya, Irina; Nazimuddin, Farzana; Gupta, Minnal; Chen, Fang; Everett, John K; Alexander, Katherine A; Lin-Shiao, Enrique; Gee, Marvin H; Liu, Xiaojun; Young, Regina M; Ambrose, David; Wang, Yan; Xu, Jun; Jordan, Martha S; Marcucci, Katherine T; Levine, Bruce L; Garcia, K Christopher; Zhao, Yangbing; Kalos, Michael; Porter, David L; Kohli, Rahul M; Lacey, Simon F; Berger, Shelley L; Bushman, Frederic D; June, Carl H; Melenhorst, J Joseph

2018-06-01

Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies 1-3 . In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells 4,5 . Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.

Mapping nonlinear receptive field structure in primate retina at single cone resolution

PubMed Central

Li, Peter H; Greschner, Martin; Gunning, Deborah E; Mathieson, Keith; Sher, Alexander; Litke, Alan M; Paninski, Liam

2015-01-01

The function of a neural circuit is shaped by the computations performed by its interneurons, which in many cases are not easily accessible to experimental investigation. Here, we elucidate the transformation of visual signals flowing from the input to the output of the primate retina, using a combination of large-scale multi-electrode recordings from an identified ganglion cell type, visual stimulation targeted at individual cone photoreceptors, and a hierarchical computational model. The results reveal nonlinear subunits in the circuity of OFF midget ganglion cells, which subserve high-resolution vision. The model explains light responses to a variety of stimuli more accurately than a linear model, including stimuli targeted to cones within and across subunits. The recovered model components are consistent with known anatomical organization of midget bipolar interneurons. These results reveal the spatial structure of linear and nonlinear encoding, at the resolution of single cells and at the scale of complete circuits. DOI: http://dx.doi.org/10.7554/eLife.05241.001 PMID:26517879

A New Cell Separation Method Based on Antibody-Immobilized Nanoneedle Arrays for the Detection of Intracellular Markers.

PubMed

Kawamura, Ryuzo; Miyazaki, Minami; Shimizu, Keita; Matsumoto, Yuta; Silberberg, Yaron R; Sathuluri, Ramachandra Rao; Iijima, Masumi; Kuroda, Shun'ichi; Iwata, Futoshi; Kobayashi, Takeshi; Nakamura, Chikashi

2017-11-08

Focusing on intracellular targets, we propose a new cell separation technique based on a nanoneedle array (NNA) device, which allows simultaneous insertion of multiple needles into multiple cells. The device is designed to target and lift ("fish") individual cells from a mixed population of cells on a substrate using an antibody-functionalized NNA. The mechanics underlying this approach were validated by force analysis using an atomic force microscope. Accurate high-throughput separation was achieved using one-to-one contacts between the nanoneedles and the cells by preparing a single-cell array in which the positions of the cells were aligned with 10,000 nanoneedles in the NNA. Cell-type-specific separation was realized by controlling the adhesion force so that the cells could be detached in cell-type-independent manner. Separation of nestin-expressing neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) was demonstrated using the proposed technology, and successful differentiation to neuronal cells was confirmed.

Integrated QSAR study for inhibitors of hedgehog signal pathway against multiple cell lines:a collaborative filtering method

PubMed Central

2012-01-01

Background The Hedgehog Signaling Pathway is one of signaling pathways that are very important to embryonic development. The participation of inhibitors in the Hedgehog Signal Pathway can control cell growth and death, and searching novel inhibitors to the functioning of the pathway are in a great demand. As the matter of fact, effective inhibitors could provide efficient therapies for a wide range of malignancies, and targeting such pathway in cells represents a promising new paradigm for cell growth and death control. Current research mainly focuses on the syntheses of the inhibitors of cyclopamine derivatives, which bind specifically to the Smo protein, and can be used for cancer therapy. While quantitatively structure-activity relationship (QSAR) studies have been performed for these compounds among different cell lines, none of them have achieved acceptable results in the prediction of activity values of new compounds. In this study, we proposed a novel collaborative QSAR model for inhibitors of the Hedgehog Signaling Pathway by integration the information from multiple cell lines. Such a model is expected to substantially improve the QSAR ability from single cell lines, and provide useful clues in developing clinically effective inhibitors and modifications of parent lead compounds for target on the Hedgehog Signaling Pathway. Results In this study, we have presented: (1) a collaborative QSAR model, which is used to integrate information among multiple cell lines to boost the QSAR results, rather than only a single cell line QSAR modeling. Our experiments have shown that the performance of our model is significantly better than single cell line QSAR methods; and (2) an efficient feature selection strategy under such collaborative environment, which can derive the commonly important features related to the entire given cell lines, while simultaneously showing their specific contributions to a specific cell-line. Based on feature selection results, we have proposed several possible chemical modifications to improve the inhibitor affinity towards multiple targets in the Hedgehog Signaling Pathway. Conclusions Our model with the feature selection strategy presented here is efficient, robust, and flexible, and can be easily extended to model large-scale multiple cell line/QSAR data. The data and scripts for collaborative QSAR modeling are available in the Additional file 1. PMID:22849868

Integrated QSAR study for inhibitors of Hedgehog Signal Pathway against multiple cell lines:a collaborative filtering method.

PubMed

Gao, Jun; Che, Dongsheng; Zheng, Vincent W; Zhu, Ruixin; Liu, Qi

2012-07-31

The Hedgehog Signaling Pathway is one of signaling pathways that are very important to embryonic development. The participation of inhibitors in the Hedgehog Signal Pathway can control cell growth and death, and searching novel inhibitors to the functioning of the pathway are in a great demand. As the matter of fact, effective inhibitors could provide efficient therapies for a wide range of malignancies, and targeting such pathway in cells represents a promising new paradigm for cell growth and death control. Current research mainly focuses on the syntheses of the inhibitors of cyclopamine derivatives, which bind specifically to the Smo protein, and can be used for cancer therapy. While quantitatively structure-activity relationship (QSAR) studies have been performed for these compounds among different cell lines, none of them have achieved acceptable results in the prediction of activity values of new compounds. In this study, we proposed a novel collaborative QSAR model for inhibitors of the Hedgehog Signaling Pathway by integration the information from multiple cell lines. Such a model is expected to substantially improve the QSAR ability from single cell lines, and provide useful clues in developing clinically effective inhibitors and modifications of parent lead compounds for target on the Hedgehog Signaling Pathway. In this study, we have presented: (1) a collaborative QSAR model, which is used to integrate information among multiple cell lines to boost the QSAR results, rather than only a single cell line QSAR modeling. Our experiments have shown that the performance of our model is significantly better than single cell line QSAR methods; and (2) an efficient feature selection strategy under such collaborative environment, which can derive the commonly important features related to the entire given cell lines, while simultaneously showing their specific contributions to a specific cell-line. Based on feature selection results, we have proposed several possible chemical modifications to improve the inhibitor affinity towards multiple targets in the Hedgehog Signaling Pathway. Our model with the feature selection strategy presented here is efficient, robust, and flexible, and can be easily extended to model large-scale multiple cell line/QSAR data. The data and scripts for collaborative QSAR modeling are available in the Additional file 1.

Whole organism lineage tracing by combinatorial and cumulative genome editing

PubMed Central

McKenna, Aaron; Findlay, Gregory M.; Gagnon, James A.; Horwitz, Marshall S.; Schier, Alexander F.; Shendure, Jay

2016-01-01

Multicellular systems develop from single cells through distinct lineages. However, current lineage tracing approaches scale poorly to whole, complex organisms. Here we use genome editing to progressively introduce and accumulate diverse mutations in a DNA barcode over multiple rounds of cell division. The barcode, an array of CRISPR/Cas9 target sites, marks cells and enables the elucidation of lineage relationships via the patterns of mutations shared between cells. In cell culture and zebrafish, we show that rates and patterns of editing are tunable, and that thousands of lineage-informative barcode alleles can be generated. By sampling hundreds of thousands of cells from individual zebrafish, we find that most cells in adult organs derive from relatively few embryonic progenitors. In future analyses, genome editing of synthetic target arrays for lineage tracing (GESTALT) can be used to generate large-scale maps of cell lineage in multicellular systems for normal development and disease. PMID:27229144

Tracking targeted bimodal nanovaccines: immune responses and routing in cells, tissue, and whole organism.

PubMed

Cruz, Luis J; Tacken, Paul J; Zeelenberg, Ingrid S; Srinivas, Mangala; Bonetto, Fernando; Weigelin, Bettina; Eich, Christina; de Vries, I Jolanda; Figdor, Carl G

2014-12-01

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs), involved in the induction of immunity and currently exploited for antitumor immunotherapies. An optimized noninvasive imaging modality capable of determining and quantifying DC-targeted nanoparticle (NP) trajectories could provide valuable information regarding therapeutic vaccine outcome. Here, targeted poly(d,l-lactide-co-glycolide) nanoparticles (PLGA NPs) recognizing DC receptors were equipped with superparamagnetic iron oxide particles (SPIO) or gold nanoparticles with fluorescently labeled antigen. The fluorescent label allowed for rapid analysis and quantification of DC-specific uptake of targeted PLGA NPs in comparison to uptake by other cells. Transmission electron microscopy (TEM) showed that a fraction of the encapsulated antigen reached the lysosomal compartment of DCs, where SPIO and gold were already partially released. However, part of the PLGA NPs localized within the cytoplasm, as confirmed by confocal microscopy. DCs targeted with NPs carrying SPIO or fluorescent antigen were detected within lymph nodes as early as 1 h after injection by magnetic resonance imaging (MRI). Despite the fact that targeting did not markedly affect PLGA NP biodistribution on organism and tissue level, it increased delivery of NPs to DCs residing in peripheral lymph nodes and resulted in enhanced T cell proliferation. In conclusion, two imaging agents within a single carrier allows tracking of targeted PLGA NPs at the subcellular, cellular, and organismal levels, thereby facilitating the rational design of in vivo targeted vaccination strategies.

Depleting tumor-specific Tregs at a single site eradicates disseminated tumors

PubMed Central

Marabelle, Aurélien; Kohrt, Holbrook; Sagiv-Barfi, Idit; Ajami, Bahareh; Axtell, Robert C.; Zhou, Gang; Rajapaksa, Ranjani; Green, Michael R.; Torchia, James; Brody, Joshua; Luong, Richard; Rosenblum, Michael D.; Steinman, Lawrence; Levitsky, Hyam I.; Tse, Victor; Levy, Ronald

2013-01-01

Activation of TLR9 by direct injection of unmethylated CpG nucleotides into a tumor can induce a therapeutic immune response; however, Tregs eventually inhibit the antitumor immune response and thereby limit the power of cancer immunotherapies. In tumor-bearing mice, we found that Tregs within the tumor preferentially express the cell surface markers CTLA-4 and OX40. We show that intratumoral coinjection of anti–CTLA-4 and anti-OX40 together with CpG depleted tumor-infiltrating Tregs. This in situ immunomodulation, which was performed with low doses of antibodies in a single tumor, generated a systemic antitumor immune response that eradicated disseminated disease in mice. Further, this treatment modality was effective against established CNS lymphoma with leptomeningeal metastases, sites that are usually considered to be tumor cell sanctuaries in the context of conventional systemic therapy. These results demonstrate that antitumor immune effectors elicited by local immunomodulation can eradicate tumor cells at distant sites. We propose that, rather than using mAbs to target cancer cells systemically, mAbs could be used to target the tumor infiltrative immune cells locally, thereby eliciting a systemic immune response. PMID:23728179

Targeting single-walled carbon nanotubes for the treatment of breast cancer using photothermal therapy

NASA Astrophysics Data System (ADS)

Neves, Luís F. F.; Krais, John J.; Van Rite, Brent D.; Ramesh, Rajagopal; Resasco, Daniel E.; Harrison, Roger G.

2013-09-01

This paper focuses on the targeting of single-walled carbon nanotubes (SWNTs) for the treatment of breast cancer with minimal side effects using photothermal therapy. The human protein annexin V (AV) binds specifically to anionic phospholipids expressed externally on the surface of tumour cells and endothelial cells that line the tumour vasculature. A 2 h incubation of the SWNT-AV conjugate with proliferating endothelial cells followed by washing and near-infrared (NIR) irradiation at a wavelength of 980 nm was enough to induce significant cell death; there was no significant cell death with irradiation or the conjugate alone. Administration of the same conjugate i.v. in BALB/c female mice with implanted 4T1 murine mammary at a dose of 0.8 mg SWNT kg-1 and followed one day later by NIR irradiation of the tumour at a wavelength of 980 nm led to complete disappearance of implanted 4T1 mouse mammary tumours for the majority of the animals by 11 days since the irradiation. The combination of the photothermal therapy with the immunoadjuvant cyclophosphamide resulted in increased survival. The in vivo results suggest the SWNT-AV/NIR treatment is a promising approach to treat breast cancer.

Double activity imaging reveals distinct cellular targets of haloperidol, clozapine and dopamine D(3) receptor selective RGH-1756.

PubMed

Kovács, K J; Csejtei, M; Laszlovszky, I

2001-03-01

Acute administration of typical (haloperidol) and atypical (clozapine) antipsychotics results in distinct and overlapping regions of immediate-early gene expression in the rat brain. RGH-1756 is a recently developed atypical antipsychotic with high affinity to dopamine D(3) receptors that results in a unique pattern of c-Fos induction. A single injection of either antipsychotic results in c-fos mRNA expression that peaks around 30 min after drug administration, while the maximum of c-Fos protein induction is seen 2 h after challenge. The transient and distinct temporal inducibility of c-fos mRNA and c-Fos protein was exploited to reveal and compare cellular targets of different antipsychotic drugs by concomitant localization of c-fos mRNA and c-Fos immunoreactivity in brain sections of rats that were timely challenged with two different antipsychotics. Double activity imaging revealed that haloperidol, clozapine and RGH-1756 share cellular targets in the nucleus accumbens, where 40% of all labeled neurons displayed both c-fos mRNA and c-Fos protein. Haloperidol activates cells in the caudate putamen, while clozapine-responsive, single labeled neurons were dominant in the prefrontal cortex and major island of Calleja. RGH-1756 targets haloperidol-sensitive cells in the caudate putamen, but cells that are activated by clozapine and RGH-1756 in the major island of Calleja are different.

Two-photon targeted recording of GFP-expressing neurons for light responses and live cell imaging in the mouse retina

PubMed Central

Wei, Wei; Elstrott, Justin; Feller, Marla B.

2015-01-01

Cell type-specific GFP expression in the retina has been achieved in an expanding repertoire of transgenic mouse lines, which are valuable tools for dissecting the retinal circuitry. However, measuring light responses from GFP-labeled cells is challenging because single-photon excitation of GFP easily bleaches the photoreceptors. To circumvent this problem, we used two-photon excitation at 920 nm to target GFP-expressing cells, followed by electrophysiological recording of light responses using conventional infrared optics. This protocol offers fast and sensitive detection of GFP while preserving the light sensitivity of the retina, and can be used to obtain the light responses as well as the detailed morphology of a GFP-expressing cell. Targeting of a GFP-expressing neuron takes less than 3 minutes, and the retina preparation remains light sensitive and suitable for recording for at least 8 hours. This protocol can also be applied to study retinal neurons labeled with other two-photon-excitable fluorophores. PMID:20595962

Simultaneous Detection of Human C-Terminal p53 Isoforms by Single Template Molecularly Imprinted Polymers (MIPs) Coupled with Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)-Based Targeted Proteomics.

PubMed

Jiang, Wenting; Liu, Liang; Chen, Yun

2018-03-06

Abnormal expression of C-terminal p53 isoforms α, β, and γ can cause the development of cancers including breast cancer. To date, much evidence has demonstrated that these isoforms can differentially regulate target genes and modulate their expression. Thus, quantification of individual isoforms may help to link clinical outcome to p53 status and to improve cancer patient treatment. However, there are few studies on accurate determination of p53 isoforms, probably due to sequence homology of these isoforms and also their low abundance. In this study, a targeted proteomics assay combining molecularly imprinted polymers (MIPs) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for simultaneous quantification of C-terminal p53 isoforms. Isoform-specific surrogate peptides (i.e., KPLDGEYFTLQIR (peptide-α) for isoform α, KPLDGEYFTLQDQTSFQK (peptide-β) for isoform β, and KPLDGEYFTLQMLLDLR (peptide-γ) for isoform γ) were first selected and used in both MIPs enrichment and mass spectrometric detection. The common sequence KPLDGEYFTLQ of these three surrogate peptides was used as single template in MIPs. In addition to optimization of imprinting conditions and characterization of the prepared MIPs, binding affinity and cross-reactivity of the MIPs for each surrogate peptide were also evaluated. As a result, a LOQ of 5 nM was achieved, which was >15-fold more sensitive than that without MIPs. Finally, the assay was validated and applied to simultaneous quantitative analysis of C-terminal p53 isoforms α, β, and γ in several human breast cell lines (i.e., MCF-10A normal cells, MCF-7 and MDA-MB-231 cancer cells, and drug-resistant MCF-7/ADR cancer cells). This study is among the first to employ single template MIPs and cross-reactivity phenomenon to select isoform-specific surrogate peptides and enable simultaneous quantification of protein isoforms in LC-MS/MS-based targeted proteomics.

Non-biased and efficient global amplification of a single-cell cDNA library

PubMed Central

Huang, Huan; Goto, Mari; Tsunoda, Hiroyuki; Sun, Lizhou; Taniguchi, Kiyomi; Matsunaga, Hiroko; Kambara, Hideki

2014-01-01

Analysis of single-cell gene expression promises a more precise understanding of molecular mechanisms of a living system. Most techniques only allow studies of the expressions for limited numbers of gene species. When amplification of cDNA was carried out for analysing more genes, amplification biases were frequently reported. A non-biased and efficient global-amplification method, which uses a single-cell cDNA library immobilized on beads, was developed for analysing entire gene expressions for single cells. Every step in this analysis from reverse transcription to cDNA amplification was optimized. By removing degrading excess primers, the bias due to the digestion of cDNA was prevented. Since the residual reagents, which affect the efficiency of each subsequent reaction, could be removed by washing beads, the conditions for uniform and maximized amplification of cDNAs were achieved. The differences in the amplification rates for randomly selected eight genes were within 1.5-folds, which could be negligible for most of the applications of single-cell analysis. The global amplification gives a large amount of amplified cDNA (>100 μg) from a single cell (2-pg mRNA), and that amount is enough for downstream analysis. The proposed global-amplification method was used to analyse transcript ratios of multiple cDNA targets (from several copies to several thousand copies) quantitatively. PMID:24141095

Reversible Aptamer-Au Plasmon Rulers for Secreted Single Molecules

DOE PAGES

Lee, Somin Eunice; Chen, Qian; Bhat, Ramray; ...

2015-06-03

Plasmon rulers, consisting of pairs of gold nanoparticles, allow single-molecule analysis without photobleaching or blinking; however, current plasmon rulers are irreversible, restricting detection to only single events. Here, we present a reversible plasmon ruler, comprised of coupled gold nanoparticles linked by a single aptamer, capable of binding individual secreted molecules with high specificity. We show that the binding of target secreted molecules to the reversible plasmon ruler is characterized by single-molecule sensitivity, high specificity, and reversibility. Lastly, such reversible plasmon rulers should enable dynamic and adaptive live-cell measurement of secreted single molecules in their local microenvironment.

Endogenous miRNA and Target Concentrations Determine Susceptibility to Potential ceRNA Competition

PubMed Central

Bosson, Andrew D.; Zamudio, Jesse R.; Sharp, Phillip A.

2016-01-01

SUMMARY Target competition (ceRNA crosstalk) within miRNA-regulated gene networks has been proposed to influence biological systems. To assess target competition, we characterize and quantitate miRNA networks in two cell types. Argonaute iCLIP reveals that hierarchical binding of high- to low-affinity miRNA targets is a key characteristic of in vivo activity. Quantification of cellular miRNA and mRNA/ncRNA target pool levels indicates that miRNA:target pool ratios and an affinity partitioned target pool accurately predict in vivo Ago binding profiles and miRNA susceptibility to target competition. Using single-cell reporters, we directly test predictions and estimate that ~3,000 additional high-affinity target sites can affect active miRNA families with low endogenous miRNA:target ratios, such as miR-92/25. In contrast, the highly expressed miR-294 and let-7 families are not susceptible to increases of nearly 10,000 sites. These results show differential susceptibility based on endogenous miRNA:target pool ratios and provide a physiological context for ceRNA competition in vivo. PMID:25449132

Visualization of phage DNA degradation by a type I CRISPR-Cas system at the single-cell level.

PubMed

Guan, Jingwen; Shi, Xu; Burgos, Roberto; Zeng, Lanying

2017-03-01

The CRISPR-Cas system is a widespread prokaryotic defense system which targets and cleaves invasive nucleic acids, such as plasmids or viruses. So far, a great number of studies have focused on the components and mechanisms of this system, however, a direct visualization of CRISPR-Cas degrading invading DNA in real-time has not yet been studied at the single-cell level. In this study, we fluorescently label phage lambda DNA in vivo , and track the labeled DNA over time to characterize DNA degradation at the single-cell level. At the bulk level, the lysogenization frequency of cells harboring CRISPR plasmids decreases significantly compared to cells with a non-CRISPR control. At the single-cell level, host cells with CRISPR activity are unperturbed by phage infection, maintaining normal growth like uninfected cells, where the efficiency of our anti-lambda CRISPR system is around 26%. During the course of time-lapse movies, the average fluorescence of invasive phage DNA in cells with CRISPR activity, decays more rapidly compared to cells without, and phage DNA is fully degraded by around 44 minutes on average. Moreover, the degradation appears to be independent of cell size or the phage DNA ejection site suggesting that Cas proteins are dispersed in sufficient quantities throughout the cell. With the CRISPR-Cas visualization system we developed, we are able to examine and characterize how a CRISPR system degrades invading phage DNA at the single-cell level. This work provides direct evidence and improves the current understanding on how CRISPR breaks down invading DNA.

A Monte Carlo study of macroscopic and microscopic dose descriptors for kilovoltage cellular dosimetry

NASA Astrophysics Data System (ADS)

Oliver, P. A. K.; Thomson, Rowan M.

2017-02-01

This work investigates how doses to cellular targets depend on cell morphology, as well as relations between cellular doses and doses to bulk tissues and water. Multicellular models of five healthy and cancerous soft tissues are developed based on typical values of cell compartment sizes, elemental compositions and number densities found in the literature. Cells are modelled as two concentric spheres with nucleus and cytoplasm compartments. Monte Carlo simulations are used to calculate the absorbed dose to the nucleus and cytoplasm for incident photon energies of 20-370 keV, relevant for brachytherapy, diagnostic radiology, and out-of-field radiation in higher-energy external beam radiotherapy. Simulations involving cell clusters, single cells and single nuclear cavities are carried out for cell radii between 5 and 10~μ m, and nuclear radii between 2 and 9~μ m. Seven nucleus and cytoplasm elemental compositions representative of animal cells are considered. The presence of a cytoplasm, extracellular matrix and surrounding cells can affect the nuclear dose by up to 13 % . Differences in cell and nucleus size can affect dose to the nucleus (cytoplasm) of the central cell in a cluster of 13 cells by up to 13 % (8 % ). Furthermore, the results of this study demonstrate that neither water nor bulk tissue are reliable substitutes for subcellular targets for incident photon energies  <50 keV: nuclear (cytoplasm) doses differ from dose-to-medium by up to 32 % (18 % ), and from dose-to-water by up to 21 % (8 % ). The largest differences between dose descriptors are seen for the lowest incident photon energies; differences are less than 3 % for energies ≥slant 90 keV. The sensitivity of results with regard to the parameters of the microscopic tissue structure model and cell model geometry, and the importance of the nucleus and cytoplasm as targets for radiation-induced cell death emphasize the importance of accurate models for cellular dosimetry studies.

A DNA Vaccine That Targets Hemagglutinin to Antigen-Presenting Cells Protects Mice against H7 Influenza

PubMed Central

Andersen, Tor Kristian; Zhou, Fan; Cox, Rebecca; Bogen, Bjarne

2017-01-01

ABSTRACT Zoonotic influenza H7 viral infections have a case fatality rate of about 40%. Currently, no or limited human to human spread has occurred, but we may be facing a severe pandemic threat if the virus acquires the ability to transmit between humans. Novel vaccines that can be rapidly produced for global distribution are urgently needed, and DNA vaccines may be the only type of vaccine that allows for the speed necessary to quench an emerging pandemic. Here, we constructed DNA vaccines encoding the hemagglutinin (HA) from influenza A/chicken/Italy/13474/99 (H7N1). In order to increase the efficacy of DNA vaccination, HA was targeted to either major histocompatibility complex class II molecules or chemokine receptors 1, 3, and 5 (CCR1/3/5) that are expressed on antigen-presenting cells (APC). A single DNA vaccination with APC-targeted HA significantly increased antibody levels in sera compared to nontargeted control vaccines. The antibodies were confirmed neutralizing in an H7 pseudotype-based neutralization assay. Furthermore, the APC-targeted vaccines increased the levels of antigen-specific cytotoxic T cells, and a single DNA vaccination could confer protection against a lethal challenge with influenza A/turkey/Italy/3889/1999 (H7N1) in mice. In conclusion, we have developed a vaccine that rapidly could contribute protection against a pandemic threat from avian influenza. IMPORTANCE Highly pathogenic avian influenza H7 constitute a pandemic threat that can cause severe illness and death in infected individuals. Vaccination is the main method of prophylaxis against influenza, but current vaccine strategies fall short in a pandemic situation due to a prolonged production time and insufficient production capabilities. In contrast, a DNA vaccine can be rapidly produced and deployed to prevent the potential escalation of a highly pathogenic influenza pandemic. We here demonstrate that a single DNA delivery of hemagglutinin from an H7 influenza could mediate full protection against a lethal challenge with H7N1 influenza in mice. Vaccine efficacy was contingent on targeting of the secreted vaccine protein to antigen-presenting cells. PMID:28931687

Visible light-sensitive APTES-bound ZnO nanowire toward a potent nanoinjector sensing biomolecules in a living cell

NASA Astrophysics Data System (ADS)

Lee, Jooran; Choi, Sunyoung; Bae, Seon Joo; Yoon, Seok Min; Choi, Joon Sig; Yoon, Minjoong

2013-10-01

Nanoscale cell injection techniques combined with nanoscopic photoluminescence (PL) spectroscopy have been important issues in high-resolution optical biosensing, gene and drug delivery and single-cell endoscopy for medical diagnostics and therapeutics. However, the current nanoinjectors remain limited for optical biosensing and communication at the subwavelength level, requiring an optical probe such as semiconductor quantum dots, separately. Here, we show that waveguided red emission is observed at the tip of a single visible light-sensitive APTES-modified ZnO nanowire (APTES-ZnO NW) and it exhibits great enhancement upon interaction with a complementary sequence-based double stranded (ds) DNA, whereas it is not significantly affected by non-complementary ds DNA. Further, the tip of a single APTES-ZnO NW can be inserted into the subcellular region of living HEK 293 cells without significant toxicity, and it can also detect the enhancement of the tip emission from subcellular regions with high spatial resolution. These results indicate that the single APTES-ZnO NW would be useful as a potent nanoinjector which can guide visible light into intracellular compartments of mammalian cells, and can also detect nanoscopic optical signal changes induced by interaction with the subcellular specific target biomolecules without separate optical probes.Nanoscale cell injection techniques combined with nanoscopic photoluminescence (PL) spectroscopy have been important issues in high-resolution optical biosensing, gene and drug delivery and single-cell endoscopy for medical diagnostics and therapeutics. However, the current nanoinjectors remain limited for optical biosensing and communication at the subwavelength level, requiring an optical probe such as semiconductor quantum dots, separately. Here, we show that waveguided red emission is observed at the tip of a single visible light-sensitive APTES-modified ZnO nanowire (APTES-ZnO NW) and it exhibits great enhancement upon interaction with a complementary sequence-based double stranded (ds) DNA, whereas it is not significantly affected by non-complementary ds DNA. Further, the tip of a single APTES-ZnO NW can be inserted into the subcellular region of living HEK 293 cells without significant toxicity, and it can also detect the enhancement of the tip emission from subcellular regions with high spatial resolution. These results indicate that the single APTES-ZnO NW would be useful as a potent nanoinjector which can guide visible light into intracellular compartments of mammalian cells, and can also detect nanoscopic optical signal changes induced by interaction with the subcellular specific target biomolecules without separate optical probes. Electronic supplementary information (ESI) available: Synthesis of APTES-modified ZnO nanowires, DNA functionalization and spectroscopic measurements with additional fluorescence image ad fluorescence decay times, cell culture, injection of a single nanowire into living cells, subcellular imaging and determination of cytotoxicity. See DOI: 10.1039/c3nr03042c

Lineage-related cytotoxicity and clonogenic profile of 1,4-benzoquinone-exposed hematopoietic stem and progenitor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chow, Paik Wah; Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur; Abdul Hamid, Zariyantey, E-mail: zyantey@ukm.edu.my

Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are sensitive targets for benzene-induced hematotoxicity and leukemogenesis. The impact of benzene exposure on the complex microenvironment of HSCs and HPCs remains elusive. This study aims to investigate the mechanism linking benzene exposure to targeting HSCs and HPCs using phenotypic and clonogenic analyses. Mouse bone marrow (BM) cells were exposed ex vivo to the benzene metabolite, 1,4-benzoquinone (1,4-BQ), for 24 h. Expression of cellular surface antigens for HSC (Sca-1), myeloid (Gr-1, CD11b), and lymphoid (CD45, CD3e) populations were confirmed by flow cytometry. The clonogenicity of cells was studied using the colony-formingmore » unit (CFU) assay for multilineage (CFU-GM and CFU-GEMM) and single-lineage (CFU-E, BFU-E, CFU-G, and CFU-M) progenitors. 1,4-BQ demonstrated concentration-dependent cytotoxicity in mouse BM cells. The percentage of apoptotic cells increased (p < 0.05) following 1,4-BQ exposure. Exposure to 1,4-BQ showed no significant effect on CD3e{sup +} cells but reduced the total counts of Sca-1{sup +}, CD11b{sup +}, Gr-1{sup +}, and CD45{sup +} cells at 7 and 12 μM (p < 0.05). Furthermore, the CFU assay showed reduced (p < 0.05) clonogenicity in 1,4-BQ-treated cells. 1,4-BQ induced CFU-dependent cytotoxicity by significantly inhibiting colony growth for CFU-E, BFU-E, CFU-G, and CFU-M starting at a low concentration of exposure (5 μM); whereas for the CFU-GM and CFU-GEMM, the inhibition of colony growth was remarkable only at 7 and 12 μM of 1,4-BQ, respectively. Taken together, 1,4-BQ caused lineage-related cytotoxicity in mouse HPCs, demonstrating greater toxicity in single-lineage progenitors than in those of multi-lineage. - Highlights: • We examine 1,4-BQ toxicity targeting mouse hematopoietic cell lineages. • 1,4-BQ induces concentration-dependent cytotoxicity in bone marrow (BM) cells. • 1,4-BQ shows lineage-related toxicity on hematopoietic stem and progenitors. • 1,4-BQ toxicity is greater in single- than multilineage committed progenitors.« less

Antibiotic regimen based on population analysis of residing persister cells eradicates Staphylococcus epidermidis biofilms

PubMed Central

Yang, Shoufeng; Hay, Iain D.; Cameron, David R.; Speir, Mary; Cui, Bintao; Su, Feifei; Peleg, Anton Y.; Lithgow, Trevor; Deighton, Margaret A.; Qu, Yue

2015-01-01

Biofilm formation is a major pathogenicity strategy of Staphylococcus epidermidis causing various medical-device infections. Persister cells have been implicated in treatment failure of such infections. We sought to profile bacterial subpopulations residing in S. epidermidis biofilms, and to establish persister-targeting treatment strategies to eradicate biofilms. Population analysis was performed by challenging single biofilm cells with antibiotics at increasing concentrations ranging from planktonic minimum bactericidal concentrations (MBCs) to biofilm MBCs (MBCbiofilm). Two populations of “persister cells” were observed: bacteria that survived antibiotics at MBCbiofilm for 24/48 hours were referred to as dormant cells; those selected with antibiotics at 8 X MICs for 3 hours (excluding dormant cells) were defined as tolerant-but-killable (TBK) cells. Antibiotic regimens targeting dormant cells were tested in vitro for their efficacies in eradicating persister cells and intact biofilms. This study confirmed that there are at least three subpopulations within a S. epidermidis biofilm: normal cells, dormant cells, and TBK cells. Biofilms comprise more TBK cells and dormant cells than their log-planktonic counterparts. Using antibiotic regimens targeting dormant cells, i.e. effective antibiotics at MBCbiofilm for an extended period, might eradicate S. epidermidis biofilms. Potential uses for this strategy are in antibiotic lock techniques and inhaled aerosolized antibiotics. PMID:26687035

T Cell Receptor Engineering and Analysis Using the Yeast Display Platform

PubMed Central

Smith, Sheena N.; Harris, Daniel T.; Kranz, David M.

2017-01-01

The αβ heterodimeric T cell receptor (TCR) recognizes peptide antigens that are transported to the cell surface as a complex with a protein encoded by the major histocompatibility complex (MHC). T cells thus evolved a strategy to sense these intracellular antigens, and to respond either by eliminating the antigen-presenting cell (e.g. a virus-infected cell) or by secreting factors that recruit the immune system to the site of the antigen. The central role of the TCR in the binding of antigens as peptide-MHC (pepMHC) ligands has now been studied thoroughly. Interestingly, despite their exquisite sensitivity (e.g. T cell activation by as few as 1 to 3 pepMHC complexes on a single target cell), TCRs are known to have relatively low affinities for pepMHC, with KD values in the micromolar range. There has been interest in engineering the affinity of TCRs in order to use this class of molecules in ways similar to now done with antibodies. By doing so, it would be possible to harness the potential of TCRs as therapeutics against a much wider array of antigens that include essentially all intracellular targets. To engineer TCRs, and to analyze their binding features more rapidly, we have used a yeast display system as a platform. Expression and engineering of a single-chain form of the TCR, analogous to scFv fragments from antibodies, allow the TCR to be affinity matured with a variety of possible pepMHC ligands. In addition, the yeast display platform allows one to rapidly generate TCR variants with diverse binding affinities and to analyze specificity and affinity without the need for purification of soluble forms of the TCRs. The present chapter describes the methods for engineering and analyzing single-chain TCRs using yeast display. PMID:26060072

“Combination-oriented molecular-targeting prevention” of cancer: a model involving the combination of TRAIL and a DR5 inducer

PubMed Central

Yoshida, Tatsushi; Horinaka, Mano

2010-01-01

Malignant tumors carry a high risk of death, and the prevention of malignant tumors is a crucial issue in preventive medicine. To this end, many chemopreventive agents have been tested, but the effects of single agents have been found to be insufficient to justify clinical trials. We have therefore hypothesized that combinations of different chemopreventive agents may synergistically enhance the preventive effect of chemopreventive agents used singly. To provide the treating physician with some guideline by which to choose the most effective agents to be combined, we propose a strategy which we have termed the “combination-oriented molecular-targeting prevention” of cancer. As the molecular target of our model, we focused on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which specifically causes apoptosis in malignant tumor cells. Many of these agents were found to up-regulate the expression of death receptor 5, a TRAIL receptor. They were also found to synergistically induce apoptosis in malignant tumor cells when combined with TRAIL. Here, we strongly advocate that the strategy of “combination-oriented molecular-targeting prevention” of cancer will be a practical approach for chemoprevention against human malignant tumors. PMID:21432546

microMS: A Python Platform for Image-Guided Mass Spectrometry Profiling

NASA Astrophysics Data System (ADS)

Comi, Troy J.; Neumann, Elizabeth K.; Do, Thanh D.; Sweedler, Jonathan V.

2017-09-01

Image-guided mass spectrometry (MS) profiling provides a facile framework for analyzing samples ranging from single cells to tissue sections. The fundamental workflow utilizes a whole-slide microscopy image to select targets of interest, determine their spatial locations, and subsequently perform MS analysis at those locations. Improving upon prior reported methodology, a software package was developed for working with microscopy images. microMS, for microscopy-guided mass spectrometry, allows the user to select and profile diverse samples using a variety of target patterns and mass analyzers. Written in Python, the program provides an intuitive graphical user interface to simplify image-guided MS for novice users. The class hierarchy of instrument interactions permits integration of new MS systems while retaining the feature-rich image analysis framework. microMS is a versatile platform for performing targeted profiling experiments using a series of mass spectrometers. The flexibility in mass analyzers greatly simplifies serial analyses of the same targets by different instruments. The current capabilities of microMS are presented, and its application for off-line analysis of single cells on three distinct instruments is demonstrated. The software has been made freely available for research purposes. [Figure not available: see fulltext.

microMS: A Python Platform for Image-Guided Mass Spectrometry Profiling.

PubMed

Comi, Troy J; Neumann, Elizabeth K; Do, Thanh D; Sweedler, Jonathan V

2017-09-01

Image-guided mass spectrometry (MS) profiling provides a facile framework for analyzing samples ranging from single cells to tissue sections. The fundamental workflow utilizes a whole-slide microscopy image to select targets of interest, determine their spatial locations, and subsequently perform MS analysis at those locations. Improving upon prior reported methodology, a software package was developed for working with microscopy images. microMS, for microscopy-guided mass spectrometry, allows the user to select and profile diverse samples using a variety of target patterns and mass analyzers. Written in Python, the program provides an intuitive graphical user interface to simplify image-guided MS for novice users. The class hierarchy of instrument interactions permits integration of new MS systems while retaining the feature-rich image analysis framework. microMS is a versatile platform for performing targeted profiling experiments using a series of mass spectrometers. The flexibility in mass analyzers greatly simplifies serial analyses of the same targets by different instruments. The current capabilities of microMS are presented, and its application for off-line analysis of single cells on three distinct instruments is demonstrated. The software has been made freely available for research purposes. Graphical Abstract ᅟ.

The evolving role of targeted drugs in the treatment of Hodgkin lymphoma.

PubMed

Eichenauer, Dennis A; Engert, Andreas

2017-09-01

Hodgkin lymphoma (HL) is a B-cell-derived malignancy mostly affecting young adults. More than 80% of patients are cured after stage-adapted first-line treatment with chemotherapy and/or radiotherapy. About 50% of patients with disease recurrence achieve long-term remission with second-line treatment consisting of high-dose chemotherapy and autologous stem cell transplantation. However, HL treatment is often associated with acute toxicity and in part life-threatening late effects. Implementing targeted drugs may reduce toxicity and potentially further optimize efficacy. In recent years, the CD30-directed antibody-drug conjugate brentuximab vedotin (BV) and anti-PD-1 antibodies, nivolumab and pembrolizumab, underwent extensive evaluation in HL. They have exhibited encouraging single agent activity and a favorable toxicity profile in patients with multiple relapses. Therefore, they are currently under investigation in different additional indications. Areas covered: This article gives an overview over clinical trials evaluating targeted drugs either as single agent or as part of combination therapies in HL patients. Expert commentary: A multitude of targeted drugs are investigated in HL. Promising data have particularly emerged from studies with BV and anti-PD-1 antibodies. However, mature data needed for final conclusions are still pending.

Metuzumab enhanced chemosensitivity and apoptosis in non-small cell lung carcinoma

PubMed Central

Feng, Fei; Wang, Bin; Sun, Xiuxuan; Zhu, Yumeng; Tang, Hao; Nan, Gang; Wang, Lijuan; Wu, Bo; Huhe, Muren; Liu, Shuangshuang; Diao, Tengyue; Hou, Rong; Zhang, Yang; Zhang, Zheng

2017-01-01

ABSTRACT Targeted therapeutics is used as an alternative treatment of non-small cell lung cancer (NSCLC); however, treatment effect is far from being satisfactory, and therefore identification of new targets is needed. We have previously shown that metuzumab inhibit tumor growth in vivo. The present study was performed to investigate the anti-tumor efficacy of metuzumab combined with gemcitabine and cisplatin (GP), paclitaxel and cisplatin (TP) or navelbine and cisplatin (NP) regimens in multiple NSCLC cell lines. Our results demonstrate that, in comparison to single agent metuzumab or GP treated cells, metuzumab combined with GP display inhibitory effects on tumor growth. Furthermore, we found that metuzumab elevated the sensitivity of cell lines to gemcitabine, which was identified by MTT assay. Flow cytometric analysis showed that metuzumab combined with gemcitabine (GEM) treatment led to an obvious G1 arrest and an elevated apoptosis in A549, NCI-H460 and NCI-H520 cells. Western blot analysis also demonstrated a significantly reduced level of cyclin D1, Bcl-2, and an obviously increase level of Bax and full-length caspase-3 in A549, NCI-H460 and NCI-H520 cells treated with metuzumab/gemcitabine combination in comparison with single agent treated cells. In addition, metuzumab/gemcitabine treated A549, NCI-H460 and NCI-H520 cells also demonstrated a significantly increase in deoxycytidine kinase (dCK) protein level compared with single agent metuzumab or gemcitabine treated cells. Xenograft models also demonstrated that this metuzumab/gemcitabine combination led to upregulation of dCK. Taken together, the mechanisms of metuzumab combined with GP repress tumor growth were that the combined treatment significantly inhibited the tumor cell proliferation, apoptosis and cell cycle in vitro and in vivo and at least partially by induction of dCK expression. Our results suggested that metuzumab could significantly enhance chemosensitivity of human NSCLC cells to gemcitabine. Metuzumab/gemcitabine combination treatment may be a potentially useful therapeutic regimen for NSCLC patients. PMID:28055291

Leukotriene signaling via ALOX5 and cysteinyl leukotriene receptor 1 is dispensable for in vitro growth of CD34+CD38- stem and progenitor cells in chronic myeloid leukemia.

PubMed

Dolinska, Monika; Piccini, Alexandre; Wong, Wan Man; Gelali, Eleni; Johansson, Anne-Sofie; Klang, Johannis; Xiao, Pingnan; Yektaei-Karin, Elham; Strömberg, Ulla Olsson; Mustjoki, Satu; Stenke, Leif; Ekblom, Marja; Qian, Hong

2017-08-19

Tyrosine kinase inhibitors targeting the BCR-ABL oncoprotein in chronic myeloid leukemia (CML) are remarkably effective inducing deep molecular remission in most patients. However, they are less effective to eradicate the leukemic stem cells (LSC), resulting in disease persistence. Therefore, there is great need to develop novel therapeutic strategies to specifically target the LSC. In an experimental mouse CML model system, the leukotriene pathway, and specifically, the expression ALOX5, encoding 5-lipoxygenase (5-LO), has been reported as a critical regulator of the LSC. Based on these results, the 5-LO inhibitor zileuton has been introduced in clinical trials as a therapeutic option to target the LSC although its effect on primary human CML LSC has not been studied. We have here by using multiplex single cell PCR analyzed the expression of the mediators of the leukotriene pathway in bone marrow (BM) BCR-ABL + CD34 + CD38 - cells at diagnosis, and found low or undetectable expression of ALOX5. In line with this, zileuton did not exert significant overall growth inhibition in the long-term culture-initiating cell (LTC-IC) and colony (CFU-C) assays of BM CD34 + CD38 - cells from 7 CML patients. The majority of the single leukemic BCR-ABL + CD34 + CD38 - cells expressed cysteinyl leukotriene receptors CYSLT1 and CYSLT2. However, montelukast, an inhibitor of CYSLT1, also failed to significantly suppress CFU-C and LTC-IC growth. These findings indicate that targeting ALOX5 or CYSLT1 signaling with leukotriene antagonists, introduced into the clinical practice primarily as prophylaxis and treatment for asthma, may not be a promising pharmacological strategy to eradicate persisting LSC in CML patients. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Selective inhibitors of zinc-dependent histone deacetylases. Therapeutic targets relevant to cancer.

PubMed

Kollar, Jakub; Frecer, Vladimir

2015-01-01

Histone deacetylases (HDACs), which act on acetylated histones and/or other non-histone protein substrates, represent validated epigenetic targets for the treatment of cancer and other human diseases. The inhibition of HDAC activity was shown to induce cell cycle arrest, differentiation, apoptosis as well as a decrease in proliferation, angiogenesis, migration, and cell resistance to chemotherapy. Targeting single HDAC isoforms with selective inhibitors will help to reveal the role of individual HDACs in cancer development or uncover further biological consequences of protein acetylation. This review focuses on conventional zinc-containing HDACs. In its first part, the biological role of individual HDACs in various types of cancer is summarized. In the second part, promising HDAC inhibitors showing activity both in enzymatic and cell-based assays are surveyed with an emphasis on the inhibitors selective to the individual HDACs.

Modification of Expanded NK Cells with Chimeric Antigen Receptor mRNA for Adoptive Cellular Therapy.

PubMed

Chu, Yaya; Flower, Allyson; Cairo, Mitchell S

2016-01-01

NK cells are bone marrow-derived cytotoxic lymphocytes that play a major role in the rejection of tumors and cells infected by viruses. The regulation of NK activation vs inhibition is regulated by the expression of a variety of NK receptors (NKRs) and specific NKRs' ligands expressed on their targets. However, factors limiting NK therapy include small numbers of active NK cells in unexpanded peripheral blood and lack of specific tumor targeting. Chimeric antigen receptors (CAR) usually include a single-chain Fv variable fragment from a monoclonal antibody, a transmembrane hinge region, and a signaling domain such as CD28, CD3-zeta, 4-1BB (CD137), or 2B4 (CD244) endodimers. Redirecting NK cells with a CAR will circumvent the limitations of the lack of NK targeting specificity. This chapter focuses on the methods to expand human NK cells from peripheral blood by co-culturing with feeder cells and to modify the expanded NK cells efficiently with the in vitro transcribed CAR mRNA by electroporation and to test the functionality of the CAR-modified expanded NK cells for use in adoptive cellular immunotherapy.

Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors.

PubMed

Song, Erwei; Zhu, Pengcheng; Lee, Sang-Kyung; Chowdhury, Dipanjan; Kussman, Steven; Dykxhoorn, Derek M; Feng, Yi; Palliser, Deborah; Weiner, David B; Shankar, Premlata; Marasco, Wayne A; Lieberman, Judy

2005-06-01

Delivery of small interfering RNAs (siRNAs) into cells is a key obstacle to their therapeutic application. We designed a protamine-antibody fusion protein to deliver siRNA to HIV-infected or envelope-transfected cells. The fusion protein (F105-P) was designed with the protamine coding sequence linked to the C terminus of the heavy chain Fab fragment of an HIV-1 envelope antibody. siRNAs bound to F105-P induced silencing only in cells expressing HIV-1 envelope. Additionally, siRNAs targeted against the HIV-1 capsid gene gag, inhibited HIV replication in hard-to-transfect, HIV-infected primary T cells. Intratumoral or intravenous injection of F105-P-complexed siRNAs into mice targeted HIV envelope-expressing B16 melanoma cells, but not normal tissue or envelope-negative B16 cells; injection of F105-P with siRNAs targeting c-myc, MDM2 and VEGF inhibited envelope-expressing subcutaneous B16 tumors. Furthermore, an ErbB2 single-chain antibody fused with protamine delivered siRNAs specifically into ErbB2-expressing cancer cells. This study demonstrates the potential for systemic, cell-type specific, antibody-mediated siRNA delivery.

Polymorphism in liver-stage malaria vaccine candidate proteins: immune evasion and implications for vaccine design.

PubMed

Flanagan, Katie L; Wilson, Kirsty L; Plebanski, Magdalena

2016-01-01

The pre-erythrocytic stage of infection by malaria parasites represents a key target for vaccines that aim to eradicate malaria. Two important broad immune evasion strategies that can interfere with vaccine efficacy include the induction of dendritic cell (DC) dysfunction and regulatory T cells (Tregs) by blood-stage malaria parasites, leading to inefficient priming of T cells targeting liver-stage infections. The parasite also uses 'surgical strike' strategies, whereby polymorphism in pre-erythrocytic antigens can interfere with host immunity. Specifically, we review how even single amino acid changes in T cell epitopes can lead to loss of binding to major histocompatibility complex (MHC), lack of cross-reactivity, or antagonism and immune interference, where simultaneous or sequential stimulation with related variants of the same T cell epitope can cause T cell anergy or the conversion of effector to immunosuppressive T cell phenotypes.

Stem cell based anti-HIV Gene therapy

PubMed Central

Kitchen, Scott G.; Shimizu, Saki; An, Dong Sung

2011-01-01

Human stem cell-based therapeutic intervention strategies for treating HIV infection have recently undergone a renaissance as a major focus of investigation. Unlike most conventional antiviral therapies, genetically engineered hematopoietic stem cells possess the capacity for prolonged self-renewal that would continuously produce protected immune cells to fight against HIV. A successful strategy therefore has the potential to stably control and ultimately eradicate HIV from patients by a single or minimal treatment. Recent progress in the development of new technologies and clinical trials sets the stage for the current generation of gene therapy approaches to combat HIV infection. In this review, we will discuss two major approaches that are currently underway in the development of stem cell-based gene therapy to target HIV: One that focuses on the protection of cells from productive infection with HIV, and the other that focuses on targeting immune cells to directly combat HIV infection. PMID:21247612

Dynamics of different-sized solid-state nanocrystals as tracers for a drug-delivery system in the interstitium of a human tumor xenograft

PubMed Central

Kawai, Masaaki; Higuchi, Hideo; Takeda, Motohiro; Kobayashi, Yoshio; Ohuchi, Noriaki

2009-01-01

Introduction Recent anticancer drugs have been made larger to pass selectively through tumor vessels and stay in the interstitium. Understanding drug movement in association with its size at the single-molecule level and estimating the time needed to reach the targeted organ is indispensable for optimizing drug delivery because single cell-targeted therapy is the ongoing paradigm. This report describes the tracking of single solid nanoparticles in tumor xenografts and the estimation of arrival time. Methods Different-sized nanoparticles measuring 20, 40, and 100 nm were injected into the tail vein of the female Balb/c nu/nu mice bearing human breast cancer on their backs. The movements of the nanoparticles were visualized through the dorsal skin-fold chamber with the high-speed confocal microscopy that we manufactured. Results An analysis of the particle trajectories revealed diffusion to be inversely related to the particle size and position in the tumor, whereas the velocity of the directed movement was related to the position. The difference in the velocity was the greatest for 40-nm particles in the perivascular to the intercellular region: difference = 5.8 nm/s. The arrival time of individual nanoparticles at tumor cells was simulated. The estimated times for the 20-, 40-, and 100-nm particles to reach the tumor cells were 158.0, 218.5, and 389.4 minutes, respectively, after extravasation. Conclusions This result suggests that the particle size can be individually designed for each goal. These data and methods are also important for understanding drug pharmacokinetics. Although this method may be subject to interference by surface molecules attached on the particles, it has the potential to elucidate the pharmacokinetics involved in constructing novel drug-delivery systems involving cell-targeted therapy. PMID:19575785

A Simple Method for Visualization of Locus-Specific H4K20me1 Modifications in Living Caenorhabditis elegans Single Cells.

PubMed

Shinkai, Yoichi; Kuramochi, Masahiro; Doi, Motomichi

2018-05-03

Recently, advances in next-generation sequencing technologies have enabled genome-wide analyses of epigenetic modifications; however, it remains difficult to analyze the states of histone modifications at a single-cell resolution in living multicellular organisms because of the heterogeneity within cellular populations. Here we describe a simple method to visualize histone modifications on the specific sequence of target locus at a single-cell resolution in living Caenorhabditis elegans , by combining the LacO/LacI system and a genetically-encoded H4K20me1-specific probe, "mintbody". We demonstrate that Venus-labeled mintbody and mTurquoise2-labeled LacI can co-localize on an artificial chromosome carrying both the target locus and LacO sequences, where H4K20me1 marks the target locus. We demonstrate that our visualization method can precisely detect H4K20me1 depositions on the her-1 gene sequences on the artificial chromosome, to which the dosage compensation complex binds to regulate sex determination. The degree of H4K20me1 deposition on the her-1 sequences on the artificial chromosome correlated strongly with sex, suggesting that, using the artificial chromosome, this method can reflect context-dependent changes of H4K20me1 on endogenous genomes. Furthermore, we demonstrate live imaging of H4K20me1 depositions on the artificial chromosome. Combined with ChIP assays, this mintbody-LacO/LacI visualization method will enable analysis of developmental and context-dependent alterations of locus-specific histone modifications in specific cells and elucidation of the underlying molecular mechanisms. Copyright © 2018, G3: Genes, Genomes, Genetics.

CCR5 Gene Disruption via Lentiviral Vectors Expressing Cas9 and Single Guided RNA Renders Cells Resistant to HIV-1 Infection

PubMed Central

Liu, Jingjing; Zhang, Di; Kimata, Jason T.; Zhou, Paul

2014-01-01

CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1. PMID:25541967

Combination of the MEK inhibitor pimasertib with BTK or PI3K-delta inhibitors is active in preclinical models of aggressive lymphomas.

PubMed

Gaudio, E; Tarantelli, C; Kwee, I; Barassi, C; Bernasconi, E; Rinaldi, A; Ponzoni, M; Cascione, L; Targa, A; Stathis, A; Goodstal, S; Zucca, E; Bertoni, F

2016-06-01

Lymphomas are among the most common human cancers and represent the cause of death for still too many patients. The B-cell receptor with its downstream signaling pathways represents an important therapeutic target for B-cell lymphomas. Here, we evaluated the activity of the MEK1/2 inhibitor pimasertib as single agent and in combination with other targeted drugs in lymphoma preclinical models. Cell lines derived mature B-cell lymphomas were exposed to increasing doses of pimasertib alone. Immunoblotting and gene expression profiling were performed. Combination of pimasertib with idelalisib or ibrutinib was assessed. Pimasertib as single agent exerted a dose-dependent antitumor activity across a panel of 23 lymphoma cell lines, although at concentrations higher than reported for solid tumors. Strong synergism was observed with pimasertib combined with the PI3K inhibitor idelalisib and the BTK inhibitor ibrutinib in cell lines derived from diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma. The data were confirmed in an in vivo experiment treating DLBCL xenografts with pimasertib and ibrutinib. The data presented here provide the basis for further investigation of regimens including pimasertib in relapsed and refractory lymphomas. © The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

A miniaturized multipurpose platform for rapid, label-free, and simultaneous separation, patterning, and in vitro culture of primary and rare cells.

PubMed

Didar, Tohid Fatanat; Bowey, Kristen; Almazan, Guillermina; Tabrizian, Maryam

2014-02-01

Given that current cell isolation techniques are expensive, time consuming, yield low isolation purities, and/or alter target cell properties, a versatile, cost effective, and easy-to-operate microchip with the capability to simultaneously separate, capture, pattern, and culture rare and primary cells in vitro is developed. The platform is based on target cell adhesion onto the micro-fabricated interfaces produced by microcontact printing of cell-specific antibodies. Results show over 95% separation efficiency in less than 10 min for the separation of oligodendrocyte progenitor cells (OPCs) and cardiomyocytes from rat brain and heart mixtures, respectively. Target cell attachment and single cell spreading can be precisely controlled on the basis of the designed patterns. Both cell types can maintain their biofunctionality. Indeed, isolated OPCs can proliferate and differentiate into mature oligodendrocytes, while isolated cardiomyocytes retain their contractile properties on the separation platform. Successful separation of two dissimilar cell types present in varying concentrations in their respective cell mixtures and the demonstration of their integrity after separation open new avenues for time and cost-effective sorting of various cell types using the developed miniaturized platform. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lipid body accumulation alters calcium signaling dynamics in immune cells

PubMed Central

Greineisen, William E.; Speck, Mark; Shimoda, Lori M.N.; Sung, Carl; Phan, Nolwenn; Maaetoft-Udsen, Kristina; Stokes, Alexander J.; Turner, Helen

2014-01-01

Summary There is well-established variability in the numbers of lipid bodies (LB) in macrophages, eosinophils, and neutrophils. Similarly to the steatosis observed in adipocytes and hepatocytes during hyperinsulinemia and nutrient overload, immune cell LB hyper-accumulate in response to bacterial and parasitic infection and inflammatory presentations. Recently we described that hyperinsulinemia, both in vitro and in vivo, drives steatosis and phenotypic changes in primary and transformed mast cells and basophils. LB reach high numbers in these steatotic cytosols, and here we propose that they could dramatically impact the transcytoplasmic signaling pathways. We compared calcium release and influx responses at the population and single cell level in normal and steatotic model mast cells. At the population level, all aspects of FcεRI-dependent calcium mobilization, as well as activation of calcium-dependent downstream signalling targets such as NFATC1 phosphorylation are suppressed. At the single cell level, we demonstrate that LB are both sources and sinks of calcium following FcεRI cross-linking. Unbiased analysis of the impact of the presence of LB on the rate of trans-cytoplasmic calcium signals suggest that LB enrichment accelerates calcium propagation, which may reflect a Bernoulli effect. LB abundance thus impacts this fundamental signalling pathway and its downstream targets. PMID:25016314

Prodrug-based nano-drug delivery system for co-encapsulate paclitaxel and carboplatin for lung cancer treatment.

PubMed

Zhang, Wen; Li, Changzheng; Shen, Chengwu; Liu, Yuguo; Zhao, Xiaoting; Liu, Ying; Zou, Dongna; Gao, Zhenfa; Yue, Chunwen

2016-09-01

Paclitaxel (PTX) and carboplatin (CBP) are widely used for the combined chemotherapy of non-small cell lung cancer (NSCLC). However, the development of multidrug resistance of cancer cells, as well as systemic toxic side effects resulting from nonspecific localization of anticancer drugs to non-tumor areas are major obstacles to the success of chemotherapy in treating cancers. This study aimed to engineer a prodrug-based nano-drug delivery system for co-encapsulate hydrophilic (CBP) and hydrophobic anti-tumor drugs (PTX). This system was expected to resolve the multidrug resistance cause by single drug, and the dual-drug-loaded liposome was also planned to specifically target the cancer cells without obvious influence on normal cells and tissues. In this paper, PLGA-PEG-CBP was synthesized by the conjugation between the carboxylic group of PLGA-PEG-COOH and the amino group of CBP. Then, self-assembled nanoparticles for combination delivery of PTX and PLGA-PEG-CBP (PTX/CBP NPs) were prepared by solvent displacement technique. The in vitro and in vivo anti-tumor efficacy was assessed in NCL-H460 human non-small cell lung carcinoma cell line. PTX/CBP NPs achieved the highest cytotoxic effect among all formulations in vitro, as compared with single drug delivery NPs. In vivo investigation on NSCLC animal models showed that co-delivery of PTX and CBP possessed high tumor-targeting capacity and strong anti-tumor activity. The PTX/CBP NPs constructed in this research offers an effective strategy for targeted combinational lung cancer therapy.

Single-Domain Antibodies and the Promise of Modular Targeting in Cancer Imaging and Treatment.

PubMed

Iezzi, María Elena; Policastro, Lucía; Werbajh, Santiago; Podhajcer, Osvaldo; Canziani, Gabriela Alicia

2018-01-01

Monoclonal antibodies and their fragments have significantly changed the outcome of cancer in the clinic, effectively inhibiting tumor cell proliferation, triggering antibody-dependent immune effector cell activation and complement mediated cell death. Along with a continued expansion in number, diversity, and complexity of validated tumor targets there is an increasing focus on engineering recombinant antibody fragments for lead development. Single-domain antibodies (sdAbs), in particular those engineered from the variable heavy-chain fragment (VHH gene) found in Camelidae heavy-chain antibodies (or IgG2 and IgG3), are the smallest fragments that retain the full antigen-binding capacity of the antibody with advantageous properties as drugs. For similar reasons, growing attention is being paid to the yet smaller variable heavy chain new antigen receptor (VNAR) fragments found in Squalidae. sdAbs have been selected, mostly from immune VHH libraries, to inhibit or modulate enzyme activity, bind soluble factors, internalize cell membrane receptors, or block cytoplasmic targets. This succinct review is a compilation of recent data documenting the application of engineered, recombinant sdAb in the clinic as epitope recognition "modules" to build monomeric, dimeric and multimeric ligands that target, tag and stall solid tumor growth in vivo . Size, affinity, specificity, and the development profile of sdAbs drugs are seemingly consistent with desirable clinical efficacy and safety requirements. But the hepatotoxicity of the tetrameric anti-DR5-VHH drug in patients with pre-existing anti-drug antibodies halted the phase I clinical trial and called for a thorough pre-screening of the immune and poly-specific reactivities of the sdAb leads.

Human Parainfluenza Virus-3 can be Targeted by Rapidly ex vivo Expanded T-Lymphocytes

PubMed Central

McLaughlin, Lauren P.; Lang, Haili; Williams, Elizabeth; Wright, Kaylor E.; Powell, Allison; Cruz, Conrad R; Colberg-Poley, Anamaris M.; Barese, Cecilia; Hanley, Patrick J.; Bollard, Catherine M.; Keller, Michael D.

2016-01-01

Background Human Parainfluenza virus-3 (HPIV) is a common cause of respiratory infection in immunocompromised patients, and presently has no effective therapies. Virus-specific T-cell therapy has been successful for the treatment or prevention of viral infections in immunocompromised patients, but requires determination of T-cell antigens on targeted viruses. Methods HPIV3-specific T cells were expanded from peripheral blood of healthy donors using a rapid generation protocol targeting four HPIV3 proteins. Immunophenotyping was performed by flow cytometry. Viral specificity was determined by IFN-γ ELISpot, intracellular cytokine staining, and cytokine measurements from culture supernatants by Luminex assay. Cytotoxic activity was tested by 51Cr release and CD107a mobilization assays. Virus-specific T-cells targeting 6 viruses were then produced by rapid protocol, and the phenotype of HPIV3-specific T-cells was determined by immunomagnetic sorting for IFN-γ producing cells. Results HPIV3-specific T cells were expanded from 13 healthy donors. HPIV3-specific T-cells showed a CD4+ predominance (mean CD4:CD8 ratio 2.89), and demonstrated specificity for multiple HPIV3 antigens. The expanded T-cells were polyfunctional based on cytokine production, but only had a minor cytotoxic component. T cells targeting six viruses in a single product similarly showed HPIV3 specificity, with a predominant effector memory phenotype (CD3+/CD45RA-/CCR7-) in responder cells. Discussion HPIV3-specific T cells can be produced using a rapid ex vivo protocol from healthy donors and are predominantly CD4+ T-cells with Th1 activity. HPIV3 epitopes can also be successfully targeted alongside multiple other viral epitopes in production of 6-virus T-cells, without loss of HPIV3 specificity. These products may be clinically beneficial to combat HPIV3 infections by adoptive T-cell therapy in immune compromised patients. PMID:27692559

Selective Blockade of Human Natural Killer Cells by a Monoclonal Antibody

NASA Astrophysics Data System (ADS)

Newman, Walter

1982-06-01

A murine monoclonal antibody, 13.1, which blocks human natural killer (NK) cell-mediated lysis, has been developed. Hybridoma 13.1 was derived by fusion of NS-1 cells with spleen cells from mice immunized with an enriched population of NK cells. Supernatants of growing hybridomas were screened for their ability to block NK cell-mediated lysis of K562 targets. Antibody 13.1 is an IgG1 with a single light chain type and it does not fix complement. The 13.1 antigen is expressed on all peripheral blood mononuclear cells, with an antigen density approximately 1/30th that of HLA antigen heavy chain. Pretreatment and washing experiments revealed that inhibition of cytotoxicity occurred at the effector cell level only. Significant blocking was achieved with nanogram quantities of antibody and was not due to toxic effects on NK cells. Likewise, controls with other antibodies of the same subclass demonstrated that blocking was not a consequence of mere binding to NK cells. When a panel of 17 NK cell-susceptible targets was tested, the lysis of only 5 of these was blocked, namely K562, HL-60, KG-1, Daudi, and HEL, a human erythroleukemic cell line. The lysis of 12 human B cell and T cell line targets was not inhibited. In addition to the demonstration that the 13.1 antigen is a crucial cell surface structure involved in NK lysis, a heterogeneity of target cell recognition has been revealed that argues for the proposition that individual NK cells have multiple recognitive capabilities.

A resource of vectors and ES cells for targeted deletion of microRNAs in mice

PubMed Central

Prosser, Haydn M.; Koike-Yusa, Hiroko; Cooper, James D.; Law, Frances C.; Bradley, Allan

2011-01-01

The 21-23 nucleotide single-stranded RNAs classified as microRNAs (miRNA) perform fundamental roles in a wide range of cellular and developmental processes. miRNAs regulate protein expression through sequence-specific base pairing with target messenger RNAs (mRNA) reducing both their stability and the process of protein translation1, 2. At least 30% of protein coding genes appear to be conserved targets for miRNAs1. In contrast to the protein coding genes3, 4, no public resource of miRNA mouse mutant alleles exists. We have generated a library of highly germ-line transmissible C57BL/6N mouse mutant embryonic stem (ES) cells with targeted deletions for the majority of miRNA genes currently annotated within the miRBase registry5. These alleles have been designed to be highly adaptable research tools that can be efficiently altered to create reporter, conditional and other allelic variants. This ES cell resource can be searched electronically and is available from ES cell repositories for distribution to the scientific community6. PMID:21822254

Measles virus–specific plasma cells are prominent in subacute sclerosing panencephalitis CSF

PubMed Central

Owens, G.P.; Ritchie, A.M.; Gilden, D.H.; Burgoon, M.P.; Becker, D.; Bennett, J.L.

2012-01-01

Objective To demonstrate the specificity of expanded CD138+ plasma cell clones recovered from the CSF of a patient with subacute sclerosing panencephalitis (SSPE) for measles virus (MV). Methods IgG variable region sequences of single-antibody-secreting CD138+ cells sorted from SSPE CSF were amplified by single-cell PCR and analyzed. Human IgG1 recombinant antibodies (rAbs) were produced from four expanded CD138+ clones and assayed for immunoreactivity against MV proteins. Results Clonal expansion was a prominent feature of the SSPE plasma cell repertoire, and each of the four rAbs assayed was specific for either the MV fusion or the MV nucleocapsid protein. Conclusions Expanded plasma cell clones in the CSF of patients with subacute sclerosing panencephalitis produce disease-relevant antibodies. Recombinant antibodies derived from CSF B cells could provide a tool to identify target antigens in idiopathic inflammatory disorders. PMID:17515543

Impact of Diverse Immune Evasion Mechanisms of Cancer Cells on T Cells Engaged by EpCAM/CD3-Bispecific Antibody Construct AMG 110

PubMed Central

Deisting, Wibke; Raum, Tobias; Kufer, Peter; Baeuerle, Patrick A.; Münz, Markus

2015-01-01

Background Bispecific T cell engager (BiTE®) are single-chain bispecific antibody constructs with dual specificity for CD3 on T cells and a surface antigen on target cells. They can elicit a polyclonal cytotoxic T cell response that is not restricted by T cell receptor (TCR) specificity, and surface expression of MHC class I/peptide antigen complexes. Using human EpCAM/CD3-bispecific BiTE® antibody construct AMG 110, we here assessed to what extent surface expression of PD-L1, cytoplasmic expression of indoleamine-2,3-deoxygenase type 1, Bcl-2 and serpin PI-9, and the presence of transforming growth factor beta (TGF-β), interleukin-10 (IL-10) and adenosine in culture medium can impact redirected lysis by AMG 110-engaged T cells. Methods The seven factors, which are all involved in inhibiting T cell functions by cancer cells, were tested with human EpCAM-expressing Chinese hamster ovary (CHO) target cells at levels that in most cases exceeded those observed in a number of human cancer cell lines. Co-culture experiments were used to determine the impact of the evasion mechanisms on EC50 values and amplitude of redirected lysis by AMG 110, and on BiTE®-induced proliferation of previously resting human peripheral T cells. Findings An inhibitory effect on redirected lysis by AMG 110-engaged T cells was seen upon overexpression of serpin PI-9, Bcl-2, TGF-βand PD-L1. An inhibitory effect on induction of T cell proliferation was only seen with CHO cells overexpressing IDO. In no case, a single evasion mechanism rendered target cells completely resistant to BiTE®-induced lysis, and even various combinations could not. Conclusions Our data suggest that diverse mechanisms employed by cancer cells to fend off T cells cannot inactivate AMG 110-engaged T cells, and that inhibitory effects observed in vitro may be overcome by increased concentrations of the BiTE® antibody construct. PMID:26510188

Hypoxia-Targeting Drug Evofosfamide (TH-302) Enhances Sunitinib Activity in Neuroblastoma Xenograft Models.

PubMed

Kumar, Sushil; Sun, Jessica D; Zhang, Libo; Mokhtari, Reza Bayat; Wu, Bing; Meng, Fanying; Liu, Qian; Bhupathi, Deepthi; Wang, Yan; Yeger, Herman; Hart, Charles; Baruchel, Sylvain

2018-05-23

Antiangiogenic therapy has shown promising results in preclinical and clinical trials. However, tumor cells acquire resistance to this therapy by gaining ability to survive and proliferate under hypoxia induced by antiangiogenic therapy. Combining antiangiogenic therapy with hypoxia-activated prodrugs can overcome this limitation. Here, we have tested the combination of antiangiogenic drug sunitinib in combination with hypoxia-activated prodrug evofosfamide in neuroblastoma. In vitro, neuroblastoma cell line SK-N-BE(2) was 40-folds sensitive to evofosfamide under hypoxia compared to normoxia. In IV metastatic model, evofosfamide significantly increased mice survival compared to the vehicle (P=.02). In SK-N-BE(2) subcutaneous xenograft model, we tested two different treatment regimens using 30 mg/kg sunitinib and 50 mg/kg evofosfamide. Here, sunitinib therapy when started along with evofosfamide treatment showed higher efficacy compared to single agents in subcutaneous SK-N-BE(2) xenograft model, whereas sunitinib when started 7 days after evofosfamide treatment did not have any advantage compared to treatment with either single agent. Immunofluorescence of tumor sections revealed higher number of apoptotic cells and hypoxic areas compared to either single agent when both treatments were started together. Treatment with 80 mg/kg sunitinib with 50 mg/kg evofosfamide was significantly superior to single agents in both xenograft and metastatic models. This study confirms the preclinical efficacy of sunitinib and evofosfamide in murine models of aggressive neuroblastoma. Sunitinib enhances the efficacy of evofosfamide by increasing hypoxic areas, and evofosfamide targets hypoxic tumor cells. Consequently, each drug enhances the activity of the other. Copyright © 2018. Published by Elsevier Inc.

Targeted Nanoparticles for Image-guided Treatment of Triple Negative Breast Cancer: Clinical Significance and Technological Advances

PubMed Central

Miller-Kleinhenz, Jasmine M.; Bozeman, Erica N.

2015-01-01

Effective treatment of triple negative breast cancer (TNBC) with its aggressive tumor biology, highly heterogeneous tumor cells, and poor prognosis requires an integrated therapeutic approach that addresses critical issues in cancer therapy. Multifunctional nanoparticles with the abilities of targeted drug delivery and non-invasive imaging for monitoring drug delivery and responses to therapy, such as theranostic nanoparticles, hold great promise towards the development of novel therapeutic approaches for the treatment of TNBC using a single therapeutic platform. The biological and pathological characteristics of TNBC provide insight into several potential molecular targets for current and future nanoparticle based therapeutics. Extensive tumor stroma, highly proliferative cells, and a high rate of drug-resistance are all barriers that must be appropriately addressed in order for these nanotherapeutic platforms to be effective. Utilization of the enhanced permeability and retention (EPR) effect coupled with active targeting of cell surface receptors expressed by TNBC cells, and tumor associated endothelial cells, stromal fibroblasts and macrophages is likely to overcome such barriers to facilitate more effective drug delivery. An in depth summary of current studies investigating targeted nanoparticles in preclinical TNBC mouse and human xenograft models is presented. This review aims to outline the current status of nanotherapeutic options for TNBC patients, identification of promising molecular targets, challenges associated with the development of targeted nanotherapeutics, the research done by our group as well as others and future perspectives on the nanomedicine field and ways to translate current preclinical studies into the clinic. PMID:25966677

Applications of particle microbeams in space radiation research.

PubMed

Durante, Marco

2009-03-01

Galactic cosmic radiation is acknowledged as one of the major barriers to human space exploration. In space, astronauts are exposed to charged particles from Z = 1 (H) up to Z = 28 (Ni), but the probability of a hit to a specific single cell in the human body is low. Particle microbeams can deliver single charged particles of different charge and energy to single cells from different tissues, and microbeam studies are therefore very useful for improving current risk estimates for long-term space travel. 2D in vitro cell cultures can be very useful for establishing basic molecular mechanisms, but they are not sufficient to extrapolate risk, given the substantial evidence proving tissue effects are key in determining the response to radiation insult. 3D tissue or animal systems represent a more promising target for space radiobiology using microbeams.

Single cell–resolution western blotting

PubMed Central

Kang, Chi-Chih; Yamauchi, Kevin A; Vlassakis, Julea; Sinkala, Elly; Duncombe, Todd A; Herr, Amy E

2017-01-01

This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). the gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. to extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. once the microdevice has been fabricated, the assay can be completed in 4–6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. the technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine. PMID:27466711

Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity

PubMed Central

Guldevall, Karolin; Brandt, Ludwig; Forslund, Elin; Olofsson, Karl; Frisk, Thomas W.; Olofsson, Per E.; Gustafsson, Karin; Manneberg, Otto; Vanherberghen, Bruno; Brismar, Hjalmar; Kärre, Klas; Uhlin, Michael; Önfelt, Björn

2016-01-01

Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon–glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy. PMID:27092139

Recognition of Antigen-Specific B Cell Receptors From Chronic Lymphocytic Leukemia Patients By Synthetic Antigen Surrogates

PubMed Central

Sarkar, Mohosin; Liu, Yun; Morimoto, Jumpei; Peng, Haiyong; Aquino, Claudio; Rader, Christoph; Chiorazzi, Nicholas

2014-01-01

In patients with chronic lymphocytic leukemia (CLL), a single neoplastic antigen-specific B cell accumulates and overgrows other B cells, leading to immune deficiency. CLL is often treated with drugs that ablate all B cells, leading to further weakening of humoral immunity, and a more focused therapeutic strategy capable of targeting only the pathogenic B cells would represent a significant advance. One approach to this would be to develop synthetic surrogates of the CLL antigens allowing differentiation of the CLL cells and healthy B cells in a patient. Here, we describe discovery of non-peptidic molecules capable of targeting antigen-specific B cell receptors with good affinity and selectivity using a combinatorial library screen. We demonstrate that our hit compounds act as synthetic antigen surrogates and recognize CLL cells and not healthy B cells. Additionally, we argue that the technology we developed can be used for discovery of other classes of antigen surrogates. PMID:25467125

Recognition of antigen-specific B-cell receptors from chronic lymphocytic leukemia patients by synthetic antigen surrogates.

PubMed

Sarkar, Mohosin; Liu, Yun; Morimoto, Jumpei; Peng, Haiyong; Aquino, Claudio; Rader, Christoph; Chiorazzi, Nicholas; Kodadek, Thomas

2014-12-18

In patients with chronic lymphocytic leukemia (CLL), a single neoplastic antigen-specific B cell accumulates and overgrows other B cells, leading to immune deficiency. CLL is often treated with drugs that ablate all B cells, leading to further weakening of humoral immunity, and a more focused therapeutic strategy capable of targeting only the pathogenic B cells would represent a significant advance. One approach to this would be to develop synthetic surrogates of the CLL antigens allowing differentiation of the CLL cells and healthy B cells in a patient. Here, we describe nonpeptidic molecules capable of targeting antigen-specific B cell receptors with good affinity and selectivity using a combinatorial library screen. We demonstrate that our hit compounds act as synthetic antigen surrogates and recognize CLL cells and not healthy B cells. Additionally, we argue that the technology we developed can be used to identify other classes of antigen surrogates. Copyright © 2014 Elsevier Ltd. All rights reserved.

Insertion of targeting domains into the envelope glycoprotein of Moloney murine leukemia virus (MoMLV)-based vectors modulates the route of mCAT-1-mediated viral entry.

PubMed

Viejo-Borbolla, A; Pizzato, M; Blair, E D; Schulz, T F

2005-03-01

Several groups have inserted targeting domains into the envelope glycoprotein (Env) of Moloney murine leukemia virus (MoMLV) in an attempt to produce targeted retroviral vectors for human gene therapy. While binding of these modified Envs to the target molecule expressed on the surface of human cells was observed, specific high-titer infection of human cells expressing the target molecule was not achieved. Here we investigate the initial steps in the entry process of targeted MoMLV vectors both in murine and human cells expressing the MoMLV receptor, the mouse cationic amino acid transporter-1 (mCAT-1). We show that insertion of a small ligand targeted to E-selectin and of a single chain antibody (scFv) targeted to folate-binding protein (FBP) into the N-terminus of MoMLV Env results in the reduction of the infectivity and the kinetics of entry of the MoMLV vectors. The use of soluble receptor-binding domain (sRBD), bafilomycin A1 (BafA1) and methyl-beta-cyclodextrin (MbetaC) increase the infectivity of the MoMLV vectors targeted to FBP (MoMLV-FBP) suggesting that the scFv targeted to FBP increases the threshold for fusion and might re-route entry of the targeted MoMLV-FBP vector towards an endocytic, non-productive pathway.

Converting adult pancreatic islet α-cells into β-cells by targeting both Dnmt1 and Arx

PubMed Central

Chakravarthy, Harini; Gu, Xueying; Enge, Martin; Dai, Xiaoqing; Wang, Yong; Damond, Nicolas; Downie, Carolina; Liu, Kathy; Wang, Jing; Xing, Yuan; Chera, Simona; Thorel, Fabrizio; Quake, Stephen; Oberholzer, Jose; MacDonald, Patrick E.; Herrera, Pedro L.; Kim, Seung K.

2017-01-01

Summary Insulin-producing pancreatic β-cells in mice can slowly regenerate from glucagon-producing α-cells in settings like β-cell loss, but the basis of this conversion is unknown. Moreover it remains unclear if this intra-islet cell conversion is relevant to diseases like type 1 diabetes (T1D). We show that the α-cell regulators Aristaless-related homeobox (Arx) and DNA methyltransferase 1 (Dnmt1) maintain α-cell identity in mice. Within 3 months of Dnmt1 and Arx loss, lineage tracing and single cell RNA sequencing revealed extensive α-cell conversion into progeny resembling native β-cells. Physiological studies demonstrated that converted α-cells acquire hallmark β-cell electrophysiology, and show glucose-stimulated insulin secretion. In T1D patients, subsets of Glucagon-expressing cells show loss of DNMT1 and ARX, and produce Insulin and other β-cell factors, suggesting that DNMT1 and ARX maintain α-cell identity in humans. Our work reveals pathways regulated by Arx and Dnmt1 sufficient for achieving targeted generation of β-cells from adult pancreatic α-cells. PMID:28215845

Accelerated killing of cancer cells using a multifunctional single-walled carbon nanotube-based system for targeted drug delivery in combination with photothermal therapy.

PubMed

Jeyamohan, Prashanti; Hasumura, Takashi; Nagaoka, Yutaka; Yoshida, Yasuhiko; Maekawa, Toru; Kumar, D Sakthi

2013-01-01

The photothermal effect of single-walled carbon nanotubes (SWCNTs) in combination with the anticancer drug doxorubicin (DOX) for targeting and accelerated destruction of breast cancer cells is demonstrated in this paper. A targeted drug-delivery system was developed for selective killing of breast cancer cells with polyethylene glycol biofunctionalized and DOX-loaded SWCNTs conjugated with folic acid. In our work, in vitro drug-release studies showed that the drug (DOX) binds at physiological pH (pH 7.4) and is released only at a lower pH, ie, lysosomal pH (pH 4.0), which is the characteristic pH of the tumor environment. A sustained release of DOX from the SWCNTs was observed for a period of 3 days. SWCNTs have strong optical absorbance in the near-infrared (NIR) region. In this special spectral window, biological systems are highly transparent. Our study reports that under laser irradiation at 800 nm, SWCNTs exhibited strong light-heat transfer characteristics. These optical properties of SWCNTs open the way for selective photothermal ablation in cancer therapy. It was also observed that internalization and uptake of folate-conjugated NTs into cancer cells was achieved by a receptor-mediated endocytosis mechanism. Results of the in vitro experiments show that laser was effective in destroying the cancer cells, while sparing the normal cells. When the above laser effect was combined with DOX-conjugated SWCNTs, we found enhanced and accelerated killing of breast cancer cells. Thus, this nanodrug-delivery system, consisting of laser, drug, and SWCNTs, looks to be a promising selective modality with high treatment efficacy and low side effects for cancer therapy.

Ion mediated targeting of cells with nanoparticles

NASA Astrophysics Data System (ADS)

Maheshwari, Vivek; Fu, Jinlong

2010-03-01

In eukaryotic cells, Ca^2+ ions are necessary for intracellular signaling, in activity of mitochondria and a variety of other cellular process that have been linked to cell apoptosis, proteins synthesis and cell-cycle regulation. Here we show that Ca^2+ ions, serving as the bio-compatible interface can be used to target Saccharomyces cerevisiae (SaC, baker's yeast), a model eukaryotic cell, with Au nanoparticles (10 nm). The Ca^2+ ions bind to the carboxylic acid groups in the citrate functionalized Au nanoparticles. This transforms the nanoparticles into micron long 1-D branched chain assemblies due to inter-particle dipole-dipole interaction and inter-particle bonding due to the divalent nature of the Ca^2+ ion. A similar transformation is observed with the use of divalent ions Mg^2+, Cd^2+ and Fe^2+. The 1-D assembly aids the interfacing of ion-nanoparticles on the cell by providing multiple contact points. Further monovalent ions such as Na^+ are also effective for the targeting of the cell with nanoparticles. However Na-Au nanoparticles are limited in their deposition as they exist in solution as single particles. The cells remain alive after the deposition process and their vitality is unaffected by the interfacing with ion-nanoparticles.

Computational discovery of pathway-level genetic vulnerabilities in non-small-cell lung cancer

PubMed Central

Young, Jonathan H.; Peyton, Michael; Seok Kim, Hyun; McMillan, Elizabeth; Minna, John D.; White, Michael A.; Marcotte, Edward M.

2016-01-01

Motivation: Novel approaches are needed for discovery of targeted therapies for non-small-cell lung cancer (NSCLC) that are specific to certain patients. Whole genome RNAi screening of lung cancer cell lines provides an ideal source for determining candidate drug targets. Results: Unsupervised learning algorithms uncovered patterns of differential vulnerability across lung cancer cell lines to loss of functionally related genes. Such genetic vulnerabilities represent candidate targets for therapy and are found to be involved in splicing, translation and protein folding. In particular, many NSCLC cell lines were especially sensitive to the loss of components of the LSm2-8 protein complex or the CCT/TRiC chaperonin. Different vulnerabilities were also found for different cell line subgroups. Furthermore, the predicted vulnerability of a single adenocarcinoma cell line to loss of the Wnt pathway was experimentally validated with screening of small-molecule Wnt inhibitors against an extensive cell line panel. Availability and implementation: The clustering algorithm is implemented in Python and is freely available at https://bitbucket.org/youngjh/nsclc_paper. Contact: marcotte@icmb.utexas.edu or jon.young@utexas.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26755624

Computational discovery of pathway-level genetic vulnerabilities in non-small-cell lung cancer.

PubMed

Young, Jonathan H; Peyton, Michael; Seok Kim, Hyun; McMillan, Elizabeth; Minna, John D; White, Michael A; Marcotte, Edward M

2016-05-01

Novel approaches are needed for discovery of targeted therapies for non-small-cell lung cancer (NSCLC) that are specific to certain patients. Whole genome RNAi screening of lung cancer cell lines provides an ideal source for determining candidate drug targets. Unsupervised learning algorithms uncovered patterns of differential vulnerability across lung cancer cell lines to loss of functionally related genes. Such genetic vulnerabilities represent candidate targets for therapy and are found to be involved in splicing, translation and protein folding. In particular, many NSCLC cell lines were especially sensitive to the loss of components of the LSm2-8 protein complex or the CCT/TRiC chaperonin. Different vulnerabilities were also found for different cell line subgroups. Furthermore, the predicted vulnerability of a single adenocarcinoma cell line to loss of the Wnt pathway was experimentally validated with screening of small-molecule Wnt inhibitors against an extensive cell line panel. The clustering algorithm is implemented in Python and is freely available at https://bitbucket.org/youngjh/nsclc_paper marcotte@icmb.utexas.edu or jon.young@utexas.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

Heterodimeric bispecific single chain variable fragments (scFv) killer engagers (BiKEs) enhance NK-cell activity against CD133+ colorectal cancer cells

PubMed Central

JU, Schmohl; MK, Gleason; PR, Dougherty; JS, Miller; DA, Vallera

2015-01-01

Background Natural killer (NK) cells are potent cytotoxic lymphocytes that play a critical role in tumor immunosurveillance and control. Cancer stem cells (CSC) initiate and sustain tumor cell growth, mediate drug refractory cancer relapse and express the well-known surface marker CD133. Methods DNA fragments from two fully humanized single chain fragment variable (scFv) antibody recognizing CD16 on NK-cells and CD133 on CSC were genetically spliced forming a novel drug, 16 × 133 BiKE that simultaneously recognizes these antigen to facilitate an immunologic synapse. The anti-CD133 was created using a fusion protein prepared by fusing DNA fragments encoding the two extracellular domains of CD133. Immunization of mice with the resulting fusion protein generated an unique antibody that recognized the molecular framework and was species cross-reactive. Results In vitro 51chromium release cytotoxicity assays at both high and low effector:target ratios demonstrated the ability of the heterodimeric biological drug to greatly enhance NK-cell killing of human Caco-2 colorectal carcinoma cells known to overexpress CD133. The tumor associated antigen specificity of the drug for CD133 even enhanced NK-cell cytotoxicity against the NK-resistant human Burkitt's lymphoma Daudi cell line, which has less than 5% CD133 surface expression. Flow cytometry analysis revealed increases in NK-cell degranulation and Interferon-γ production upon co-culture with Caco-2 targets in the presence of the drug. Conclusion These studies demonstrate that the innate immune system can be effectively recruited to kill CSC using bispecific antibodies targeting CD133, and that this anti-CD133 scFv may be useful in this bispecific platform or, perhaps, in the design of more complex trispecific molecules for carcinoma therapy. PMID:26566946

Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution

PubMed Central

Ludlow, Andrew T.; Robin, Jerome D.; Sayed, Mohammed; Litterst, Claudia M.; Shelton, Dawne N.; Shay, Jerry W.; Wright, Woodring E.

2014-01-01

The telomere repeat amplification protocol (TRAP) for the human reverse transcriptase, telomerase, is a PCR-based assay developed two decades ago and is still used for routine determination of telomerase activity. The TRAP assay can only reproducibly detect ∼2-fold differences and is only quantitative when compared to internal standards and reference cell lines. The method generally involves laborious radioactive gel electrophoresis and is not conducive to high-throughput analyzes. Recently droplet digital PCR (ddPCR) technologies have become available that allow for absolute quantification of input deoxyribonucleic acid molecules following PCR. We describe the reproducibility and provide several examples of a droplet digital TRAP (ddTRAP) assay for telomerase activity, including quantitation of telomerase activity in single cells, telomerase activity across several common telomerase positive cancer cells lines and in human primary peripheral blood mononuclear cells following mitogen stimulation. Adaptation of the TRAP assay to digital format allows accurate and reproducible quantification of the number of telomerase-extended products (i.e. telomerase activity; 57.8 ± 7.5) in a single HeLa cell. The tools developed in this study allow changes in telomerase enzyme activity to be monitored on a single cell basis and may have utility in designing novel therapeutic approaches that target telomerase. PMID:24861623

Cancer cell-selective, clathrin-mediated endocytosis of aptamer decorated nanoparticles

PubMed Central

Engelberg, Shira; Modrejewski, Julia; Walter, Johanna G.; Livney, Yoav D.; Assaraf, Yehuda G.

2018-01-01

Lung cancer is the leading cause of cancer mortality worldwide, resulting in 88% deaths of all diagnosed patients. Hence, novel therapeutic modalities are urgently needed. Single-stranded oligonucleotide-based aptamers (APTs) are excellent ligands for tumor cell targeting. However, the molecular mechanisms underlying their internalization into living cells have been poorly studied. Towards the application of APTs for active drug targeting to cancer cells, we herein studied the mechanism underlying S15-APT internalization into human non-small cell lung cancer A549 cells. We thus delineated the mode of entry of a model nanomedical system based on quantum dots (QDs) decorated with S15-APTs as a selective targeting moiety for uptake by A549 cells. These APT-decorated QDs displayed selective binding to, and internalization by target A549 cells, but not by normal human bronchial epithelial BEAS2B, cervical carcinoma (HeLa) and colon adenocarcinoma CaCo-2 cells, hence demonstrating high specificity. Flow cytometric analysis revealed a remarkably low dissociation constant of S15-APTs-decorated QDs to A549 cells (Kd = 13.1 ± 1.6 nM). Through the systematic application of a series of established inhibitors of known mechanisms of endocytosis, we show that the uptake of S15-APTs proceeds via a classical clathrin-dependent receptor-mediated endocytosis. This cancer cell-selective mode of entry could possibly be used in the future to evade plasma membrane-localized multidrug resistance efflux pumps, thereby overcoming an important mechanism of cancer multidrug resistance. PMID:29765515

Cancer cell-selective, clathrin-mediated endocytosis of aptamer decorated nanoparticles.

PubMed

Engelberg, Shira; Modrejewski, Julia; Walter, Johanna G; Livney, Yoav D; Assaraf, Yehuda G

2018-04-20

Lung cancer is the leading cause of cancer mortality worldwide, resulting in 88% deaths of all diagnosed patients. Hence, novel therapeutic modalities are urgently needed. Single-stranded oligonucleotide-based aptamers (APTs) are excellent ligands for tumor cell targeting. However, the molecular mechanisms underlying their internalization into living cells have been poorly studied. Towards the application of APTs for active drug targeting to cancer cells, we herein studied the mechanism underlying S15-APT internalization into human non-small cell lung cancer A549 cells. We thus delineated the mode of entry of a model nanomedical system based on quantum dots (QDs) decorated with S15-APTs as a selective targeting moiety for uptake by A549 cells. These APT-decorated QDs displayed selective binding to, and internalization by target A549 cells, but not by normal human bronchial epithelial BEAS2B, cervical carcinoma (HeLa) and colon adenocarcinoma CaCo-2 cells, hence demonstrating high specificity. Flow cytometric analysis revealed a remarkably low dissociation constant of S15-APTs-decorated QDs to A549 cells (K d = 13.1 ± 1.6 nM). Through the systematic application of a series of established inhibitors of known mechanisms of endocytosis, we show that the uptake of S15-APTs proceeds via a classical clathrin-dependent receptor-mediated endocytosis. This cancer cell-selective mode of entry could possibly be used in the future to evade plasma membrane-localized multidrug resistance efflux pumps, thereby overcoming an important mechanism of cancer multidrug resistance.

Functional kinomics identifies candidate therapeutic targets in head and neck cancer

PubMed Central

Moser, Russell; Xu, Chang; Kao, Michael; Annis, James; Lerma, Luisa Angelica; Schaupp, Christopher M.; Gurley, Kay E.; Jang, In Sock; Biktasova, Asel; Yarbrough, Wendell G.; Margolin, Adam A.; Grandori, Carla; Kemp, Christopher J.; Méndez, Eduardo

2014-01-01

Purpose To identify novel therapeutic drug targets for p53 mutant head and neck squamous cell carcinoma (HNSCC). Experimental Design RNAi kinome viability screens were performed on HNSCC cells including autologous pairs from primary tumor and recurrent/metastatic lesions, and in parallel on murine squamous cell carcinoma (MSCC) cells derived from tumors of inbred mice bearing germline mutations in Trp53, and p53 regulatory genes: Atm, Prkdc, and p19Arf. Cross-species analysis of cell lines stratified by p53 mutational status and metastatic phenotype was utilized to select 38 kinase targets. Both primary and secondary RNAi validation assays were performed on additional HNSCC cell lines to credential these kinase targets utilizing multiple phenotypic endpoints. Kinase targets were also examined via chemical inhibition utilizing a panel of kinase inhibitors. A preclinical study was conducted on the WEE1 kinase inhibitor, MK-1775. Results Our functional kinomics approach identified novel survival kinases in HNSCC involved in G2/M cell cycle checkpoint, SFK, PI3K and FAK pathways. RNAi mediated knockdown and chemical inhibition of the WEE1 kinase with a specific inhibitor, MK-1775, had a significant effect on both viability and apoptosis. Sensitivity to the MK-1775 kinase inhibitor is in part determined by p53 mutational status, and due to unscheduled mitotic entry. MK-1775 displays single-agent activity and potentiates the efficacy of cisplatin in a p53 mutant HNSCC xenograft model. Conclusions WEE1 kinase is a potential therapeutic drug target for HNSCC. This study supports the application of a functional kinomics strategy to identify novel therapeutic targets for cancer. PMID:25125259

Functional kinomics identifies candidate therapeutic targets in head and neck cancer.

PubMed

Moser, Russell; Xu, Chang; Kao, Michael; Annis, James; Lerma, Luisa Angelica; Schaupp, Christopher M; Gurley, Kay E; Jang, In Sock; Biktasova, Asel; Yarbrough, Wendell G; Margolin, Adam A; Grandori, Carla; Kemp, Christopher J; Méndez, Eduardo

2014-08-15

To identify novel therapeutic drug targets for p53-mutant head and neck squamous cell carcinoma (HNSCC). RNAi kinome viability screens were performed on HNSCC cells, including autologous pairs from primary tumor and recurrent/metastatic lesions, and in parallel on murine squamous cell carcinoma (MSCC) cells derived from tumors of inbred mice bearing germline mutations in Trp53, and p53 regulatory genes: Atm, Prkdc, and p19(Arf). Cross-species analysis of cell lines stratified by p53 mutational status and metastatic phenotype was used to select 38 kinase targets. Both primary and secondary RNAi validation assays were performed on additional HNSCC cell lines to credential these kinase targets using multiple phenotypic endpoints. Kinase targets were also examined via chemical inhibition using a panel of kinase inhibitors. A preclinical study was conducted on the WEE1 kinase inhibitor, MK-1775. Our functional kinomics approach identified novel survival kinases in HNSCC involved in G2-M cell-cycle checkpoint, SFK, PI3K, and FAK pathways. RNAi-mediated knockdown and chemical inhibition of the WEE1 kinase with a specific inhibitor, MK-1775, had a significant effect on both viability and apoptosis. Sensitivity to the MK-1775 kinase inhibitor is in part determined by p53 mutational status, and due to unscheduled mitotic entry. MK-1775 displays single-agent activity and potentiates the efficacy of cisplatin in a p53-mutant HNSCC xenograft model. WEE1 kinase is a potential therapeutic drug target for HNSCC. This study supports the application of a functional kinomics strategy to identify novel therapeutic targets for cancer. ©2014 American Association for Cancer Research.

Targeting Fibroblast Activation Protein in Tumor Stroma with Chimeric Antigen Receptor T Cells Can Inhibit Tumor Growth and Augment Host Immunity Without Severe Toxicity

PubMed Central

Wang, Liang-Chuan S; Lo, Albert; Scholler, John; Sun, Jing; Majumdar, Rajrupa S; Kapoor, Veena; Antzis, Michael; Cotner, Cody E.; Johnson, Laura A; Durham, Amy C; Solomides, Charalambos C.; June, Carl H; Puré, Ellen; Albelda, Steven M

2013-01-01

The majority of chimeric antigen receptor (CAR) T cell research has focused on attacking cancer cells. Here we show that targeting the tumor-promoting, non-transformed stromal cells using CAR T cells may offer several advantages. We developed a retroviral CAR construct specific for the mouse fibroblast activation protein (FAP), comprising a single chain Fv FAP (mAb 73.3) with the CD8α hinge and transmembrane regions, and the human CD3ζ and 4-1BB activation domains. The transduced muFAP-CAR mouse T cells secreted IFNγ and killed FAP-expressing 3T3 target cells specifically. Adoptively transferred 73.3-FAP-CAR mouse T cells selectively reduced FAPhi stromal cells and inhibited the growth of multiple types of subcutaneously transplanted tumors in wild-type, but not FAP-null immune-competent syngeneic mice. The antitumor effects could be augmented by multiple injections of the CAR T cells, by using CAR T cells with a deficiency in diacylglycerol kinase, or by combination with a vaccine. A major mechanism of action of the muFAP-CAR T cells was the augmentation of the endogenous CD8+ T cell antitumor responses. Off-tumor toxicity in our models was minimal following muFAP-CAR T cell therapy. In summary, inhibiting tumor growth by targeting tumor stroma with adoptively transferred CAR T cells directed to FAP can be safe and effective suggesting that further clinical development of anti-human FAP-CAR is warranted. PMID:24778279

Chapter 17. Extension of endogenous primers as a tool to detect micro-RNA targets.

PubMed

Vatolin, Sergei; Weil, Robert J

2008-01-01

Mammalian cells express a large number of small, noncoding RNAs, including micro-RNAs (miRNAs), that can regulate both the level of a target mRNA and the protein produced by the target mRNA. Recognition of miRNA targets is a complicated process, as a single target mRNA may be regulated by several miRNAs. The potential for combinatorial miRNA-mediated regulation of miRNA targets complicates diagnostic and therapeutic applications of miRNAs. Despite significant progress in understanding the biology of miRNAs and advances in computational predictions of miRNA targets, methods that permit direct physical identification of miRNA-mRNA complexes in eukaryotic cells are still required. Several groups have utilized coimmunoprecipitation of RNA associated with a protein(s) that is part of the RNA silencing macromolecular complex. This chapter describes a detailed but straightforward strategy that identifies miRNA targets based on the assumption that small RNAs base paired with a complementary target mRNA can be used as a primer to synthesize cDNA that may be used for cloning, identification, and functional analysis.

RNAi of COL1A1 in mesenchymal progenitor cells.

PubMed

Millington-Ward, Sophia; McMahon, Helena P; Allen, Danny; Tuohy, Gearóid; Kiang, Anna-Sophia; Palfi, Arpad; Kenna, Paul F; Humphries, Peter; Farrar, G Jane

2004-10-01

Given that mutant COL1A1 is known to cause Osteogenesis Imperfecta (OI), tools to modulate COL1A1 expression are likely to be of significant therapeutic value. In this context, we have evaluated RNA interference (RNAi) as a means to downregulate COL1A1 expression in Cos-7 cells and in human mesenchymal progenitor stem cells (MPCs), the latter cells giving rise to bone and therefore representing a target cell type for collagen-related disorders. In addition, allele-specificity, a key factor to the success of RNAi-based suppression, was explored with a view to developing a mutation-independent RNAi-based therapeutic for OI by targeting an intragenic SNP within transcripts derived from the COL1A1 gene. Preferential suppression of individual polymorphic alleles that differed by a single nucleotide was observed.

Evaluation of a new continuous mononuclear cell collection procedure in a single transplant center cohort enriched for AL amyloidosis patients.

PubMed

Pudusseri, Anita; Smith, India; Sarnacki, Diane; Brauneis, Dina; Shelton, Anthony; Sanchorawala, Vaishali; Sloan, J Mark; Sarosiek, Shayna; Quillen, Karen

2018-04-26

The Spectra Optia continuous mononuclear cell (CMNC) program is newly available, and herein validated in a single-center cohort enriched with AL amyloidosis patients to collect a target CD34+ yield of 2.5 × 10 6 cells/kg within 2 days. Consecutive autologous transplant patients in 2016 are included. Patients undergo leukapheresis with Optia CMNC and Spectra v4.7 over a 2-day cycle. Data collection includes collection efficiency, adverse events and engraftment kinetics. 36 leukapheresis procedures on 18 patients are included. The diagnoses are AL amyloidosis (9), myeloma (7), lymphoma (2), and scleroderma (1). Median age is 60; 12 are men. Plerixafor was employed pre-emptively in 6 cycles. Median blood CD34+ on Day 1 of leukapheresis was 46 cells/uL. Median number of blood volumes processed on Day 1 was 3.1. All collection cycles were completed within 2 days; only one in a heavily pretreated lymphoma patient did not reach the target requiring a second mobilization attempt. Mean collection efficiencies were comparable between the two devices. There were 2 adverse events: tubing rupture on the Optia; and one case of hypotension. All 18 patients underwent high-dose chemotherapy: median cell dose infused was 7.7 × 10 6 CD34+ cells/kg. Median days to neutrophil and platelet engraftment were 10 and 13 respectively. The Optia CMNC collection protocol is safe and effective in a small single-center autologous stem cell transplant cohort enriched for high-risk patients with AL amyloidosis and cardiac involvement. Caution is needed for tubing setup because there is less cumulative experience with Optia. Copyright © 2018 Elsevier Ltd. All rights reserved.

Genome Editing in Mice Using TALE Nucleases.

PubMed

Wefers, Benedikt; Brandl, Christina; Ortiz, Oskar; Wurst, Wolfgang; Kühn, Ralf

2016-01-01

Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as sequence-specific nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step without the need for embryonic stem cells. By embryo microinjection of TALEN mRNAs and targeting vectors, knockout and knock-in alleles can be generated fast and efficiently. In this chapter we provide protocols for the application of TALENs in mouse zygotes.

Chimeric Antigen Receptor-Engineered NK-92 Cells: An Off-the-Shelf Cellular Therapeutic for Targeted Elimination of Cancer Cells and Induction of Protective Antitumor Immunity.

PubMed

Zhang, Congcong; Oberoi, Pranav; Oelsner, Sarah; Waldmann, Anja; Lindner, Aline; Tonn, Torsten; Wels, Winfried S

2017-01-01

Significant progress has been made in recent years toward realizing the potential of natural killer (NK) cells for cancer immunotherapy. NK cells can respond rapidly to transformed and stressed cells and have the intrinsic potential to extravasate and reach their targets in almost all body tissues. In addition to donor-derived primary NK cells, also the established NK cell line NK-92 is being developed for adoptive immunotherapy, and general safety of infusion of irradiated NK-92 cells has been established in phase I clinical trials with clinical responses observed in some of the cancer patients treated. To enhance their therapeutic utility, NK-92 cells have been modified to express chimeric antigen receptors (CARs) composed of a tumor-specific single chain fragment variable antibody fragment fused via hinge and transmembrane regions to intracellular signaling moieties such as CD3ζ or composite signaling domains containing a costimulatory protein together with CD3ζ. CAR-mediated activation of NK cells then bypasses inhibitory signals and overcomes NK resistance of tumor cells. In contrast to primary NK cells, CAR-engineered NK-92 cell lines suitable for clinical development can be established from molecularly and functionally well-characterized single cell clones following good manufacturing practice-compliant procedures. In preclinical in vitro and in vivo models, potent antitumor activity of NK-92 variants targeted to differentiation antigens expressed by hematologic malignancies, and overexpressed or mutated self-antigens associated with solid tumors has been found, encouraging further development of CAR-engineered NK-92 cells. Importantly, in syngeneic mouse tumor models, induction of endogenous antitumor immunity after treatment with CAR-expressing NK-92 cells has been demonstrated, resulting in cures and long-lasting immunological memory protecting against tumor rechallenge at distant sites. Here, we summarize the current status and future prospects of CAR-engineered NK-92 cells as off-the-shelf cellular therapeutics, with special emphasis on ErbB2 (HER2)-specific NK-92 cells that are approaching clinical application.

Chimeric Antigen Receptor-Engineered NK-92 Cells: An Off-the-Shelf Cellular Therapeutic for Targeted Elimination of Cancer Cells and Induction of Protective Antitumor Immunity

PubMed Central

Zhang, Congcong; Oberoi, Pranav; Oelsner, Sarah; Waldmann, Anja; Lindner, Aline; Tonn, Torsten; Wels, Winfried S.

2017-01-01

Significant progress has been made in recent years toward realizing the potential of natural killer (NK) cells for cancer immunotherapy. NK cells can respond rapidly to transformed and stressed cells and have the intrinsic potential to extravasate and reach their targets in almost all body tissues. In addition to donor-derived primary NK cells, also the established NK cell line NK-92 is being developed for adoptive immunotherapy, and general safety of infusion of irradiated NK-92 cells has been established in phase I clinical trials with clinical responses observed in some of the cancer patients treated. To enhance their therapeutic utility, NK-92 cells have been modified to express chimeric antigen receptors (CARs) composed of a tumor-specific single chain fragment variable antibody fragment fused via hinge and transmembrane regions to intracellular signaling moieties such as CD3ζ or composite signaling domains containing a costimulatory protein together with CD3ζ. CAR-mediated activation of NK cells then bypasses inhibitory signals and overcomes NK resistance of tumor cells. In contrast to primary NK cells, CAR-engineered NK-92 cell lines suitable for clinical development can be established from molecularly and functionally well-characterized single cell clones following good manufacturing practice-compliant procedures. In preclinical in vitro and in vivo models, potent antitumor activity of NK-92 variants targeted to differentiation antigens expressed by hematologic malignancies, and overexpressed or mutated self-antigens associated with solid tumors has been found, encouraging further development of CAR-engineered NK-92 cells. Importantly, in syngeneic mouse tumor models, induction of endogenous antitumor immunity after treatment with CAR-expressing NK-92 cells has been demonstrated, resulting in cures and long-lasting immunological memory protecting against tumor rechallenge at distant sites. Here, we summarize the current status and future prospects of CAR-engineered NK-92 cells as off-the-shelf cellular therapeutics, with special emphasis on ErbB2 (HER2)-specific NK-92 cells that are approaching clinical application. PMID:28572802

Protease activated receptor-2 (PAR2): possible target of phytochemicals.

PubMed

Kakarala, Kavita Kumari; Jamil, Kaiser

2015-09-01

The use of phytochemicals either singly or in combination with other anticancer drugs comes with an advantage of less toxicity and minimal side effects. Signaling pathways play central role in cell cycle, cell growth, metabolism, etc. Thus, the identification of phytochemicals with promising antagonistic effect on the receptor/s playing key role in single transduction may have better therapeutic application. With this background, phytochemicals were screened against protease-activated receptor 2 (PAR2). PAR2 belongs to the superfamily of GPCRs and is an important target for breast cancer. Using in silico methods, this study was able to identify the phytochemicals with promising binding affinity suggesting their therapeutic potential in the treatment of breast cancer. The findings from this study acquires importance as the information on the possible agonists and antagonists of PAR2 is limited due its unique mechanism of activation.

Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation.

PubMed

Zhyvoloup, Alexander; Melamed, Anat; Anderson, Ian; Planas, Delphine; Lee, Chen-Hsuin; Kriston-Vizi, Janos; Ketteler, Robin; Merritt, Andy; Routy, Jean-Pierre; Ancuta, Petronela; Bangham, Charles R M; Fassati, Ariberto

2017-07-01

HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.

Development of Raman Spectroscopy as a Clinical Diagnostic Tool

NASA Astrophysics Data System (ADS)

Borel, Santa

Raman spectroscopy is the collection of inelastically scattered light in which the spectra contain biochemical information of the probed cells or tissue. This work presents both targeted and untargeted ways that the technique can be exploited in biological samples. First, surface enhanced Raman scattering (SERS) gold nanoparticles conjugated to targeting antibodies were shown to be successful for multiplexed detection of overexpressed surface antigens in lung cancer cell lines. Further work will need to optimize the conjugation technique to preserve the strong binding affinity of the antibodies. Second, untargeted Raman microspectroscopy combined with multivariate statistical analysis was able to successfully differentiate mouse ovarian surface epithelial (MOSE) cells and spontaneously transformed ovarian surface epithelial (STOSE) cells with high accuracy. The differences between the two groups were associated with increased nucleic acid content in the STOSE cells. This shows potential for single cell detection of ovarian cancer.

Dual-targeting Wnt and uPA receptors using peptide conjugated ultra-small nanoparticle drug carriers inhibited cancer stem-cell phenotype in chemo-resistant breast cancer.

PubMed

Miller-Kleinhenz, Jasmine; Guo, Xiangxue; Qian, Weiping; Zhou, Hongyu; Bozeman, Erica N; Zhu, Lei; Ji, Xin; Wang, Y Andrew; Styblo, Toncred; O'Regan, Ruth; Mao, Hui; Yang, Lily

2018-01-01

Heterogeneous tumor cells, high incidence of tumor recurrence, and decrease in overall survival are the major challenges for the treatment of chemo-resistant breast cancer. Results of our study showed differential chemotherapeutic responses among breast cancer patient derived xenograft (PDX) tumors established from the same patients. All doxorubicin (Dox)-resistant tumors expressed higher levels of cancer stem-like cell biomarkers, including CD44, Wnt and its receptor LRP5/6, relative to Dox-sensitive tumors. To effectively treat resistant tumors, we developed an ultra-small magnetic iron oxide nanoparticle (IONP) drug carrier conjugated with peptides that are dually targeted to Wnt/LRP5/6 and urokinase plasminogen activator receptor (uPAR). Our results showed that simultaneous binding to LRP5/6 and uPAR by the dual receptor targeted IONPs was required to inhibit breast cancer cell invasion. Molecular analysis revealed that the dual receptor targeted IONPs significantly inhibited Wnt/β-catenin signaling and cancer stem-like phenotype of tumor cells, with marked reduction of Wnt ligand, CD44 and uPAR. Systemic administration of the dual targeted IONPs led to nanoparticle-drug delivery into PDX tumors, resulting in stronger tumor growth inhibition compared to non-targeted or single-targeted IONP-Dox in a human breast cancer PDX model. Therefore, co-targeting Wnt/LRP and uPAR using IONP drug carriers is a promising therapeutic approach for effective drug delivery to chemo-resistant breast cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

A triplex ribozyme expression system based on a single hairpin ribozyme.

PubMed

Aquino-Jarquin, Guillermo; Benítez-Hess, María Luisa; DiPaolo, Joseph A; Alvarez-Salas, Luis M

2008-09-01

Triplex ribozyme (RZ) configurations allow for the individual activity of trans-acting RZs in multiple expression cassettes (multiplex), thereby increasing target cleavage relative to conventionally expressed RZs. Although hairpin RZs have been advantageously compared to hammerhead RZs, their longer size and structural features complicated triplex design. We present a triplex expression system based on a single hairpin RZ with transcleavage capability and simple engineering. The system was tested in vitro using cis- and trans-cleavage kinetic assays against a known target RNA from HPV-16 E6/E7 mRNA. Single and multiplex triplex RZ constructs were more efficient in cleaving the target than tandem-cloned hairpin RZs, suggesting that the release of individual RZs enhanced trans-cleavage kinetics. Multiplex systems constructed with two different hairpin RZs resulted in better trans-cleavage compared to standard double-RZ constructs. In addition, the triplex RZ performed cis- and trans-cleavage in cervical cancer cells. The use of triplex configurations with multiplex RZs permit differential targeting of the same or different RNA, thus improving potential use against unstable targets. This prototype will provide the basis for the development of future RZ-based therapies and technologies.

SIMSISH Technique Does Not Alter the Apparent Isotopic Composition of Bacterial Cells

PubMed Central

Chapleur, Olivier; Wu, Ting-Di; Guerquin-Kern, Jean-Luc; Mazéas, Laurent; Bouchez, Théodore

2013-01-01

In order to identify the function of uncultured microorganisms in their environment, the SIMSISH method, combining in situ hybridization (ISH) and nanoscale secondary ion mass spectrometry (nanoSIMS) imaging, has been proposed to determine the quantitative uptake of specific labelled substrates by uncultured microbes at the single cell level. This technique requires the hybridization of rRNA targeted halogenated DNA probes on fixed and permeabilized microorganisms. Exogenous atoms are introduced into cells and endogenous atoms removed during the experimental procedures. Consequently differences between the original and the apparent isotopic composition of cells may occur. In the present study, the influence of the experimental procedures of SIMSISH on the isotopic composition of carbon in E. coli cells was evaluated with nanoSIMS and compared to elemental analyser-isotopic ratio mass spectrometer (EA-IRMS) measurements. Our results show that fixation and hybridization have a very limited, reproducible and homogeneous influence on the isotopic composition of cells. Thereby, the SIMSISH procedure minimizes the contamination of the sample by exogenous atoms, thus providing a means to detect the phylogenetic identity and to measure precisely the carbon isotopic composition at the single cell level. This technique was successfully applied to a complex sample with double bromine – iodine labelling targeting a large group of bacteria and a specific archaea to evaluate their specific 13C uptake during labelled methanol anaerobic degradation. PMID:24204855

Single-cell analysis of dihydroartemisinin-induced apoptosis through reactive oxygen species-mediated caspase-8 activation and mitochondrial pathway in ASTC-a-1 cells using fluorescence imaging techniques

NASA Astrophysics Data System (ADS)

Lu, Ying-Ying; Chen, Tong-Sheng; Wang, Xiao-Ping; Li, Li

2010-07-01

Dihydroartemisinin (DHA), a front-line antimalarial herbal compound, has been shown to possess promising anticancer activity with low toxicity. We have previously reported that DHA induced caspase-3-dependent apoptosis in human lung adenocarcinoma cells. However, the cellular target and molecular mechanism of DHA-induced apoptosis is still poorly defined. We use confocal fluorescence microscopy imaging, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching techniques to explore the roles of DHA-elicited reactive oxygen species (ROS) in the DHA-induced Bcl-2 family proteins activation, mitochondrial dysfunction, caspase cascade, and cell death. Cell Counting Kit-8 assay and flow cytometry analysis showed that DHA induced ROS-mediated apoptosis. Confocal imaging analysis in a single living cell and Western blot assay showed that DHA triggered ROS-dependent Bax translocation, mitochondrial membrane depolarization, alteration of mitochondrial morphology, cytochrome c release, caspase-9, caspase-8, and caspase-3 activation, indicating the coexistence of ROS-mediated mitochondrial and death receptor pathway. Collectively, our findings demonstrate for the first time that DHA induces cell apoptosis by triggering ROS-mediated caspase-8/Bid activation and the mitochondrial pathway, which provides some novel insights into the application of DHA as a potential anticancer drug and a new therapeutic strategy by targeting ROS signaling in lung adenocarcinoma therapy in the future.

CELL-SELEX: Novel Perspectives of Aptamer-Based Therapeutics

PubMed Central

Guo, Ke-Tai; Paul, Angela; Schichor, Christian; Ziemer, Gerhard; Wendel, Hans P.

2008-01-01

Aptamers, single stranded DNA or RNA molecules, generated by a method called SELEX (systematic evolution of ligands by exponential enrichment) have been widely used in various biomedical applications. The newly developed Cell-SELEX (cell based-SELEX) targeting whole living cells has raised great expectations for cancer biology, -therapy and regenerative medicine. Combining nanobiotechnology with aptamers, this technology opens the way to more sophisticated applications in molecular diagnosis. This paper gives a review of recent developments in SELEX technologies and new applications of aptamers. PMID:19325777

Coherent synchrotron emission in transmission with double foil target

NASA Astrophysics Data System (ADS)

Xu, X. R.; Qiao, B.; Chang, H. X.; Zhang, Y. X.; Zhang, H.; Zhong, C. L.; Zhou, C. T.; Zhu, S. P.; He, X. T.

2018-04-01

Generation of intense single attosecond pulses from coherent synchrotron emission (CSE), in the transmitted direction of the laser-irradiated double foil targets, has been investigated theoretically and numerically. Unlike conventional CSE in the single foil target case, here the dense electron nanobunch is formed in the vacuum gap between two foils, which is composed of the electrons blown out from the first ultrathin foil. Owing to the existence of the vacuum gap, the electron nanobunch can be accelerated to more energy. In addition, more laser energy can penetrate through the nanobunch and get reflected from the second foil. These reflected lasers and electron nanobunches interact with each other and results in enhanced CSE and consequently, the generation of intense attosecond pulses. Particle-in-cell simulations show that a single attosecond pulse with duration of 18 {as}, photon energy > 0.16 {keV} and peak intensity of 1.7× {10}20 {{W}}/{cm}}2 can be obtained from the double-foil targets irradiated by a laser at intensity of 7.7× {10}21 {{W}}/{cm}}2.

Fundamental trade-offs between information flow in single cells and cellular populations.

PubMed

Suderman, Ryan; Bachman, John A; Smith, Adam; Sorger, Peter K; Deeds, Eric J

2017-05-30

Signal transduction networks allow eukaryotic cells to make decisions based on information about intracellular state and the environment. Biochemical noise significantly diminishes the fidelity of signaling: networks examined to date seem to transmit less than 1 bit of information. It is unclear how networks that control critical cell-fate decisions (e.g., cell division and apoptosis) can function with such low levels of information transfer. Here, we use theory, experiments, and numerical analysis to demonstrate an inherent trade-off between the information transferred in individual cells and the information available to control population-level responses. Noise in receptor-mediated apoptosis reduces information transfer to approximately 1 bit at the single-cell level but allows 3-4 bits of information to be transmitted at the population level. For processes such as eukaryotic chemotaxis, in which single cells are the functional unit, we find high levels of information transmission at a single-cell level. Thus, low levels of information transfer are unlikely to represent a physical limit. Instead, we propose that signaling networks exploit noise at the single-cell level to increase population-level information transfer, allowing extracellular ligands, whose levels are also subject to noise, to incrementally regulate phenotypic changes. This is particularly critical for discrete changes in fate (e.g., life vs. death) for which the key variable is the fraction of cells engaged. Our findings provide a framework for rationalizing the high levels of noise in metazoan signaling networks and have implications for the development of drugs that target these networks in the treatment of cancer and other diseases.

Stable expression and purification of a functional processed Fab' fragment from a single nascent polypeptide in CHO cells expressing the mCAT-1 retroviral receptor.

PubMed

Camper, Nicolas; Byrne, Teresa; Burden, Roberta E; Lowry, Jenny; Gray, Breena; Johnston, James A; Migaud, Marie E; Olwill, Shane A; Buick, Richard J; Scott, Christopher J

2011-09-30

Monoclonal antibodies and derivative formats such as Fab' fragments are used in a broad range of therapeutic, diagnostic and research applications. New systems and methodologies that can improve the production of these proteins are consequently of much interest. Here we present a novel approach for the rapid production of processed Fab' fragments in a CHO cell line that has been engineered to express the mouse cationic amino acid transporter receptor 1 (mCAT-1). This facilitated the introduction of the target antibody gene through retroviral transfection, rapidly producing stable expression. Using this system, we designed a single retroviral vector construct for the expression of a target Fab' fragment as a single polypeptide with a furin cleavage site and a FMDV 2A self-cleaving peptide introduced to bridge the light and truncated heavy chain regions. The introduction of these cleavage motifs ensured equimolar expression and processing of the heavy and light domains as exemplified by the production of an active chimeric Fab' fragment against the Fas receptor, routinely expressed in 1-2mg/L yield in spinner-flask cell cultures. These results demonstrate that this method could have application in the facile production of bioactive Fab' fragments. Copyright © 2011 Elsevier B.V. All rights reserved.

Cell selection and characterization of a novel human endothelial cell specific nanobody.

PubMed

Ahmadvand, Davoud; Rasaee, Mohammad J; Rahbarizadeh, Fatemeh; Kontermann, Roland E; Sheikholislami, Farzaneh

2009-05-01

Antibody-based targeting of angiogenesis and vascular targeting therapy of cancer are extremely attractive conceptually and open new important diagnostic and therapeutic opportunities. Compelling evidence suggests that CD105 represents an ideal target for anti-angiogenic therapy and its presence in solid tumor vasculature has prognostic value. Camelids produce functional antibodies devoid of light chains and constant heavy chain domain (CH1). Nanobodies, the antigen-binding fragments of such heavy chain antibodies, are therefore comprised in one single domain. The aim of this study was to explore the possibilities of using anti-endoglin nanobody as an angiogenesis inhibitor. The anti-CD105 nanobody (AR-86a) was isolated from immune library by selections on purified antigens and target cells. Immunocytochemistry and FACS analysis showed that the purified nanobody reacted specifically with human umbilical vein endothelial cells (HUVECs) but not with other cell lines such as MDA-MB-453, Mel III, T-47D, MCF-7, AGO and HT 29. Further, selected nanobody potently inhibited proliferation of human endothelial cells and formation of capillary-like structures. This selected high affinity anti-endoglin nanobody may offer high specificity towards tumors with reduced side effects, and may be less likely to elicit drug resistance compared to conventional therapy.

Exosome and microvesicle mediated phene transfer in mammalian cells.

PubMed

Christianson, Helena C; Svensson, Katrin J; Belting, Mattias

2014-10-01

Extracellular vesicles (EVs), e.g. exosomes and microvesicles, emerge as new signaling organelles in the exchange of information between cells at the paracrine and systemic level. It is clear that these virus like particles carry complex biological information that can elicit a pleiotropic response in recipient cells with potential relevance in physiology as well as in cancer and other pathological conditions. Numerous studies convincingly show that the molecular composition of EVs closely reflects their cell or tissue of origin. Thus, the signaling status of donor cells, more specifically their endosomal compartments, may largely determine the biological output in recipient cells, a process that we then may conceptualize as vesicle mediated phene transfer. Whereas more conventional modes of cell-cell communication mostly depend on extracellular ligand concentration and cell-surface receptor availability, the magnitude of the EV signaling response relies on the capture and uptake by target cells, allowing release of the EV content. Numerous reports point at the intriguing possibility that, among thousands of mRNAs, miRNAs, and proteins, single EV constituents effectuate the biological response, e.g. stimulation of angiogenesis and cancer cell metastasis, in recipient cells; however, we find it conceivable that strategies targeted at general mechanisms of EV function should provide more rational avenues for therapeutic intervention directed at the EV system. Such strategies include manipulation of EV formation in the endolysosomal system, EV stability in the extracellular milieu, and EV entry into target cells. Here, we provide important insights into potential mechanisms of EV transport in mammalian cells and how these may be targeted. Copyright © 2014 Elsevier Ltd. All rights reserved.

Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells.

PubMed

Hoban, Megan D; Cost, Gregory J; Mendel, Matthew C; Romero, Zulema; Kaufman, Michael L; Joglekar, Alok V; Ho, Michelle; Lumaquin, Dianne; Gray, David; Lill, Georgia R; Cooper, Aaron R; Urbinati, Fabrizia; Senadheera, Shantha; Zhu, Allen; Liu, Pei-Qi; Paschon, David E; Zhang, Lei; Rebar, Edward J; Wilber, Andrew; Wang, Xiaoyan; Gregory, Philip D; Holmes, Michael C; Reik, Andreas; Hollis, Roger P; Kohn, Donald B

2015-04-23

Sickle cell disease (SCD) is characterized by a single point mutation in the seventh codon of the β-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells would allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrate efficient targeted cleavage at the β-globin locus with minimal off-target modification. By co-delivering a homologous donor template (either an integrase-defective lentiviral vector or a DNA oligonucleotide), high levels of gene modification were achieved in CD34(+) hematopoietic stem and progenitor cells. Modified cells maintained their ability to engraft NOD/SCID/IL2rγ(null) mice and to produce cells from multiple lineages, although with a reduction in the modification levels relative to the in vitro samples. Importantly, ZFN-driven gene correction in CD34(+) cells from the bone marrow of patients with SCD resulted in the production of wild-type hemoglobin tetramers. © 2015 by The American Society of Hematology.

The detection of cancer in living tissue with single-cell precision and the development of a system for targeted drug delivery to cancer

NASA Astrophysics Data System (ADS)

Fields, Adam; Pi, Sean; Ramek, Alex; Bernheim, Taylor; Fields, Jessica; Pernodet, Nadine; Rafailovich, Miriam

2007-03-01

The development of innovations in the field of cancer diagnostics is imperative to improve the early identification of malignant cells within the human body. Two novel techniques are presented for the detection of cancer cells in living tissue. First, shear modulation force microscopy (SMFM) was employed to measure cell mechanics of normal and cancer cells in separate and mixed tissue cultures. We found that the moduli of normal keratinocytes were twice as high as the moduli of SCC cancerous keratinocytes, and that the cancer cells were unambiguously identifiable from a mixture of both kinds of cells. Second, confocal microscopy and the BIAcore 2000 were used to demonstrate the preferential adhesion of glass micro-beads impregnated with fluorescent dye to the membranes of cancer cells as compared to those of normal cells. In addition to their use as a cancer detection system, these hollow and porous beads present a model system for targeted drug delivery in the treatment of cancer.

Single organelle dynamics linked to 3D structure by correlative live-cell imaging and 3D electron microscopy.

PubMed

Fermie, Job; Liv, Nalan; Ten Brink, Corlinda; van Donselaar, Elly G; Müller, Wally H; Schieber, Nicole L; Schwab, Yannick; Gerritsen, Hans C; Klumperman, Judith

2018-05-01

Live-cell correlative light-electron microscopy (live-cell-CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3-dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB-SEM) in a modular live-cell-CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal-associated membrane protein 1-green fluorescent protein (LAMP-1-GFP), analyzed the dynamics of individual GFP-positive spots, and correlated these to their corresponding fine-architecture and immediate cellular environment. By FIB-SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB-SEM, which significantly reduces time required for image acquisition and data processing. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Cellular imaging by targeted assembly of hot-spot SERS and photoacoustic nanoprobes using split-fluorescent protein scaffolds.

PubMed

Köker, Tuğba; Tang, Nathalie; Tian, Chao; Zhang, Wei; Wang, Xueding; Martel, Richard; Pinaud, Fabien

2018-02-09

The in cellulo assembly of plasmonic nanomaterials into photo-responsive probes is of great interest for many bioimaging and nanophotonic applications but remains challenging with traditional nucleic acid scaffolds-based bottom-up methods. Here, we address this quandary using split-fluorescent protein (FP) fragments as molecular glue and switchable Raman reporters to assemble gold or silver plasmonic nanoparticles (NPs) into photonic clusters directly in live cells. When targeted to diffusing surface biomarkers in cancer cells, the NPs self-assemble into surface-enhanced Raman-scattering (SERS) nanoclusters having hot spots homogenously seeded by the reconstruction of full-length FPs. Within plasmonic hot spots, autocatalytic activation of the FP chromophore and near-field amplification of its Raman fingerprints enable selective and sensitive SERS imaging of targeted cells. This FP-driven assembly of metal colloids also yields enhanced photoacoustic signals, allowing the hybrid FP/NP nanoclusters to serve as contrast agents for multimodal SERS and photoacoustic microscopy with single-cell sensitivity.

Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection

PubMed Central

Eyquem, Justin; Mansilla-Soto, Jorge; Giavridis, Theodoros; van der Stegen, Sjoukje J. C.; Hamieh, Mohamad; Cunanan, Kristen M.; Odak, Ashlesha; Gönen, Mithat; Sadelain, Michel

2017-01-01

Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumour rejection1. The most successful CARs used to date are those targeting CD19 (ref. 2), which offer the prospect of complete remission in patients with chemorefractory or relapsed B-cell malignancies3. CARs are typically transduced into the T cells of a patient using γ-retroviral4 vectors or other randomly integrating vectors5, which may result in clonal expansion, oncogenic transformation, variegated transgene expression and transcriptional silencing6–8. Recent advances in genome editing enable efficient sequence-specific interventions in human cells9,10, including targeted gene delivery to the CCR5 and AAVS1 loci11,12. Here we show that directing a CD19-specific CAR to the T-cell receptor α constant (TRAC) locus not only results in uniform CAR expression in human peripheral blood T cells, but also enhances T-cell potency, with edited cells vastly outperforming conventionally generated CAR T cells in a mouse model of acute lymphoblastic leukaemia. We further demonstrate that targeting the CAR to the TRAC locus averts tonic CAR signalling and establishes effective internalization and re-expression of the CAR following single or repeated exposure to antigen, delaying effector T-cell differentiation and exhaustion. These findings uncover facets of CAR immunobiology and underscore the potential of CRISPR/Cas9 genome editing to advance immunotherapies. PMID:28225754

Preclinical development of a bispecific antibody that safely and effectively targets CD19 and CD47 for the treatment of B cell lymphoma and leukemia.

PubMed

Buatois, Vanessa; Johnson, Zoë; Salgado-Pires, Susana; Papaïoannou, Anne; Hatterer, Eric; Chauchet, Xavier; Richard, Françoise; Barba, Leticia; Daubeuf, Bruno; Cons, Laura; Broyer, Lucile; D'Asaro, Matilde; Matthes, Thomas; LeGallou, Simon; Fest, Thierry; Tarte, Karin; Clarke Hinojosa, Robert K; Genescà Ferrer, Eulàlia; Ribera, José María; Dey, Aditi; Bailey, Katharine; Fielding, Adele K; Eissenberg, Linda; Ritchey, Julie; Rettig, Michael; DiPersio, John F; Kosco-Vilbois, Marie H; Masternak, Krzysztof; Fischer, Nicolas; Shang, Limin; Ferlin, Walter G

2018-05-09

CD47, a ubiquitously expressed innate immune checkpoint receptor that serves as a universal "don't eat me" signal of phagocytosis, is often up-regulated by hematological and solid cancers to evade immune surveillance. Development of CD47-targeted modalities is hindered by the ubiquitous expression of the target, often leading to rapid drug elimination and hemotoxicity including anemia. To overcome such liabilities, we have developed a fully human bispecific antibody, NI-1701, designed to co-engage CD47 and CD19 selectively on B cells. NI-1701 demonstrates favorable elimination kinetics with no deleterious effects seen on hematological parameters following single or multiple administrations to non-human primates. Potent in vitro and in vivo activity is induced by NI-1701 to kill cancer cells across a plethora of B cell malignancies and control tumor growth in xenograft mouse models. The mechanism affording maximal tumor growth inhibition by NI-1701 is dependent on the co-engagement of CD47/CD19 on B cells inducing potent antibody dependent cellular phagocytosis of the targeted cells. NI-1701-induced control of tumor growth in immunodeficient NOD/SCID mice was more effective than that achieved with the anti-CD20 targeted antibody, rituximab. Interestingly, a synergistic effect was seen when tumor-implanted mice were co-administered NI-1701 and rituximab leading to significantly improved tumor growth inhibition and regression in some animals. We describe herein, a novel bispecific antibody approach aimed at sensitizing B cells to become more readily phagocytosed and eliminated thus offering an alternative or adjunct therapeutic option to patients with B cell malignancies refractory/resistant to anti-CD20 targeted therapy. Copyright ©2018, American Association for Cancer Research.

Targeting the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway: an emerging treatment strategy for squamous cell lung carcinoma.

PubMed

Beck, Joseph Thaddeus; Ismail, Amen; Tolomeo, Christina

2014-09-01

Squamous cell lung carcinoma accounts for approximately 30% of all non-small cell lung cancers (NSCLCs). Despite progress in the understanding of the biology of cancer, cytotoxic chemotherapy remains the standard of care for patients with squamous cell lung carcinoma, but the prognosis is generally poor. The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway is one of the most commonly activated signaling pathways in cancer, leading to cell proliferation, survival, and differentiation. It has therefore become a major focus of clinical research. Various alterations in the PI3K/AKT/mTOR pathway have been identified in squamous cell lung carcinoma and a number of agents targeting these alterations are in clinical development for use as single agents and in combination with other targeted and conventional treatments. These include pan-PI3K inhibitors, isoform-specific PI3K inhibitors, AKT inhibitors, mTOR inhibitors, and dual PI3K/mTOR inhibitors. These agents have demonstrated antitumor activity in preclinical models of NSCLC and preliminary clinical evidence is also available for some agents. This review will discuss the role of the PI3K/AKT/mTOR pathway in cancer and how the discovery of genetic alterations in this pathway in patients with squamous cell lung carcinoma can inform the development of targeted therapies for this disease. An overview of ongoing clinical trials investigating PI3K/AKT/mTOR pathway inhibitors in squamous cell lung carcinoma will also be included. Copyright © 2014 Elsevier Ltd. All rights reserved.

Generation and genetic engineering of human induced pluripotent stem cells using designed zinc finger nucleases.

PubMed

Ramalingam, Sivaprakash; London, Viktoriya; Kandavelou, Karthikeyan; Cebotaru, Liudmila; Guggino, William; Civin, Curt; Chandrasegaran, Srinivasan

2013-02-15

Zinc finger nucleases (ZFNs) have become powerful tools to deliver a targeted double-strand break at a pre-determined chromosomal locus in order to insert an exogenous transgene by homology-directed repair. ZFN-mediated gene targeting was used to generate both single-allele chemokine (C-C motif) receptor 5 (CCR5)-modified human induced pluripotent stem cells (hiPSCs) and biallele CCR5-modified hiPSCs from human lung fibroblasts (IMR90 cells) and human primary cord blood mononuclear cells (CBMNCs) by site-specific insertion of stem cell transcription factor genes flanked by LoxP sites into the endogenous CCR5 locus. The Oct4 and Sox2 reprogramming factors, in combination with valproic acid, induced reprogramming of human lung fibroblasts to form CCR5-modified hiPSCs, while 5 factors, Oct4/Sox2/Klf4/Lin28/Nanog, induced reprogramming of CBMNCs. Subsequent Cre recombinase treatment of the CCR5-modified IMR90 hiPSCs resulted in the removal of the Oct4 and Sox2 transgenes. Further genetic engineering of the single-allele CCR5-modified IMR90 hiPSCs was achieved by site-specific addition of the large CFTR transcription unit to the remaining CCR5 wild-type allele, using CCR5-specific ZFNs and a donor construct containing tdTomato and CFTR transgenes flanked by CCR5 homology arms. CFTR was expressed efficiently from the endogenous CCR5 locus of the CCR5-modified tdTomato/CFTR hiPSCs. These results suggest that it might be feasible to use ZFN-evoked strategies to (1) generate precisely targeted genetically well-defined patient-specific hiPSCs, and (2) then to reshape their function by targeted addition and expression of therapeutic genes from the CCR5 chromosomal locus for autologous cell-based transgene-correction therapy to treat various recessive monogenic human diseases in the future.

Spatial and temporal controls target pal-1 blastomere-specification activity to a single blastomere lineage in C. elegans embryos.

PubMed

Hunter, C P; Kenyon, C

1996-10-18

The early asymmetric cleavages of Caenorhabditis elegans embryos produce blastomeres with distinct developmental potentials. Here, we show that the caudal-like homeodomain protein PAL-1 is required to specify the somatic identity of one posterior blastomere in the 4 cell embryo. We find that pal-1 activity is sequentially restricted to this blastomere. First, at the 4 cell stage, it is translated only in the two posterior blastomeres. Then, its function is restricted to one of these blastomeres. This second targeting step is dependent on the activities of the posteriorly localized SKN-1 and asymmetrically segregated PIE-1 proteins. We propose that the segregation of PIE-1, combined with the temporal decay of SKN-1, targets pal-1 activity to this posterior lineage, thus coupling the regulation of this conserved posterior patterning gene to asymmetric cell cleavages.

Planar optical waveguide based sandwich assay sensors and processes for the detection of biological targets including early detection of cancers

DOEpatents

Martinez, Jennifer S [Santa Fe, NM; Swanson, Basil I [Los Alamos, NM; Shively, John E [Arcadia, CA; Li, Lin [Monrovia, CA

2009-06-02

An assay element is described including recognition ligands adapted for binding to carcinoembryonic antigen (CEA) bound to a film on a single mode planar optical waveguide, the film from the group of a membrane, a polymerized bilayer membrane, and a self-assembled monolayer containing polyethylene glycol or polypropylene glycol groups therein and an assay process for detecting the presence of CEA is described including injecting a possible CEA-containing sample into a sensor cell including the assay element, maintaining the sample within the sensor cell for time sufficient for binding to occur between CEA present within the sample and the recognition ligands, injecting a solution including a reporter ligand into the sensor cell; and, interrogating the sample within the sensor cell with excitation light from the waveguide, the excitation light provided by an evanescent field of the single mode penetrating into the biological target-containing sample to a distance of less than about 200 nanometers from the waveguide thereby exciting any bound reporter ligand within a distance of less than about 200 nanometers from the waveguide and resulting in a detectable signal.

Remote excitation fluorescence correlation spectroscopy using silver nanowires

NASA Astrophysics Data System (ADS)

Su, Liang; Yuan, Haifeng; Lu, Gang; Hofkens, Johan; Roeffaers, Maarten; Uji-i, Hiroshi

2014-11-01

Fluorescence correlation spectroscopy (FCS), a powerful tool to resolve local properties, dynamical process of molecules, rotational and translational diffusion motions, relies on the fluctuations of florescence observables in the observation volume. In the case of rare transition events or small dynamical fluctuations, FCS requires few molecules or even single molecules in the observation volume at a time to minimize the background signals. Metal nanoparticle which possess unique localized surface plasmon resonance (LSPR) have been used to reduce the observation volume down to sub-diffraction limited scale while maintain at high analyst concentration up to tens of micromolar. Nevertheless, the applications of functionalized nanoparticles in living cell are limited due to the continuous diffusion after cell uptake, which makes it difficult to target the region of interests in the cell. In this work, we demonstrate the use of silver nanowires for remote excitation FCS on fluorescent molecules in solution. By using propagation surface plasmon polaritons (SPPs) which supported by the silver nanowire to excite the fluorescence, both illumination and observation volume can be reduced simultaneously. In such a way, less perturbation is induced to the target region, and this will broaden the application scope of silver nanowire as tip in single cell endoscopy.

Aurora A kinase RNAi and small molecule inhibition of Aurora kinases with VE-465 induce apoptotic death in multiple myeloma cells.

PubMed

Evans, Robert; Naber, Claudia; Steffler, Tara; Checkland, Tamara; Keats, Jonathan; Maxwell, Christopher; Perry, Troy; Chau, Heidi; Belch, Andrew; Pilarski, Linda; Reiman, Tony

2008-03-01

The expression of RHAMM and other centrosome-associated genes are known to correlate with the extent of centrosome amplification in multiple myeloma, and with poor prognosis. RHAMM has a significant interaction with TPX2, a protein which regulates the localization and action of Aurora A kinase (AURKA) at the spindle poles. AURKA is known to be a central determinant of centrosome and spindle function and is a target for cancer therapy. Given these observations, we investigated the role of Aurora kinases as therapeutic targets in myeloma. Here we report that AURKA is expressed ubiquitously in myeloma, to varying degrees, in both cell lines and patients' bone marrow plasma cells. siRNA targeting AURKA induces apoptotic cell death in myeloma cell lines. The Aurora kinase inhibitor VE-465 also induces apoptosis and death in myeloma cell lines and primary myeloma plasma cells. The combination of VE-465 and dexamethasone improves cell killing compared with the use of either agent alone, even in cells resistant to the single agents. The phenotype of myeloma cells treated with VE-465 is consistent with published reports on the effects of Aurora kinase inhibition. Aurora kinase inhibitors should be pursued as potential treatments for myeloma.

Microfluidic devices for the controlled manipulation of small volumes

DOEpatents

Ramsey, J Michael [Knoxville, TN; Jacobson, Stephen C [Knoxville, TN

2003-02-25

A method for conducting a broad range of biochemical analyses or manipulations on a series of nano- to subnanoliter reaction volumes and an apparatus for carrying out the same are disclosed. The method and apparatus are implemented on a fluidic microchip to provide high serial throughput. The method and device of the invention also lend themselves to multiple parallel analyses and manipulation to provide greater throughput for the generation of biochemical information. In particular, the disclosed device is a microfabricated channel device that can manipulate nanoliter or subnanoliter biochemical reaction volumes in a controlled manner to produce results at rates of 1 to 10 Hz per channel. The individual reaction volumes are manipulated in serial fashion analogous to a digital shift register. The method and apparatus according to this invention have application to such problems as screening molecular or cellular targets using single beads from split-synthesis combinatorial libraries, screening single cells for RNA or protein expression, genetic diagnostic screening at the single cell level, or performing single cell signal transduction studies.

Manipulation of cell membrane using carbon nanotube scaffold as a photoresponsive stimuli generator.

PubMed

Sada, Takao; Fujigaya, Tsuyohiko; Nakashima, Naotoshi

2014-08-01

We describe, for the first time, the perforation of the cell membrane in the targeted single cell based on the nanosecond pulsed near-infrared (NIR) laser irradiation of a thin film of carbon nanotubes that act as an effective photon absorber as well as stimuli generator. When the power of NIR laser is over 17.5 μ J/pulse, the cell membrane after irradiation is irreversibly disrupted and results in cell death. In sharp contrast, the perforation of the cell membrane occurs at suitable laser power (∼15 μ J/pulse) without involving cell termination.

Overcoming CRPC Treatment Resistance via Novel Dual AKR1C3 Targeting

DTIC Science & Technology

2017-10-01

We therefore characterized another drug resistant line from C4-2B cells, C4-2B AbiR cells. C4-2B AbiR cells were resistant to Abi acetate in a...Testosterone level in C4-2B AbiR cell was 12 pg/50 million cells, similar to that in C4-2B MDVR or LNCaP-AKR1C3 cells. With the single drug resistant...cell lines on hand, we tested for their cross- resistance to Enza and Abi. While the parental line was sensitive to both drugs , the resistant lines

Manipulation of cell membrane using carbon nanotube scaffold as a photoresponsive stimuli generator

PubMed Central

Sada, Takao; Fujigaya, Tsuyohiko; Nakashima, Naotoshi

2014-01-01

We describe, for the first time, the perforation of the cell membrane in the targeted single cell based on the nanosecond pulsed near-infrared (NIR) laser irradiation of a thin film of carbon nanotubes that act as an effective photon absorber as well as stimuli generator. When the power of NIR laser is over 17.5 μJ/pulse, the cell membrane after irradiation is irreversibly disrupted and results in cell death. In sharp contrast, the perforation of the cell membrane occurs at suitable laser power (∼15 μJ/pulse) without involving cell termination. PMID:27877703

Optical cell cleaning with NIR femtosecond laser pulses

NASA Astrophysics Data System (ADS)

Uchugonova, Aisada; Breunig, Hans Georg; Batista, Ana; König, Karsten

2015-03-01

Femtosecond laser microscopes have been used as both micro and nanosurgery tools. The optical knock-out of undesired cells in multiplex cell clusters shall be further reported on in this study. Femtosecond laser-induced cell death is beneficial due to the reduced collateral side effects and therefore can be used to selectively destroy target cells within monolayers, as well as within 3D tissues, all the while preserving cells of interest. This is an important characteristic for the application in stem cell research and cancer treatment. Non-precise damage compromises the viability of neighboring cells by inducing side effects such as stress to the cells surrounding the target due to the changes in the microenvironment, resulting from both the laser and laser-exposed cells. In this study, optimum laser parameters for optical cleaning by isolating single cells and cell colonies are exploited through the use of automated software control. Physiological equilibrium and cellular responses to the laser induced damages are also investigated. Cell death dependence on laser focus, determination and selectivity of intensity/dosage, controllable damage and cell recovery mechanisms are discussed.

Ivy and neurogliaform interneurons are a major target of μ opioid receptor modulation

PubMed Central

Krook-Magnuson, Esther; Luu, Lillian; Lee, Sang-Hun; Varga, Csaba; Soltesz, Ivan

2011-01-01

Mu opioid receptors (μORs) are selectively expressed on interneurons in area CA1 of the hippocampus. Fast-spiking, parvalbumin expressing, basket cells express μORs, but circumstantial evidence suggests that another major, unidentified, GABAergic cell class must also be modulated by μORs. Here we report that the abundant, dendritically targeting, neurogliaform family of cells (Ivy and neurogliaform cells) is a previously unrecognized target of direct modulation by μORs. Ivy and neurogliaform cells are not only numerous, but also have unique properties, including promiscuous gap junctions formed with various interneuronal subtypes, volume transmission, and the ability to produce a postsynaptic GABAB response after a single presynaptic spike. Using a mouse line expressing green fluorescent protein under the neuropeptide Y promoter, we find that across all layers of CA1, activation of μORs hyperpolarizes Ivy and neurogliaform cells. Further, paired recordings between synaptically coupled Ivy and pyramidal cells show that Ivy cell terminals are dramatically inhibited by μOR-activation. Effects in Ivy and neurogliaform cells are seen at similar concentrations of agonist as those producing inhibition in fast-spiking PV basket cells. We also report that Ivy cells display the recently described phenomenon of persistent firing, a state of continued firing in the absence of continued input, and that induction of persistent firing is inhibited by μOR-activation. Together these findings identify a major, previously unrecognized, target of μOR-modulation. Given the prominence of this cell type in and beyond CA1, as well as its unique role in microcircuitry, opioid modulation of neurogliaform cells has wide implications. PMID:22016519

Ivy and neurogliaform interneurons are a major target of μ-opioid receptor modulation.

PubMed

Krook-Magnuson, Esther; Luu, Lillian; Lee, Sang-Hun; Varga, Csaba; Soltesz, Ivan

2011-10-19

μ-Opioid receptors (μORs) are selectively expressed on interneurons in area CA1 of the hippocampus. Fast-spiking, parvalbumin-expressing, basket cells express μORs, but circumstantial evidence suggests that another major, unidentified, GABAergic cell class must also be modulated by μORs. Here we report that the abundant, dendritically targeting, neurogliaform family of cells (Ivy and neurogliaform cells) is a previously unrecognized target of direct modulation by μORs. Ivy and neurogliaform cells are not only numerous but also have unique properties, including promiscuous gap junctions formed with various interneuronal subtypes, volume transmission, and the ability to produce a postsynaptic GABA(B) response after a single presynaptic spike. Using a mouse line expressing green fluorescent protein under the neuropeptide Y promoter, we find that, across all layers of CA1, activation of μORs hyperpolarizes Ivy and neurogliaform cells. Furthermore, paired recordings between synaptically coupled Ivy and pyramidal cells show that Ivy cell terminals are dramatically inhibited by μOR activation. Effects in Ivy and neurogliaform cells are seen at similar concentrations of agonist as those producing inhibition in fast-spiking parvalbumin basket cells. We also report that Ivy cells display the recently described phenomenon of persistent firing, a state of continued firing in the absence of continued input, and that induction of persistent firing is inhibited by μOR activation. Together, these findings identify a major, previously unrecognized, target of μOR modulation. Given the prominence of this cell type in and beyond CA1, as well as its unique role in microcircuitry, opioid modulation of neurogliaform cells has wide implications.

The simultaneous isolation of multiple high and low frequent T-cell populations from donor peripheral blood mononuclear cells using the major histocompatibility complex I-Streptamer isolation technology.

PubMed

Roex, Marthe C J; Hageman, Lois; Heemskerk, Matthias T; Veld, Sabrina A J; van Liempt, Ellis; Kester, Michel G D; Germeroth, Lothar; Stemberger, Christian; Falkenburg, J H Frederik; Jedema, Inge

2018-04-01

Adoptive transfer of donor-derived T cells can be applied to improve immune reconstitution in immune-compromised patients after allogeneic stem cell transplantation. The separation of beneficial T cells from potentially harmful T cells can be achieved by using the major histocompatibility complex (MHC) I-Streptamer isolation technology, which has proven its feasibility for the fast and pure isolation of T-cell populations with a single specificity. We have analyzed the feasibility of the simultaneous isolation of multiple antigen-specific T-cell populations in one procedure by combining different MHC I-Streptamers. First, the effect of combining different amounts of MHC I-Streptamers used in the isolation procedure on the isolation efficacy of target antigen-specific T cells and on the number of off-target co-isolated contaminating cells was assessed. The feasibility of this approach was demonstrated in large-scale validation procedures targeting both high and low frequent T-cell populations using the Good Manufacturing Practice (GMP)-compliant CliniMACS Plus device. T-cell products targeting up to 24 different T-cell populations could be isolated in one, simultaneous MHC I-Streptamer procedure, by adjusting the amount of MHC I- Streptamers per target antigen-specific T-cell population. Concurrently, the co-isolation of potentially harmful contaminating T cells remained below our safety limit. This technology allows the reproducible isolation of high and low frequent T-cell populations. However, the expected therapeutic relevance of direct clinical application without in vitro expansion of these low frequent T-cell populations is questionable. This study provides a feasible, fast and safe method for the generation of highly personalized MHC I-Streptamer isolated T-cell products for adoptive immunotherapy. Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells.

PubMed

Guo, Fan; Li, Lin; Li, Jingyun; Wu, Xinglong; Hu, Boqiang; Zhu, Ping; Wen, Lu; Tang, Fuchou

2017-08-01

Single-cell epigenome sequencing techniques have recently been developed. However, the combination of different layers of epigenome sequencing in an individual cell has not yet been achieved. Here, we developed a single-cell multi-omics sequencing technology (single-cell COOL-seq) that can analyze the chromatin state/nucleosome positioning, DNA methylation, copy number variation and ploidy simultaneously from the same individual mammalian cell. We used this method to analyze the reprogramming of the chromatin state and DNA methylation in mouse preimplantation embryos. We found that within < 12 h of fertilization, each individual cell undergoes global genome demethylation together with the rapid and global reprogramming of both maternal and paternal genomes to a highly opened chromatin state. This was followed by decreased openness after the late zygote stage. Furthermore, from the late zygote to the 4-cell stage, the residual DNA methylation is preferentially preserved on intergenic regions of the paternal alleles and intragenic regions of maternal alleles in each individual blastomere. However, chromatin accessibility is similar between paternal and maternal alleles in each individual cell from the late zygote to the blastocyst stage. The binding motifs of several pluripotency regulators are enriched at distal nucleosome depleted regions from as early as the 2-cell stage. This indicates that the cis-regulatory elements of such target genes have been primed to an open state from the 2-cell stage onward, long before pluripotency is eventually established in the ICM of the blastocyst. Genes may be classified into homogeneously open, homogeneously closed and divergent states based on the chromatin accessibility of their promoter regions among individual cells. This can be traced to step-wise transitions during preimplantation development. Our study offers the first single-cell and parental allele-specific analysis of the genome-scale chromatin state and DNA methylation dynamics at single-base resolution in early mouse embryos and provides new insights into the heterogeneous yet highly ordered features of epigenomic reprogramming during this process.

Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells

PubMed Central

Guo, Fan; Li, Lin; Li, Jingyun; Wu, Xinglong; Hu, Boqiang; Zhu, Ping; Wen, Lu; Tang, Fuchou

2017-01-01

Single-cell epigenome sequencing techniques have recently been developed. However, the combination of different layers of epigenome sequencing in an individual cell has not yet been achieved. Here, we developed a single-cell multi-omics sequencing technology (single-cell COOL-seq) that can analyze the chromatin state/nucleosome positioning, DNA methylation, copy number variation and ploidy simultaneously from the same individual mammalian cell. We used this method to analyze the reprogramming of the chromatin state and DNA methylation in mouse preimplantation embryos. We found that within < 12 h of fertilization, each individual cell undergoes global genome demethylation together with the rapid and global reprogramming of both maternal and paternal genomes to a highly opened chromatin state. This was followed by decreased openness after the late zygote stage. Furthermore, from the late zygote to the 4-cell stage, the residual DNA methylation is preferentially preserved on intergenic regions of the paternal alleles and intragenic regions of maternal alleles in each individual blastomere. However, chromatin accessibility is similar between paternal and maternal alleles in each individual cell from the late zygote to the blastocyst stage. The binding motifs of several pluripotency regulators are enriched at distal nucleosome depleted regions from as early as the 2-cell stage. This indicates that the cis-regulatory elements of such target genes have been primed to an open state from the 2-cell stage onward, long before pluripotency is eventually established in the ICM of the blastocyst. Genes may be classified into homogeneously open, homogeneously closed and divergent states based on the chromatin accessibility of their promoter regions among individual cells. This can be traced to step-wise transitions during preimplantation development. Our study offers the first single-cell and parental allele-specific analysis of the genome-scale chromatin state and DNA methylation dynamics at single-base resolution in early mouse embryos and provides new insights into the heterogeneous yet highly ordered features of epigenomic reprogramming during this process. PMID:28621329

A machine learning approach for the identification of key markers involved in brain development from single-cell transcriptomic data.

PubMed

Hu, Yongli; Hase, Takeshi; Li, Hui Peng; Prabhakar, Shyam; Kitano, Hiroaki; Ng, See Kiong; Ghosh, Samik; Wee, Lawrence Jin Kiat

2016-12-22

The ability to sequence the transcriptomes of single cells using single-cell RNA-seq sequencing technologies presents a shift in the scientific paradigm where scientists, now, are able to concurrently investigate the complex biology of a heterogeneous population of cells, one at a time. However, till date, there has not been a suitable computational methodology for the analysis of such intricate deluge of data, in particular techniques which will aid the identification of the unique transcriptomic profiles difference between the different cellular subtypes. In this paper, we describe the novel methodology for the analysis of single-cell RNA-seq data, obtained from neocortical cells and neural progenitor cells, using machine learning algorithms (Support Vector machine (SVM) and Random Forest (RF)). Thirty-eight key transcripts were identified, using the SVM-based recursive feature elimination (SVM-RFE) method of feature selection, to best differentiate developing neocortical cells from neural progenitor cells in the SVM and RF classifiers built. Also, these genes possessed a higher discriminative power (enhanced prediction accuracy) as compared commonly used statistical techniques or geneset-based approaches. Further downstream network reconstruction analysis was carried out to unravel hidden general regulatory networks where novel interactions could be further validated in web-lab experimentation and be useful candidates to be targeted for the treatment of neuronal developmental diseases. This novel approach reported for is able to identify transcripts, with reported neuronal involvement, which optimally differentiate neocortical cells and neural progenitor cells. It is believed to be extensible and applicable to other single-cell RNA-seq expression profiles like that of the study of the cancer progression and treatment within a highly heterogeneous tumour.

Single-Cell Transcriptomics and Fate Mapping of Ependymal Cells Reveals an Absence of Neural Stem Cell Function.

PubMed

Shah, Prajay T; Stratton, Jo A; Stykel, Morgan Gail; Abbasi, Sepideh; Sharma, Sandeep; Mayr, Kyle A; Koblinger, Kathrin; Whelan, Patrick J; Biernaskie, Jeff

2018-05-03

Ependymal cells are multi-ciliated cells that form the brain's ventricular epithelium and a niche for neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ). In addition, ependymal cells are suggested to be latent NSCs with a capacity to acquire neurogenic function. This remains highly controversial due to a lack of prospective in vivo labeling techniques that can effectively distinguish ependymal cells from neighboring V-SVZ NSCs. We describe a transgenic system that allows for targeted labeling of ependymal cells within the V-SVZ. Single-cell RNA-seq revealed that ependymal cells are enriched for cilia-related genes and share several stem-cell-associated genes with neural stem or progenitors. Under in vivo and in vitro neural-stem- or progenitor-stimulating environments, ependymal cells failed to demonstrate any suggestion of latent neural-stem-cell function. These findings suggest remarkable stability of ependymal cell function and provide fundamental insights into the molecular signature of the V-SVZ niche. Copyright © 2018 Elsevier Inc. All rights reserved.

Systems heterogeneity: An integrative way to understand cancer heterogeneity.

PubMed

Wang, Diane Catherine; Wang, Xiangdong

2017-04-01

The concept of systems heterogeneity was firstly coined and explained in the Special Issue, as a new alternative to understand the importance and complexity of heterogeneity in cancer. Systems heterogeneity can offer a full image of heterogeneity at multi-dimensional functions and multi-omics by integrating gene or protein expression, epigenetics, sequencing, phosphorylation, transcription, pathway, or interaction. The Special Issue starts with the roles of epigenetics in the initiation and development of cancer heterogeneity through the interaction between permanent genetic mutations and dynamic epigenetic alterations. Cell heterogeneity was defined as the difference in biological function and phenotypes between cells in the same organ/tissue or in different organs, as well as various challenges, as exampled in telocytes. The single cell heterogeneity has the value of identifying diagnostic biomarkers and therapeutic targets and clinical potential of single cell systems heterogeneity in clinical oncology. A number of signaling pathways and factors contribute to the development of systems heterogeneity. Proteomic heterogeneity can change the strategy and thinking of drug discovery and development by understanding the interactions between proteins or proteins with drugs in order to optimize drug efficacy and safety. The association of cancer heterogeneity with cancer cell evolution and metastasis was also overviewed as a new alternative for diagnostic biomarkers and therapeutic targets in clinical application. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cores Of Recurrent Events (CORE) | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

CORE is a statistically supported computational method for finding recurrently targeted regions in massive collections of genomic intervals, such as those arising from DNA copy number analysis of single tumor cells or bulk tumor tissues.

Clonal evolution in breast cancer revealed by single nucleus genome sequencing.

PubMed

Wang, Yong; Waters, Jill; Leung, Marco L; Unruh, Anna; Roh, Whijae; Shi, Xiuqing; Chen, Ken; Scheet, Paul; Vattathil, Selina; Liang, Han; Multani, Asha; Zhang, Hong; Zhao, Rui; Michor, Franziska; Meric-Bernstam, Funda; Navin, Nicholas E

2014-08-14

Sequencing studies of breast tumour cohorts have identified many prevalent mutations, but provide limited insight into the genomic diversity within tumours. Here we developed a whole-genome and exome single cell sequencing approach called nuc-seq that uses G2/M nuclei to achieve 91% mean coverage breadth. We applied this method to sequence single normal and tumour nuclei from an oestrogen-receptor-positive (ER(+)) breast cancer and a triple-negative ductal carcinoma. In parallel, we performed single nuclei copy number profiling. Our data show that aneuploid rearrangements occurred early in tumour evolution and remained highly stable as the tumour masses clonally expanded. In contrast, point mutations evolved gradually, generating extensive clonal diversity. Using targeted single-molecule sequencing, many of the diverse mutations were shown to occur at low frequencies (<10%) in the tumour mass. Using mathematical modelling we found that the triple-negative tumour cells had an increased mutation rate (13.3×), whereas the ER(+) tumour cells did not. These findings have important implications for the diagnosis, therapeutic treatment and evolution of chemoresistance in breast cancer.

Making cell-permeable antibodies (Transbody) through fusion of protein transduction domains (PTD) with single chain variable fragment (scFv) antibodies: potential advantages over antibodies expressed within the intracellular environment (Intrabody).

PubMed

Heng, Boon Chin; Cao, Tong

2005-01-01

Over the past decade, there has been growing interest in the use of antibodies against intracellular targets. This is currently achieved through recombinant expression of the single chain variable fragment (scFv) antibody format within the cell, which is commonly referred to as an intrabody. This possesses a number of inherent advantages over RNA interference (iRNA). Firstly, the high specificity and affinity of intrabodies to target antigens is well-established, whereas iRNA has been frequently shown to exert multiple non-specific effects. Secondly, intrabodies being proteins possess a much longer active half-life compared to iRNA. Thirdly, when the active half-life of the intracellular target molecule is long, gene silencing through iRNA would be slow to yield any effect, whereas the effects of intrabody expression would be almost instantaneous. Lastly, it is possible to design intrabodies to block certain binding interactions of a particular target molecule, while sparing others. There is, however, various technical challenges faced with intrabody expression through the application of recombinant DNA technology. In particular, protein conformational folding and structural stability of the newly-synthesized intrabody within the cell is affected by reducing conditions of the intracellular environment. Also, there are overwhelming safety concerns surrounding the application of transfected recombinant DNA in human clinical therapy, which is required to achieve intrabody expression within the cell. Of particular concern are the various viral-based vectors that are commonly-used in genetic manipulation. A novel approach around these problems would be to look at the possibility of fusing protein transduction domains (PTD) to scFv antibodies, to create a 'cell-permeable' antibody or 'Transbody'. PTD are short peptide sequences that enable proteins to translocate across the cell membrane and be internalized within the cytosol, through atypical secretory and internalization pathways. There are a number of distinct advantages that a 'Transbody' would possess over conventional intrabodies expressed within the cell. For a start, 'correct' conformational folding and disulfide bond formation can take place prior to introduction into the target cell. More importantly, the use of cell-permeable antibodies or 'Transbodies' would avoid the overwhelming safety and ethical concerns surrounding the direct application of recombinant DNA technology in human clinical therapy, which is required for intrabody expression within the cell. 'Transbodies' introduced into the cell would possess only a limited active half-life, without resulting in any permanent genetic alteration. This would allay any safety concerns with regards to their application in human clinical therapy.

Investigational CD33-targeted therapeutics for acute myeloid leukemia.

PubMed

Walter, Roland B

2018-04-01

There is long-standing interest in drugs targeting the myeloid differentiation antigen CD33 in acute myeloid leukemia (AML). Positive results from randomized trials with the antibody-drug conjugate (ADC) gemtuzumab ozogamicin (GO) validate this approach. Partly stimulated by the success of GO, several CD33-targeted therapeutics are currently in early phase testing. Areas covered: CD33-targeted therapeutics in clinical development include Fc-engineered unconjugated antibodies (BI 836858 [mAb 33.1]), ADCs (SGN-CD33A [vadastuximab talirine], IMGN779), radioimmunoconjugates ( 225 Ac-lintuzumab), bi- and trispecific antibodies (AMG 330, AMG 673, AMV564, 161533 TriKE fusion protein), and chimeric antigen receptor (CAR)-modified immune effector cells. Besides limited data on 225 Ac-lintuzumab showing modest single-agent activity, clinical data are so far primarily available for SGN-CD33A. SGN-CD33A has single-agent activity and has shown encouraging results when combined with an azanucleoside or standard chemotherapeutics. However, concerns about toxicity to the liver and normal hematopoietic cells - the latter leading to early termination of a phase 3 trial - have derailed the development of SGN-CD33A, and its future is uncertain. Expert opinion: Early results from a new generation of CD33-targeted therapeutics are anticipated in the next 2-3 years. Undoubtedly, re-approval of GO in 2017 has changed the landscape and rendered clinical development for these agents more challenging.

MiR-17-92 cluster and immunity.

PubMed

Kuo, George; Wu, Chao-Yi; Yang, Huang-Yu

2018-05-29

MicroRNAs (MiR, MiRNA) are small single-stranded non-coding RNAs that play an important role in the regulation of gene expression. MircoRNAs exert their effect by binding to complementary nucleotide sequences of the targeted messenger RNA, thus forming an RNA-induced silencing complex. The mircoRNA-17-92 cluster encoded by the miR-17-92 host gene is first found in malignant B-cell lymphoma. Recent research identifies the miR-17-92 cluster as a crucial player in the development of the immune system, the heart, the lung, and oncogenic events. In light of the miR-17-92 cluster's increasing role in regulating the immune system, our review will discuss the latest knowledge regarding its involvement in cells of both innate and adaptive immunity, including B cells, subsets of T cells such as Th1, Th2, T follicular helper cells, regulatory T cells, monocytes/macrophages, NK cells, and dendritic cells, and the possible targets that are regulated by its members. Copyright © 2018. Published by Elsevier B.V.

Impact of Blood-Brain Barrier Integrity on Tumor Growth and Therapy Response in Brain Metastases.

PubMed

Osswald, Matthias; Blaes, Jonas; Liao, Yunxiang; Solecki, Gergely; Gömmel, Miriam; Berghoff, Anna S; Salphati, Laurent; Wallin, Jeffrey J; Phillips, Heidi S; Wick, Wolfgang; Winkler, Frank

2016-12-15

The role of blood-brain barrier (BBB) integrity for brain tumor biology and therapy is a matter of debate. We developed a new experimental approach using in vivo two-photon imaging of mouse brain metastases originating from a melanoma cell line to investigate the growth kinetics of individual tumor cells in response to systemic delivery of two PI3K/mTOR inhibitors over time, and to study the impact of microregional vascular permeability. The two drugs are closely related but differ regarding a minor chemical modification that greatly increases brain penetration of one drug. Both inhibitors demonstrated a comparable inhibition of downstream targets and melanoma growth in vitro In vivo, increased BBB permeability to sodium fluorescein was associated with accelerated growth of individual brain metastases. Melanoma metastases with permeable microvessels responded similarly to equivalent doses of both inhibitors. In contrast, metastases with an intact BBB showed an exclusive response to the brain-penetrating inhibitor. The latter was true for macro- and micrometastases, and even single dormant melanoma cells. Nuclear morphology changes and single-cell regression patterns implied that both inhibitors, if extravasated, target not only perivascular melanoma cells but also those distant to blood vessels. Our study provides the first direct evidence that nonpermeable brain micro- and macrometastases can effectively be targeted by a drug designed to cross the BBB. Small-molecule inhibitors with these optimized properties are promising agents in preventing or treating brain metastases in patients. Clin Cancer Res; 22(24); 6078-87. ©2016 AACRSee related commentary by Steeg et al., p. 5953. ©2016 American Association for Cancer Research.

CbtA toxin of Escherichia coli inhibits cell division and cell elongation via direct and independent interactions with FtsZ and MreB.

PubMed

Heller, Danielle M; Tavag, Mrinalini; Hochschild, Ann

2017-09-01

The toxin components of toxin-antitoxin modules, found in bacterial plasmids, phages, and chromosomes, typically target a single macromolecule to interfere with an essential cellular process. An apparent exception is the chromosomally encoded toxin component of the E. coli CbtA/CbeA toxin-antitoxin module, which can inhibit both cell division and cell elongation. A small protein of only 124 amino acids, CbtA, was previously proposed to interact with both FtsZ, a tubulin homolog that is essential for cell division, and MreB, an actin homolog that is essential for cell elongation. However, whether or not the toxic effects of CbtA are due to direct interactions with these predicted targets is not known. Here, we genetically separate the effects of CbtA on cell elongation and cell division, showing that CbtA interacts directly and independently with FtsZ and MreB. Using complementary genetic approaches, we identify the functionally relevant target surfaces on FtsZ and MreB, revealing that in both cases, CbtA binds to surfaces involved in essential cytoskeletal filament architecture. We show further that each interaction contributes independently to CbtA-mediated toxicity and that disruption of both interactions is required to alleviate the observed toxicity. Although several other protein modulators are known to target FtsZ, the CbtA-interacting surface we identify represents a novel inhibitory target. Our findings establish CbtA as a dual function toxin that inhibits both cell division and cell elongation via direct and independent interactions with FtsZ and MreB.

CbtA toxin of Escherichia coli inhibits cell division and cell elongation via direct and independent interactions with FtsZ and MreB

PubMed Central

Heller, Danielle M.; Tavag, Mrinalini

2017-01-01

The toxin components of toxin-antitoxin modules, found in bacterial plasmids, phages, and chromosomes, typically target a single macromolecule to interfere with an essential cellular process. An apparent exception is the chromosomally encoded toxin component of the E. coli CbtA/CbeA toxin-antitoxin module, which can inhibit both cell division and cell elongation. A small protein of only 124 amino acids, CbtA, was previously proposed to interact with both FtsZ, a tubulin homolog that is essential for cell division, and MreB, an actin homolog that is essential for cell elongation. However, whether or not the toxic effects of CbtA are due to direct interactions with these predicted targets is not known. Here, we genetically separate the effects of CbtA on cell elongation and cell division, showing that CbtA interacts directly and independently with FtsZ and MreB. Using complementary genetic approaches, we identify the functionally relevant target surfaces on FtsZ and MreB, revealing that in both cases, CbtA binds to surfaces involved in essential cytoskeletal filament architecture. We show further that each interaction contributes independently to CbtA-mediated toxicity and that disruption of both interactions is required to alleviate the observed toxicity. Although several other protein modulators are known to target FtsZ, the CbtA-interacting surface we identify represents a novel inhibitory target. Our findings establish CbtA as a dual function toxin that inhibits both cell division and cell elongation via direct and independent interactions with FtsZ and MreB. PMID:28931012

CSF1R+ Macrophages Sustain Pancreatic Tumor Growth through T Cell Suppression and Maintenance of Key Gene Programs that Define the Squamous Subtype.

PubMed

Candido, Juliana B; Morton, Jennifer P; Bailey, Peter; Campbell, Andrew D; Karim, Saadia A; Jamieson, Thomas; Lapienyte, Laura; Gopinathan, Aarthi; Clark, William; McGhee, Ewan J; Wang, Jun; Escorcio-Correia, Monica; Zollinger, Raphael; Roshani, Rozita; Drew, Lisa; Rishi, Loveena; Arkell, Rebecca; Evans, T R Jeffry; Nixon, Colin; Jodrell, Duncan I; Wilkinson, Robert W; Biankin, Andrew V; Barry, Simon T; Balkwill, Frances R; Sansom, Owen J

2018-05-01

Pancreatic ductal adenocarcinoma (PDAC) is resistant to most therapies including single-agent immunotherapy and has a dense desmoplastic stroma, and most patients present with advanced metastatic disease. We reveal that macrophages are the dominant leukocyte population both in human PDAC stroma and autochthonous models, with an important functional contribution to the squamous subtype of human PDAC. We targeted macrophages in a genetic PDAC model using AZD7507, a potent selective inhibitor of CSF1R. AZD7507 caused shrinkage of established tumors and increased mouse survival in this difficult-to-treat model. Malignant cell proliferation diminished, with increased cell death and an enhanced T cell immune response. Loss of macrophages rewired other features of the TME, with global changes in gene expression akin to switching PDAC subtypes. These changes were markedly different to those elicited when neutrophils were targeted via CXCR2. These results suggest targeting the myeloid cell axis may be particularly efficacious in PDAC, especially with CSF1R inhibitors. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Simulation-based cheminformatic analysis of organelle-targeted molecules: lysosomotropic monobasic amines.

PubMed

Zhang, Xinyuan; Zheng, Nan; Rosania, Gus R

2008-09-01

Cell-based molecular transport simulations are being developed to facilitate exploratory cheminformatic analysis of virtual libraries of small drug-like molecules. For this purpose, mathematical models of single cells are built from equations capturing the transport of small molecules across membranes. In turn, physicochemical properties of small molecules can be used as input to simulate intracellular drug distribution, through time. Here, with mathematical equations and biological parameters adjusted so as to mimic a leukocyte in the blood, simulations were performed to analyze steady state, relative accumulation of small molecules in lysosomes, mitochondria, and cytosol of this target cell, in the presence of a homogenous extracellular drug concentration. Similarly, with equations and parameters set to mimic an intestinal epithelial cell, simulations were also performed to analyze steady state, relative distribution and transcellular permeability in this non-target cell, in the presence of an apical-to-basolateral concentration gradient. With a test set of ninety-nine monobasic amines gathered from the scientific literature, simulation results helped analyze relationships between the chemical diversity of these molecules and their intracellular distributions.

Optogenetic control of mitochondrial metabolism and Ca2+ signaling by mitochondria-targeted opsins.

PubMed

Tkatch, Tatiana; Greotti, Elisa; Baranauskas, Gytis; Pendin, Diana; Roy, Soumitra; Nita, Luliaoana I; Wettmarshausen, Jennifer; Prigge, Matthias; Yizhar, Ofer; Shirihai, Orian S; Fishman, Daniel; Hershfinkel, Michal; Fleidervish, Ilya A; Perocchi, Fabiana; Pozzan, Tullio; Sekler, Israel

2017-06-27

Key mitochondrial functions such as ATP production, Ca 2+ uptake and release, and substrate accumulation depend on the proton electrochemical gradient (ΔμH + ) across the inner membrane. Although several drugs can modulate ΔμH + , their effects are hardly reversible, and lack cellular specificity and spatial resolution. Although channelrhodopsins are widely used to modulate the plasma membrane potential of excitable cells, mitochondria have thus far eluded optogenetic control. Here we describe a toolkit of optometabolic constructs based on selective targeting of channelrhodopsins with distinct functional properties to the inner mitochondrial membrane of intact cells. We show that our strategy enables a light-dependent control of the mitochondrial membrane potential (Δψ m ) and coupled mitochondrial functions such as ATP synthesis by oxidative phosphorylation, Ca 2+ dynamics, and respiratory metabolism. By directly modulating Δψ m , the mitochondria-targeted opsins were used to control complex physiological processes such as spontaneous beats in cardiac myocytes and glucose-dependent ATP increase in pancreatic β-cells. Furthermore, our optometabolic tools allow modulation of mitochondrial functions in single cells and defined cell regions.

Development of an Improved Mammalian Overexpression Method for Human CD62L

PubMed Central

Brown, Haley A.; Roth, Gwynne; Holzapfel, Genevieve; Shen, Sarek; Rahbari, Kate; Ireland, Joanna; Zou, Zhongcheng; Sun, Peter D.

2014-01-01

We have previously developed a glutamine synthetase (GS)-based mammalian recombinant protein expression system that is capable of producing 5 to 30 mg/L recombinant proteins. The over expression is based on multiple rounds of target gene amplification driven by methionine sulfoximine (MSX), an inhibitor of glutamine synthetase. However, like other stable mammalian over expression systems, a major shortcoming of the GS-based expression system is its lengthy turn-around time, typically taking 4–6 months to produce. To shorten the construction time, we replaced the muti-round target gene amplifications with single-round in situ amplifications, thereby shortening the cell line construction to 2 months. The single-round in situ amplification method resulted in highest recombinant CD62L expressing CHO cell lines producing ~5mg/L soluble CD62L, similar to those derived from the multi-round amplification and selection method. In addition, we developed a MSX resistance assay as an alternative to utilizing ELISA for evaluating the expression level of stable recombinant CHO cell lines. PMID:25286402

Potent Inhibition of Human Immunodeficiency Virus Type 1 Replication by an Intracellular Anti-Rev Single-Chain Antibody

NASA Astrophysics Data System (ADS)

Duan, Lingxun; Bagasra, Omar; Laughlin, Mark A.; Oakes, Joseph W.; Pomerantz, Roger J.

1994-05-01

Human immunodeficiency virus type 1 (HIV-1) has a complex life cycle, which has made it a difficult target for conventional therapeutic modalities. A single-chain antibody moiety, directed against the HIV-1 regulatory protein Rev, which rescues unspliced viral RNA from the nucleus of infected cells, has now been developed. This anti-Rev single-chain construct (SFv) consists of both light and heavy chain variable regions of an anti-Rev monoclonal antibody, which, when expressed intracellularly within human cells, potently inhibits HIV-1 replication. This intracellular SFv molecule is demonstrated to specifically antagonize Rev function. Thus, intracellular SFv expression, against a retroviral regulatory protein, may be useful as a gene therapeutic approach to combat HIV-1 infections.

[Transient expression and characterization of intracellular single chain Fv against the nucleocapsid protein of Hantavirus].

PubMed

Bai, Wen-tao; Xu, Zhi-kai; Zhang, Fang-lin; Luo, Wen; Liu, Yong; Wu, Xing-an; Yan, Yan

2004-11-01

To transiently express an intracellular single chain Fv of monoclonal antibody 1A8 against nucleocapsid protein of Hantavirus and characterize the immunological activities of the expressed products. COS-7 cells were transfected with mammalian expression vector 1A8-scFv-Ckappa/pCI-neo via lipofectin. The expressed product was identified by indirect immunofluorescence and immunoprecipitation. A diffuse pattern fluorescence was observed in less than 1% cytoplasm of transfected COS-7 cells. The binding of intracellular antibody fragments to NP antigen was confirmed by immunoprecipitation analysis. Transiently expressed single chain intrabodies can effectively target NP antigen in the cytoplasm. The present study may provide a new approach for treatment of Hantavirus.

Plant-derived antifungal agent poacic acid targets β-1,3-glucan

PubMed Central

Piotrowski, Jeff S.; Okada, Hiroki; Lu, Fachuang; Li, Sheena C.; Hinchman, Li; Ranjan, Ashish; Smith, Damon L.; Higbee, Alan J.; Ulbrich, Arne; Coon, Joshua J.; Deshpande, Raamesh; Bukhman, Yury V.; McIlwain, Sean; Ong, Irene M.; Myers, Chad L.; Boone, Charles; Landick, Robert; Ralph, John; Kabbage, Mehdi; Ohya, Yoshikazu

2015-01-01

A rise in resistance to current antifungals necessitates strategies to identify alternative sources of effective fungicides. We report the discovery of poacic acid, a potent antifungal compound found in lignocellulosic hydrolysates of grasses. Chemical genomics using Saccharomyces cerevisiae showed that loss of cell wall synthesis and maintenance genes conferred increased sensitivity to poacic acid. Morphological analysis revealed that cells treated with poacic acid behaved similarly to cells treated with other cell wall-targeting drugs and mutants with deletions in genes involved in processes related to cell wall biogenesis. Poacic acid causes rapid cell lysis and is synergistic with caspofungin and fluconazole. The cellular target was identified; poacic acid localized to the cell wall and inhibited β-1,3-glucan synthesis in vivo and in vitro, apparently by directly binding β-1,3-glucan. Through its activity on the glucan layer, poacic acid inhibits growth of the fungi Sclerotinia sclerotiorum and Alternaria solani as well as the oomycete Phytophthora sojae. A single application of poacic acid to leaves infected with the broad-range fungal pathogen S. sclerotiorum substantially reduced lesion development. The discovery of poacic acid as a natural antifungal agent targeting β-1,3-glucan highlights the potential side use of products generated in the processing of renewable biomass toward biofuels as a source of valuable bioactive compounds and further clarifies the nature and mechanism of fermentation inhibitors found in lignocellulosic hydrolysates. PMID:25775513

Identification of lipid-phosphatidylserine (PS) as the target of unbiasedly selected cancer specific peptide-peptoid hybrid PPS1.

PubMed

Desai, Tanvi J; Toombs, Jason E; Minna, John D; Brekken, Rolf A; Udugamasooriya, Damith Gomika

2016-05-24

Phosphatidylserine (PS) is an anionic phospholipid maintained on the inner-leaflet of the cell membrane and is externalized in malignant cells. We previously launched a careful unbiased selection targeting biomolecules (e.g. protein, lipid or carbohydrate) distinct to cancer cells by exploiting HCC4017 lung cancer and HBEC30KT normal epithelial cells derived from the same patient, identifying HCC4017 specific peptide-peptoid hybrid PPS1. In this current study, we identified PS as the target of PPS1. We validated direct PPS1 binding to PS using ELISA-like assays, lipid dot blot and liposome based binding assays. In addition, PPS1 recognized other negatively charged and cancer specific lipids such as phosphatidic acid, phosphatidylinositol and phosphatidylglycerol. PPS1 did not bind to neutral lipids such as phosphatidylethanolamine found in cancer and phosphatidylcholine and sphingomyelin found in normal cells. Further we found that the dimeric version of PPS1 (PPS1D1) displayed strong cytotoxicity towards lung cancer cell lines that externalize PS, but not normal cells. PPS1D1 showed potent single agent anti-tumor activity and enhanced the efficacy of docetaxel in mice bearing H460 lung cancer xenografts. Since PS and anionic phospholipid externalization is common across many cancer types, PPS1 may be an alternative to overcome limitations of protein targeted agents.

Manipulating motions of targeted single cells in solution by an integrated double-ring magnetic tweezers imaging microscope.

PubMed

Wu, Meiling; Yadav, Rajeev; Pal, Nibedita; Lu, H Peter

2017-07-01

Controlling and manipulating living cell motions in solution hold a high promise in developing new biotechnology and biological science. Here, we developed a magnetic tweezers device that employs a combination of two permanent magnets in up-down double-ring configuration axially fitting with a microscopic objective, allowing a picoNewton (pN) bidirectional force and motion control on the sample beyond a single upward pulling direction. The experimental force calibration and magnetic field simulation using finite element method magnetics demonstrate that the designed magnetic tweezers covers a linear-combined pN force with positive-negative polarization changes in a tenability of sub-pN scale, which can be utilized to further achieve motion manipulation by shifting the force balance. We demonstrate an application of the up-down double-ring magnetic tweezers for single cell manipulation, showing that the cells with internalized paramagnetic beads can be selectively picked up and guided in a controlled fine motion.

Manipulating motions of targeted single cells in solution by an integrated double-ring magnetic tweezers imaging microscope

NASA Astrophysics Data System (ADS)

Wu, Meiling; Yadav, Rajeev; Pal, Nibedita; Lu, H. Peter

2017-07-01

Controlling and manipulating living cell motions in solution hold a high promise in developing new biotechnology and biological science. Here, we developed a magnetic tweezers device that employs a combination of two permanent magnets in up-down double-ring configuration axially fitting with a microscopic objective, allowing a picoNewton (pN) bidirectional force and motion control on the sample beyond a single upward pulling direction. The experimental force calibration and magnetic field simulation using finite element method magnetics demonstrate that the designed magnetic tweezers covers a linear-combined pN force with positive-negative polarization changes in a tenability of sub-pN scale, which can be utilized to further achieve motion manipulation by shifting the force balance. We demonstrate an application of the up-down double-ring magnetic tweezers for single cell manipulation, showing that the cells with internalized paramagnetic beads can be selectively picked up and guided in a controlled fine motion.

Dynamic ASXL1 Exon Skipping and Alternative Circular Splicing in Single Human Cells

PubMed Central

Natarajan, Sivaraman; Carter, Robert; Brown, Patrick O.

2016-01-01

Circular RNAs comprise a poorly understood new class of noncoding RNA. In this study, we used a combination of targeted deletion, high-resolution splicing detection, and single-cell sequencing to deeply probe ASXL1 circular splicing. We found that efficient circular splicing required the canonical transcriptional start site and inverted AluSx elements. Sequencing-based interrogation of isoforms after ASXL1 overexpression identified promiscuous linear splicing between all exons, with the two most abundant non-canonical linear products skipping the exons that produced the circular isoforms. Single-cell sequencing revealed a strong preference for either the linear or circular ASXL1 isoforms in each cell, and found the predominant exon skipping product is frequently co-expressed with its reciprocal circular isoform. Finally, absolute quantification of ASXL1 isoforms confirmed our findings and suggests that standard methods overestimate circRNA abundance. Taken together, these data reveal a dynamic new view of circRNA genesis, providing additional framework for studying their roles in cellular biology. PMID:27736885

Electrical analysis of c-Si/CGSe monolithic tandem solar cells by using a cell-selective light absorption scheme.

PubMed

Jeong, Ah Reum; Choi, Sung Bin; Kim, Won Mok; Park, Jong-Keuk; Choi, Jihye; Kim, Inho; Jeong, Jeung-Hyun

2017-11-16

A monolithic tandem solar cell consisting of crystalline Si (c-Si)/indium tin oxide (ITO)/CuGaSe 2 (CGSe) was demonstrated by stacking a CGSe solar cell on a c-Si/ITO solar cell to obtain a photovoltaic conversion efficiency of about 10%. Electrical analyses based on cell-selective light absorption were applied to individually characterize the photovoltaic performances of the top and bottom subcells. Illumination at a frequency that could be absorbed only by a targeted top or bottom subcell permitted measurement of the open-circuit voltage of the target subcell and the shunt resistance of the non-target subcell. The cell parameters measured from each subcell were very similar to those of the corresponding single cell, confirming the validity of the suggested method. In addition, separating the light absorption intensities at the top and bottom subcells made us measure the bias-dependent photocurrent for each subcell. The series resistance of a c-Si/ITO/CGSe cell subjected to bottom-cell limiting conditions was slightly large, implying that the tunnel junction was a little resistive or slightly beyond ohmic. This analysis demonstrated that aside from producing a slightly resistive tunnel junction, our fabrication processes were successful in monolithically integrating a CGSe cell onto a c-Si/ITO cell without degrading the performances of both cells.

Targeted cancer therapy based on single-wall carbon nanohorns with doxorubicin in vitro and in vivo

NASA Astrophysics Data System (ADS)

Ma, Xiaona; Shu, Chang; Guo, Jing; Pang, Lili; Su, Lin; Fu, Degang; Zhong, Wenying

2014-07-01

A new targeted drug delivery system (DDS) based on oxidized single-wall carbon nanohorns (oxSWCNHs) was developed. Sodium alginate (SA) was used to modify oxSWCNHs to improve its dispersibility and biocompatibility, the first time such a modification to oxSWCNHs was reported. The humanized anti-vascular endothelial growth factor (anti-VEGF) monoclonal antibody was bound to the SA as targeting group to selectively kill the tumor cells. Doxorubicin hydrochloride (DOX) was conjugated to oxSWCNHs in basic pH solution by π-π stacking, and its release was triggered by the lower pH as the micro-environment of the tumor. Quantitative analyses showed that the DOX@oxSWCNHs/SA complexes contained 1 g DOX per gram of oxSWCNHs. Cell experiment showed that the DOX@oxSWCNHs/SA-mAb effectively targeted the human breast adenocarcinoma (MCF-7) cells and rarely adhered to the human embryonic kidney 293 (HEK293) cells. And the anticancer effects of the complexes were higher than those of the free DOX. Pharmaceutical efficiency in vivo showed that the relative tumor volumes (RTV) of normal saline (NS) group, oxSWCNH/SA-mAb (2.5 mg/kg) group, DOX (2.5 mg/kg) group, and DOX@oxSWCNHs/SA-mAb (2.5 mg/kg) group were approximately 61, 56, 14, and 7.2, respectively. In addition, higher drug dose (5 mg/kg) of DOX@oxSWCNHs/SA-mAb resulted in a better antitumor activity. Histopathological studies in mice confirmed that the DOX@oxSWCNHs/SA-mAb complexes did not demonstrate any detectable hepatotoxicity, cardiotoxicity, and nephrotoxicity.

Single-Domain Antibodies and the Promise of Modular Targeting in Cancer Imaging and Treatment

PubMed Central

Iezzi, María Elena; Policastro, Lucía; Werbajh, Santiago; Podhajcer, Osvaldo; Canziani, Gabriela Alicia

2018-01-01

Monoclonal antibodies and their fragments have significantly changed the outcome of cancer in the clinic, effectively inhibiting tumor cell proliferation, triggering antibody-dependent immune effector cell activation and complement mediated cell death. Along with a continued expansion in number, diversity, and complexity of validated tumor targets there is an increasing focus on engineering recombinant antibody fragments for lead development. Single-domain antibodies (sdAbs), in particular those engineered from the variable heavy-chain fragment (VHH gene) found in Camelidae heavy-chain antibodies (or IgG2 and IgG3), are the smallest fragments that retain the full antigen-binding capacity of the antibody with advantageous properties as drugs. For similar reasons, growing attention is being paid to the yet smaller variable heavy chain new antigen receptor (VNAR) fragments found in Squalidae. sdAbs have been selected, mostly from immune VHH libraries, to inhibit or modulate enzyme activity, bind soluble factors, internalize cell membrane receptors, or block cytoplasmic targets. This succinct review is a compilation of recent data documenting the application of engineered, recombinant sdAb in the clinic as epitope recognition “modules” to build monomeric, dimeric and multimeric ligands that target, tag and stall solid tumor growth in vivo. Size, affinity, specificity, and the development profile of sdAbs drugs are seemingly consistent with desirable clinical efficacy and safety requirements. But the hepatotoxicity of the tetrameric anti-DR5-VHH drug in patients with pre-existing anti-drug antibodies halted the phase I clinical trial and called for a thorough pre-screening of the immune and poly-specific reactivities of the sdAb leads. PMID:29520274

High efficient expression of a functional humanized single-chain variable fragment (scFv) antibody against CD22 in Pichia pastoris.

PubMed

Zarei, Najmeh; Vaziri, Behrouz; Shokrgozar, Mohammad Ali; Mahdian, Reza; Fazel, Ramin; Khalaj, Vahid

2014-12-01

Single-chain variable fragments (scFvs) have recently emerged as attractive candidates in targeted immunotherapy of various malignancies. The anti-CD22 scFv is able to target CD22, on B cell surface and is being considered as a promising molecule in targeted immunotherapy of B cell malignancies. The recombinant anti-CD22 scFv has been successfully expressed in Escherichia coli; however, the insufficient production yield has been a major bottleneck for its therapeutic application. The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the production of a wide variety of recombinant proteins such as antibody fragments. In this study, we used the Pichia expression system to express a humanized scFv antibody against CD22. The full-length humanized scFv gene was codon optimized, cloned into the pPICZαA and expressed in GS115 strain. The maximum production level of the scFv (25 mg/L) were achieved at methanol concentration, 1 %; pH 6.0; inoculum density, OD600 = 3 and the induction time of 72 h. The correlation between scFv gene dosage and expression level was also investigated by real-time PCR, and the results confirmed the presence of such correlation up to five gene copies. Immunofluorescence and flow cytometry studies and Biacore analysis demonstrated binding to CD22 on the surface of human lymphoid cell line Raji and recombinant soluble CD22, respectively. Taken together, the presented data suggest that the Pichia pastoris can be considered as an efficient host for the large-scale production of anti-CD22 scFv as a promising carrier for targeted drug delivery in treatment of CD22(+) B cell malignancies.

Single-Stranded γPNAs for In Vivo Site-Specific Genome Editing via Watson-Crick Recognition

PubMed Central

Bahal, Raman; Quijano, Elias; McNeer, Nicole Ali; Liu, Yanfeng; Bhunia, Dinesh C.; López-Giráldez, Francesco; Fields, Rachel J.; Saltzman, W. Mark; Ly, Danith H.; Glazer, Peter M.

2014-01-01

Triplex-forming peptide nucleic acids (PNAs) facilitate gene editing by stimulating recombination of donor DNAs within genomic DNA via site-specific formation of altered helical structures that further stimulate DNA repair. However, PNAs designed for triplex formation are sequence restricted to homopurine sites. Herein we describe a novel strategy where next generation single-stranded gamma PNAs (γPNAs) containing miniPEG substitutions at the gamma position can target genomic DNA in mouse bone marrow at mixed-sequence sites to induce targeted gene editing. In addition to enhanced binding, γPNAs confer increased solubility and improved formulation into poly(lactic-co-glycolic acid) (PLGA) nanoparticles for efficient intracellular delivery. Single-stranded γPNAs induce targeted gene editing at frequencies of 0.8% in mouse bone marrow cells treated ex vivo and 0.1% in vivo via IV injection, without detectable toxicity. These results suggest that γPNAs may provide a new tool for induced gene editing based on Watson-Crick recognition without sequence restriction. PMID:25174576

Single-stranded γPNAs for in vivo site-specific genome editing via Watson-Crick recognition.

PubMed

Bahal, Raman; Quijano, Elias; McNeer, Nicole A; Liu, Yanfeng; Bhunia, Dinesh C; Lopez-Giraldez, Francesco; Fields, Rachel J; Saltzman, William M; Ly, Danith H; Glazer, Peter M

2014-01-01

Triplex-forming peptide nucleic acids (PNAs) facilitate gene editing by stimulating recombination of donor DNAs within genomic DNA via site-specific formation of altered helical structures that further stimulate DNA repair. However, PNAs designed for triplex formation are sequence restricted to homopurine sites. Herein we describe a novel strategy where next generation single-stranded gamma PNAs (γPNAs) containing miniPEG substitutions at the gamma position can target genomic DNA in mouse bone marrow at mixed-sequence sites to induce targeted gene editing. In addition to enhanced binding, γPNAs confer increased solubility and improved formulation into poly(lactic-co-glycolic acid) (PLGA) nanoparticles for efficient intracellular delivery. Single-stranded γPNAs induce targeted gene editing at frequencies of 0.8% in mouse bone marrow cells treated ex vivo and 0.1% in vivo via IV injection, without detectable toxicity. These results suggest that γPNAs may provide a new tool for induced gene editing based on Watson-Crick recognition without sequence restriction.

Whole-body multicolor spectrally resolved fluorescence imaging for development of target-specific optical contrast agents using genetically engineered probes

NASA Astrophysics Data System (ADS)

Kobayashi, Hisataka; Hama, Yukihiro; Koyama, Yoshinori; Barrett, Tristan; Urano, Yasuteru; Choyke, Peter L.

2007-02-01

Target-specific contrast agents are being developed for the molecular imaging of cancer. Optically detectable target-specific agents are promising for clinical applications because of their high sensitivity and specificity. Pre clinical testing is needed, however, to validate the actual sensitivity and specificity of these agents in animal models, and involves both conventional histology and immunohistochemistry, which requires large numbers of animals and samples with costly handling. However, a superior validation tool takes advantage of genetic engineering technology whereby cell lines are transfected with genes that induce the target cell to produce fluorescent proteins with characteristic emission spectra thus, identifying them as cancer cells. Multicolor fluorescence imaging of these genetically engineered probes can provide rapid validation of newly developed exogenous probes that fluoresce at different wavelengths. For example, the plasmid containing the gene encoding red fluorescent protein (RFP) was transfected into cell lines previously developed to either express or not-express specific cell surface receptors. Various antibody-based or receptor ligand-based optical contrast agents with either green or near infrared fluorophores were developed to concurrently target and validate cancer cells and their positive and negative controls, such as β-D-galactose receptor, HER1 and HER2 in a single animal/organ. Spectrally resolved fluorescence multicolor imaging was used to detect separate fluorescent emission spectra from the exogenous agents and RFP. Therefore, using this in vivo imaging technique, we were able to demonstrate the sensitivity and specificity of the target-specific optical contrast agents, thus reducing the number of animals needed to conduct these experiments.

From the Cover: Vulnerability of C6 Astrocytoma Cells After Single-Compound and Joint Exposure to Type I and Type II Pyrethroid Insecticides.

PubMed

Romero, Delfina M; Berardino, Bruno G; Wolansky, Marcelo J; Kotler, Mónica L

2017-01-01

A primary mode-of-action of all pyrethroid insecticides (PYRs) is the disruption of the voltage-gated sodium channel electrophysiology in neurons of target pests and nontarget species. The neurological actions of PYRs on non-neuronal cells of the nervous system remain poorly investigated. In the present work, we used C6 astrocytoma cells to study PYR actions (0.1-50 μM) under the hypothesis that glial cells may be targeted by and vulnerable to PYRs. To this end, we characterized the effects of bifenthrin (BF), tefluthrin (TF), α-cypermethrin (α-CYP), and deltamethrin (DM) on the integrity of nuclear, mitochondrial, and lysosomal compartments. In general, 24- to 48-h exposures produced concentration-related impairment of cell viability. In single-compound, 24-h exposure experiments, effective concentration (EC) 15 s 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT assay) were computed as follows (in μM): BF, 16.1; TF, 37.3; α-CYP, 7.8; DM, 5.0. We found concentration-related damage in several C6-cell subcellular compartments (mitochondria, nuclei, and lysosomes) at ≥ 10 -1 μM levels. Last, we examined a mixture of all PYRs (ie, Σ individual EC 15 ) using MTT assays and subcellular analyses. Our findings indicate that C6 cells are responsive to nM levels of PYRs, suggesting that astroglial susceptibility may contribute to the low-dose neurological effects caused by these insecticides. This research further suggests that C6 cells may provide relevant information as a screening platform for pesticide mixtures targeting nervous system cells by expected and unexpected toxicogenic pathways potentially contributing to clinical neurotoxicity. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A comparative analysis of conventional and pretargeted radioimmunotherapy of B-cell lymphomas by targeting CD20, CD22, and HLA-DR singly and in combinations

PubMed Central

Orgun, Nural; Hamlin, Donald K.; Wilbur, D. Scott; Gooley, Theodore A.; Gopal, Ajay K.; Park, Steven I.; Green, Damian J.; Lin, Yukang; Press, Oliver W.

2009-01-01

Relapsed B-cell lymphomas are currently incurable with conventional chemotherapy and radiation treatments. Radiolabeled antibodies directed against B-cell surface antigens have emerged as effective and safe therapies for relapsed lymphomas. We therefore investigated the potential utility of both directly radiolabeled 1F5 (anti-CD20), HD39 (anti-CD22), and Lym-1 (anti-DR) antibodies (Abs) and of pretargeted radioimmunotherapy (RIT) using Ab-streptavidin (SA) conjugates, followed by an N-acetylgalactosamine dendrimeric clearing agent and radiometal-labeled DOTA-biotin, for treatment of lymphomas in mouse models using Ramos, Raji, and FL-18 human lymphoma xenografts. This study demonstrates the marked superiority of pretargeted RIT for each of the antigenic targets with more complete tumor regressions and longer mouse survival compared with conventional one-step RIT. The Ab-SA conjugate yielding the best tumor regression and progression-free survival after pretargeted RIT varied depending upon the lymphoma cell line used, with 1F5 Ab-SA and Lym-1 Ab-SA conjugates yielding the most promising results overall. Contrary to expectations, the best rates of mouse survival were obtained using optimal single Ab-SA conjugates rather than combinations of conjugates targeting different antigens. We hypothesize that clinical implementation of pretargeted RIT methods will provide a meaningful prolongation of survival for patients with relapsed lymphomas compared with currently available treatment strategies. PMID:19124831

Enhancing venetoclax activity in acute myeloid leukemia by co-targeting MCL1.

PubMed

Teh, T-C; Nguyen, N-Y; Moujalled, D M; Segal, D; Pomilio, G; Rijal, S; Jabbour, A; Cummins, K; Lackovic, K; Blombery, P; Thompson, E; Ekert, P G; Lessene, G; Glaser, S P; Huang, D C S; Roberts, A W; Guthridge, M A; Wei, A H

2018-02-01

Targeted therapies are frequently combined with standard cytotoxic drugs to enhance clinical response. Targeting the B-cell lymphoma 2 (BCL-2) family of proteins is an attractive option to combat chemoresistance in leukemia. Preclinical and clinical studies indicate modest single-agent activity with selective BCL-2 inhibitors (for example, venetoclax). We show that venetoclax synergizes with cytarabine and idarubicin to increase antileukemic efficacy in a TP53-dependent manner. Although TP53 deficiency impaired sensitivity to combined venetoclax and chemotherapy, higher-dose idarubicin was able to suppress MCL1 and induce cell death independently of TP53. Consistent with an MCL1-specific effect, cell death from high-dose idarubicin was dependent on pro-apoptotic Bak. Combining higher-dose idarubicin with venetoclax was able to partially overcome resistance in Bak-deficient cells. Using inducible vectors and venetoclax to differentially target anti-apoptotic BCL-2 family members, BCL-2 and MCL1 emerged as critical and complementary proteins regulating cell survival in acute myeloid leukemia. Dual targeting of BCL-2 and MCL1, but not either alone, prolonged survival of leukemia-bearing mice. In conclusion, our findings support the further investigation of venetoclax in combination with standard chemotherapy, including intensified doses of idarubicin. Venetoclax should also be investigated in combination with direct inhibitors of MCL1 as a chemotherapy-free approach in the future.

Anti-HER2 antibody and ScFvEGFR-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer

PubMed Central

Chen, Hongwei; Wang, Liya; Yu, Qiqi; Qian, Weiping; Tiwari, Diana; Yi, Hong; Wang, Andrew Y; Huang, Jing; Yang, Lily; Mao, Hui

2013-01-01

Antifouling magnetic iron oxide nanoparticles (IONPs) coated with block copolymer poly(ethylene oxide)-block-poly(γ-methacryloxypropyltrimethoxysilane) (PEO-b-PγMPS) were investigated for improving cell targeting by reducing nonspecific uptake. Conjugation of a HER2 antibody, Herceptin®, or a single chain fragment (ScFv) of antibody against epidermal growth factor receptor (ScFvEGFR) to PEO-b-PγMPS-coated IONPs resulted in HER2-targeted or EGFR-targeted IONPs (anti-HER2-IONPs or ScFvEGFR-IONPs). The anti-HER2-IONPs bound specifically to SK-BR-3, a HER2-overexpressing breast cancer cell line, but not to MDA-MB-231, a HER2-underexpressing cell line. On the other hand, the ScFvEGFR-IONPs showed strong reactivity with MDA-MB-231, an EGFR-positive human breast cancer cell line, but not with MDA-MB-453, an EGFR-negative human breast cancer cell line. Transmission electron microscopy revealed internalization of the receptor-targeted nanoparticles by the targeted cancer cells. In addition, both antibody-conjugated and non-antibody-conjugated IONPs showed reduced nonspecific uptake by RAW264.7 mouse macrophages in vitro. The developed IONPs showed a long blood circulation time (serum half-life 11.6 hours) in mice and low accumulation in both the liver and spleen. At 24 hours after systemic administration of ScFvEGFR-IONPs into mice bearing EGFR-positive breast cancer 4T1 mouse mammary tumors, magnetic resonance imaging revealed signal reduction in the tumor as a result of the accumulation of the targeted IONPs. PMID:24124366

Genomic Disruption of VEGF-A Expression in Human Retinal Pigment Epithelial Cells Using CRISPR-Cas9 Endonuclease.

PubMed

Yiu, Glenn; Tieu, Eric; Nguyen, Anthony T; Wong, Brittany; Smit-McBride, Zeljka

2016-10-01

To employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells. CRISPR sequences targeting exon 1 of human VEGF-A were computationally identified based on predicted Cas9 on- and off-target probabilities. Single guide RNA (gRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the Streptococcuspyogenes Cas9 endonuclease (SpCas9) gene. The lentiviral vectors were used to infect ARPE-19 cells, a human RPE cell line. Frequency of insertion or deletion (indel) mutations was assessed by T7 endonuclease 1 mismatch detection assay; mRNA levels were assessed with quantitative real-time PCR; and VEGF-A protein levels were determined by ELISA. In vitro angiogenesis was measured using an endothelial cell tube formation assay. Five gRNAs targeting VEGF-A were selected based on the highest predicted on-target probabilities, lowest off-target probabilities, or combined average of both scores. Lentiviral delivery of the top-scoring gRNAs with SpCas9 resulted in indel formation in the VEGF-A gene at frequencies up to 37.0% ± 4.0% with corresponding decreases in secreted VEGF-A protein up to 41.2% ± 7.4% (P < 0.001), and reduction of endothelial tube formation up to 39.4% ± 9.8% (P = 0.02). No significant indel formation in the top three putative off-target sites tested was detected. The CRISPR-Cas9 endonuclease system may reduce VEGF-A secretion from human RPE cells and suppress angiogenesis, supporting the possibility of employing gene editing for antiangiogenesis therapy in ocular diseases.

Spatiotemporal Targeting of a Dual-Ligand Nanoparticle to Cancer Metastasis

PubMed Central

Doolittle, Elizabeth; Peiris, Pubudu M.; Doron, Gilad; Goldberg, Amy; Tucci, Samantha; Rao, Swetha; Shah, Shruti; Sylvestre, Meilyn; Govender, Priya; Turan, Oguz; Lee, Zhenghong; Schiemann, William P.; Karathanasis, Efstathios

2015-01-01

Various targeting strategies and ligands have been employed to direct nanoparticles to tumors that upregulate specific cell-surface molecules. However, tumors display a dynamic, heterogeneous microenvironment, which undergoes spatiotemporal changes including the expression of targetable cell-surface biomarkers. Here, we investigated a dual-ligand nanoparticle to effectively target two receptors overexpressed in aggressive tumors. By using two different chemical specificities, the dual-ligand strategy considered the spatiotemporal alterations in the expression patterns of the receptors in cancer sites. As a case study, we used two mouse models of metastasis of triple-negative breast cancer using the MDA-MB-231 and 4T1 cells. The dual-ligand system utilized two peptides targeting P-selectin and αvβ3 integrin, which are functionally linked to different stages of the development of metastatic disease at a distal site. Using in vivo multimodal imaging and post mortem histological analyses, this study shows that the dual-ligand nanoparticle effectively targeted metastatic disease that was otherwise missed by single-ligand strategies. The dual-ligand nanoparticle was capable of capturing different metastatic sites within the same animal that overexpressed either receptor or both of them. Furthermore, the highly efficient targeting resulted in 22% of the injected dual-ligand nanoparticles being deposited in early-stage metastases within 2 h after injection. PMID:26203676

Spatiotemporal Targeting of a Dual-Ligand Nanoparticle to Cancer Metastasis.

PubMed

Doolittle, Elizabeth; Peiris, Pubudu M; Doron, Gilad; Goldberg, Amy; Tucci, Samantha; Rao, Swetha; Shah, Shruti; Sylvestre, Meilyn; Govender, Priya; Turan, Oguz; Lee, Zhenghong; Schiemann, William P; Karathanasis, Efstathios

2015-08-25

Various targeting strategies and ligands have been employed to direct nanoparticles to tumors that upregulate specific cell-surface molecules. However, tumors display a dynamic, heterogeneous microenvironment, which undergoes spatiotemporal changes including the expression of targetable cell-surface biomarkers. Here, we investigated a dual-ligand nanoparticle to effectively target two receptors overexpressed in aggressive tumors. By using two different chemical specificities, the dual-ligand strategy considered the spatiotemporal alterations in the expression patterns of the receptors in cancer sites. As a case study, we used two mouse models of metastasis of triple-negative breast cancer using the MDA-MB-231 and 4T1 cells. The dual-ligand system utilized two peptides targeting P-selectin and αvβ3 integrin, which are functionally linked to different stages of the development of metastatic disease at a distal site. Using in vivo multimodal imaging and post mortem histological analyses, this study shows that the dual-ligand nanoparticle effectively targeted metastatic disease that was otherwise missed by single-ligand strategies. The dual-ligand nanoparticle was capable of capturing different metastatic sites within the same animal that overexpressed either receptor or both of them. Furthermore, the highly efficient targeting resulted in 22% of the injected dual-ligand nanoparticles being deposited in early-stage metastases within 2 h after injection.

Multifunctional quantum dot-polypeptide hybrid nanogel for targeted imaging and drug delivery

NASA Astrophysics Data System (ADS)

Yang, Jie; Yao, Ming-Hao; Wen, Lang; Song, Ji-Tao; Zhang, Ming-Zhen; Zhao, Yuan-Di; Liu, Bo

2014-09-01

A new type of multifunctional quantum dot (QD)-polypeptide hybrid nanogel with targeted imaging and drug delivery properties has been developed by metal-affinity driven self-assembly between artificial polypeptides and CdSe-ZnS core-shell QDs. On the surface of QDs, a tunable sandwich-like microstructure consisting of two hydrophobic layers and one hydrophilic layer between them was verified by capillary electrophoresis, transmission electron microscopy, and dynamic light scattering measurements. Hydrophobic and hydrophilic drugs can be simultaneously loaded in a QD-polypeptide nanogel. In vitro drug release of drug-loaded QD-polypeptide nanogels varies strongly with temperature, pH, and competitors. A drug-loaded QD-polypeptide nanogel with an arginine-glycine-aspartic acid (RGD) motif exhibited efficient receptor-mediated endocytosis in αvβ3 overexpressing HeLa cells but not in the control MCF-7 cells as analyzed by confocal microscopy and flow cytometry. In contrast, non-targeted QD-polypeptide nanogels revealed minimal binding and uptake in HeLa cells. Compared with the original QDs, the QD-polypeptide nanogels showed lower in vitro cytotoxicity for both HeLa cells and NIH 3T3 cells. Furthermore, the cytotoxicity of the targeted QD-polypeptide nanogel was lower for normal NIH 3T3 cells than that for HeLa cancer cells. These results demonstrate that the integration of imaging and drug delivery functions in a single QD-polypeptide nanogel has the potential for application in cancer diagnosis, imaging, and therapy.A new type of multifunctional quantum dot (QD)-polypeptide hybrid nanogel with targeted imaging and drug delivery properties has been developed by metal-affinity driven self-assembly between artificial polypeptides and CdSe-ZnS core-shell QDs. On the surface of QDs, a tunable sandwich-like microstructure consisting of two hydrophobic layers and one hydrophilic layer between them was verified by capillary electrophoresis, transmission electron microscopy, and dynamic light scattering measurements. Hydrophobic and hydrophilic drugs can be simultaneously loaded in a QD-polypeptide nanogel. In vitro drug release of drug-loaded QD-polypeptide nanogels varies strongly with temperature, pH, and competitors. A drug-loaded QD-polypeptide nanogel with an arginine-glycine-aspartic acid (RGD) motif exhibited efficient receptor-mediated endocytosis in αvβ3 overexpressing HeLa cells but not in the control MCF-7 cells as analyzed by confocal microscopy and flow cytometry. In contrast, non-targeted QD-polypeptide nanogels revealed minimal binding and uptake in HeLa cells. Compared with the original QDs, the QD-polypeptide nanogels showed lower in vitro cytotoxicity for both HeLa cells and NIH 3T3 cells. Furthermore, the cytotoxicity of the targeted QD-polypeptide nanogel was lower for normal NIH 3T3 cells than that for HeLa cancer cells. These results demonstrate that the integration of imaging and drug delivery functions in a single QD-polypeptide nanogel has the potential for application in cancer diagnosis, imaging, and therapy. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr03058c

Conservative site-specific and single-copy transgenesis in human LINE-1 elements

PubMed Central

Vijaya Chandra, Shree Harsha; Makhija, Harshyaa; Peter, Sabrina; Myint Wai, Cho Mar; Li, Jinming; Zhu, Jindong; Ren, Zhonglu; D'Alcontres, Martina Stagno; Siau, Jia Wei; Chee, Sharon; Ghadessy, Farid John; Dröge, Peter

2016-01-01

Genome engineering of human cells plays an important role in biotechnology and molecular medicine. In particular, insertions of functional multi-transgene cassettes into suitable endogenous sequences will lead to novel applications. Although several tools have been exploited in this context, safety issues such as cytotoxicity, insertional mutagenesis and off-target cleavage together with limitations in cargo size/expression often compromise utility. Phage λ integrase (Int) is a transgenesis tool that mediates conservative site-specific integration of 48 kb DNA into a safe harbor site of the bacterial genome. Here, we show that an Int variant precisely recombines large episomes into a sequence, termed attH4X, found in 1000 human Long INterspersed Elements-1 (LINE-1). We demonstrate single-copy transgenesis through attH4X-targeting in various cell lines including hESCs, with the flexibility of selecting clones according to transgene performance and downstream applications. This is exemplified with pluripotency reporter cassettes and constitutively expressed payloads that remain functional in LINE1-targeted hESCs and differentiated progenies. Furthermore, LINE-1 targeting does not induce DNA damage-response or chromosomal aberrations, and neither global nor localized endogenous gene expression is substantially affected. Hence, this simple transgene addition tool should become particularly useful for applications that require engineering of the human genome with multi-transgenes. PMID:26673710

Pseudotemporal Ordering of Single Cells Reveals Metabolic Control of Postnatal β Cell Proliferation.

PubMed

Zeng, Chun; Mulas, Francesca; Sui, Yinghui; Guan, Tiffany; Miller, Nathanael; Tan, Yuliang; Liu, Fenfen; Jin, Wen; Carrano, Andrea C; Huising, Mark O; Shirihai, Orian S; Yeo, Gene W; Sander, Maike

2017-05-02

Pancreatic β cell mass for appropriate blood glucose control is established during early postnatal life. β cell proliferative capacity declines postnatally, but the extrinsic cues and intracellular signals that cause this decline remain unknown. To obtain a high-resolution map of β cell transcriptome dynamics after birth, we generated single-cell RNA-seq data of β cells from multiple postnatal time points and ordered cells based on transcriptional similarity using a new analytical tool. This analysis captured signatures of immature, proliferative β cells and established high expression of amino acid metabolic, mitochondrial, and Srf/Jun/Fos transcription factor genes as their hallmark feature. Experimental validation revealed high metabolic activity in immature β cells and a role for reactive oxygen species and Srf/Jun/Fos transcription factors in driving postnatal β cell proliferation and mass expansion. Our work provides the first high-resolution molecular characterization of state changes in postnatal β cells and paves the way for the identification of novel therapeutic targets to stimulate β cell regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

Generation of Hypertension-Associated STK39 Polymorphism Knockin Cell Lines With the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 System.

PubMed

Mandai, Shintaro; Mori, Takayasu; Sohara, Eisei; Rai, Tatemitsu; Uchida, Shinichi

2015-12-01

Previous genome-wide association studies identified serine threonine kinase 39 (STK39), encoding STE20/SPS1-related proline/alanine-rich kinase, as one of a limited number of hypertension susceptibility genes. A recent meta-analysis confirmed the association of STK39 intronic polymorphism rs3754777 with essential hypertension, among previously reported hypertension-associated STK39 polymorphisms. However, the biochemical function of this polymorphism in the mechanism responsible for hypertension is yet to be clarified. We generated rs3754777G>A knockin human cell lines with clustered regularly interspaced short palindromic repeats-mediated genome engineering. Homozygous (A/A) and heterozygous (G/A) knockin human embryonic kidney cell lines were generated using a double nickase, single-guide RNAs targeting STK39 intron 5 around single-nucleotide polymorphism, and a 100-bp donor single-stranded DNA oligonucleotide. Reverse transcription polymerase chain reaction with sequencing analyses revealed the identical STK39 transcripts among the wild-type and both knockin cell lines. Quantitative reverse transcription polymerase chain reaction showed increased STK39 mRNA expression, and immunoblot analysis revealed increases in total and phosphorylated STE20/SPS1-related proline/alanine-rich kinase with increased phosphorylated Na-K-Cl cotransporter isoform 1 in both knockin cell lines. The largest increases in these molecules were observed in the homozygous cell line. These findings indicated that this intronic polymorphism increases STK39 transcription, leading to activation of the STE20/SPS1-related proline/alanine-rich kinase-solute carrier family 12A signaling cascade. Increased interactions between STE20/SPS1-related proline/alanine-rich kinase and the target cation-chloride cotransporters may be responsible for hypertension susceptibility in individuals with this polymorphism. © 2015 American Heart Association, Inc.

Cooperative Targets of Combined mTOR/HDAC Inhibition Promote MYC Degradation.

PubMed

Simmons, John K; Michalowski, Aleksandra M; Gamache, Benjamin J; DuBois, Wendy; Patel, Jyoti; Zhang, Ke; Gary, Joy; Zhang, Shuling; Gaikwad, Snehal; Connors, Daniel; Watson, Nicholas; Leon, Elena; Chen, Jin-Qiu; Kuehl, W Michael; Lee, Maxwell P; Zingone, Adriana; Landgren, Ola; Ordentlich, Peter; Huang, Jing; Mock, Beverly A

2017-09-01

Cancer treatments often require combinations of molecularly targeted agents to be effective. mTORi (rapamycin) and HDACi (MS-275/entinostat) inhibitors have been shown to be effective in limiting tumor growth, and here we define part of the cooperative action of this drug combination. More than 60 human cancer cell lines responded synergistically (CI<1) when treated with this drug combination compared with single agents. In addition, a breast cancer patient-derived xenograft, and a BCL-XL plasmacytoma mouse model both showed enhanced responses to the combination compared with single agents. Mice bearing plasma cell tumors lived an average of 70 days longer on combination treatment compared with single agents. A set of 37 genes cooperatively affected (34 downregulated; 3 upregulated) by the combination responded pharmacodynamically in human myeloma cell lines, xenografts, and a P493 model, and were both enriched in tumors, and correlated with prognostic markers in myeloma patient datasets. Genes downregulated by the combination were overexpressed in several untreated cancers (breast, lung, colon, sarcoma, head and neck, myeloma) compared with normal tissues. The MYC/E2F axis, identified by upstream regulator analyses and validated by immunoblots, was significantly inhibited by the drug combination in several myeloma cell lines. Furthermore, 88% of the 34 genes downregulated have MYC-binding sites in their promoters, and the drug combination cooperatively reduced MYC half-life by 55% and increased degradation. Cells with MYC mutations were refractory to the combination. Thus, integrative approaches to understand drug synergy identified a clinically actionable strategy to inhibit MYC/E2F activity and tumor cell growth in vivo Mol Cancer Ther; 16(9); 2008-21. ©2017 AACR . ©2017 American Association for Cancer Research.

Single-cell quantitative HER2 measurement identifies heterogeneity and distinct subgroups within traditionally defined HER2-positive patients.

PubMed

Onsum, Matthew D; Geretti, Elena; Paragas, Violette; Kudla, Arthur J; Moulis, Sharon P; Luus, Lia; Wickham, Thomas J; McDonagh, Charlotte F; MacBeath, Gavin; Hendriks, Bart S

2013-11-01

Human epidermal growth factor receptor 2 (HER2) is an important biomarker for breast and gastric cancer prognosis and patient treatment decisions. HER2 positivity, as defined by IHC or fluorescent in situ hybridization testing, remains an imprecise predictor of patient response to HER2-targeted therapies. Challenges to correct HER2 assessment and patient stratification include intratumoral heterogeneity, lack of quantitative and/or objective assays, and differences between measuring HER2 amplification at the protein versus gene level. We developed a novel immunofluorescence method for quantitation of HER2 protein expression at the single-cell level on FFPE patient samples. Our assay uses automated image analysis to identify and classify tumor versus non-tumor cells, as well as quantitate the HER2 staining for each tumor cell. The HER2 staining level is converted to HER2 protein expression using a standard cell pellet array stained in parallel with the tissue sample. This approach allows assessment of HER2 expression and heterogeneity within a tissue section at the single-cell level. By using this assay, we identified distinct subgroups of HER2 heterogeneity within traditional definitions of HER2 positivity in both breast and gastric cancers. Quantitative assessment of intratumoral HER2 heterogeneity may offer an opportunity to improve the identification of patients likely to respond to HER2-targeted therapies. The broad applicability of the assay was demonstrated by measuring HER2 expression profiles on multiple tumor types, and on normal and diseased heart tissues. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Targeted nanoparticles for image-guided treatment of triple-negative breast cancer: clinical significance and technological advances.

PubMed

Miller-Kleinhenz, Jasmine M; Bozeman, Erica N; Yang, Lily

2015-01-01

Effective treatment of triple-negative breast cancer (TNBC) with its aggressive tumor biology, highly heterogeneous tumor cells, and poor prognosis requires an integrated therapeutic approach that addresses critical issues in cancer therapy. Multifunctional nanoparticles with the abilities of targeted drug delivery and noninvasive imaging for monitoring drug delivery and responses to therapy, such as theranostic nanoparticles, hold great promise toward the development of novel therapeutic approaches for the treatment of TNBC using a single therapeutic platform. The biological and pathological characteristics of TNBC provide insight into several potential molecular targets for current and future nanoparticle-based therapeutics. Extensive tumor stroma, highly proliferative cells, and a high rate of drug resistance are all barriers that must be appropriately addressed in order for these nanotherapeutic platforms to be effective. Utilization of the enhanced permeability and retention effect coupled with active targeting of cell surface receptors expressed by TNBC cells, and tumor-associated endothelial cells, stromal fibroblasts, and macrophages is likely to overcome such barriers to facilitate more effective drug delivery. An in-depth summary of current studies investigating targeted nanoparticles in preclinical TNBC mouse and human xenograft models is presented. This review aims to outline the current status of nanotherapeutic options for TNBC patients, identification of promising molecular targets, challenges associated with the development of targeted nanotherapeutics, the research done by our group as well as by others, and future perspectives on the nanomedicine field and ways to translate current preclinical studies into the clinic. © 2015 Wiley Periodicals, Inc.

Antitumor activity of a dual cytokine/single-chain antibody fusion protein for simultaneous delivery of GM-CSF and IL-2 to Ep-CAM expressing tumor cells.

PubMed

Schanzer, Juergen M; Fichtner, Iduna; Baeuerle, Patrick A; Kufer, Peter

2006-01-01

Cytokine targeting to tumor-associated antigens via antibody cytokine fusion proteins has demonstrated potent antitumor activity in numerous animal models and has led to the clinical development of 2 antibody-interleukin-2 (IL-2) fusion proteins. We previously reported on the construction and in vitro properties of a "dual" cytokine fusion protein for simultaneous targeted delivery of human granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-2 to human tumors. The fusion protein is based on a heterodimerized core structure formed by human CH1 and Ckappa domains (heterominibody) with C-terminally fused human cytokines and N-terminally fused single-chain antibody fragments specific for the tumor-associated surface antigen epithelial cell adhesion molecule (Ep-CAM). For testing the antitumor activity in syngeneic mouse xenograft models, we developed "dual cytokine heterominibodies" with murine cytokines (mDCH). mDCH fusion proteins and, as controls, "single cytokine heterominibodies" (SCH) carrying either murine GM-CSF (mGM-CSF) or murine IL-2 (mIL-2) were constructed, of which all retained the specific activities of cytokines and binding to the Ep-CAM antigen on human Ep-CAM transfected mouse colon carcinoma CT26-KSA cells. Over a 5-day treatment course, DCH fusion proteins induced significant inhibition of established pulmonary CT26-KSA metastases in immune-competent Balb/c mice at low daily doses of 1 mug of fusion protein per mouse. However, with the tested dosing schemes, antitumor activity of mDCH was largely independent of cytokine targeting to tumors as demonstrated by a control protein with mutated Ep-CAM binding sites. Single cytokine fusion proteins mSCH-GM-CSF and mSCH-IL-2 showed similar antitumor activity as the dual cytokine fusion protein mDCH, indicating that GM-CSF and IL-2 in one molecule did not significantly synergize in tumor rejection under our experimental conditions. Our results seem to contradict the notion that IL-2 and GM-CSF can synergize in antitumor activity and that with conventional dose regimens, their specific targeting to tumors, as tested here with 2 antibodies of different affinities, enhances their antitumor activity.

Multiple-Targeted Graphene-based Nanocarrier for Intracellular Imaging of mRNAs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Ying; Li, Zhaohui; Liu, Misha

Simultaneous detection and imaging of multiple intracellular messenger RNA (mRNAs) hold great significant for early cancer diagnostics and preventive medicine development. Herein, we propose a multiple-targeted graphene oxide (GO) nanocarrier that can simultaneously detect and image different type mRNAs in living cells. First of all, in vitro detection of multiple targets have been realized successfully based on the multiple-targeted GO nanocarrier with linear relationship ranging from 3 nM to 200 nM, as well as sensitive detection limit of 1.84 nM for manganese superoxide dismutase (Mn-SOD) mRNA and 2.45 nM for β-actin mRNA. Additionally, this nanosensing platform composed of fluorescent labeledmore » single strand DNA probes and GO nanocarrier can identify Mn-SOD mRNA and endogenous mRNA of β-actin in living cancer cells, showing rapid response, high specificity, nuclease stability, and good biocompatibility during the cell imaging. Thirdly, changes of the expression levels of mRNA in living cells before or after the drug treatment can be monitored successfully. By using multiple ssDNA as probes and GO nanocarrier as the cellular delivery cargo, the proposed simultaneous multiple-targeted sensing platform will be of great potential as a powerful tool for intracellular trafficking process from basic research to clinical diagnosis.« less

SERS detection and targeted ablation of lymphoma cells using functionalized Ag nanoparticles

NASA Astrophysics Data System (ADS)

Yao, Qian; Cao, Fei; Feng, Chao; Zhao, Yan; Wang, Xiuhong

2016-03-01

Lymphoma is a heterogeneous group of malignancies of the lymphoid tissue, and is prevalent worldwide affecting both children and adults with a high mortality rate. There is in dire need of accurate and noninvasive approaches for early detection of the disease. Herein, we report a facile way to fabricate silver nanoparticle based nanoprobe by incorporating the corner-stone immunotherapeutic drug Rituxan for simultaneous detection and ablation of lymphoma cells in vitro. The fabricated nanoprobe can detect CD20 positive single lymphoma cell by surface enhanced Raman scattering technique with high specificity. The engineered nanoprobe retains the same antibody property as intact drug via Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) analysis. The nanoprobe efficiently eradicates lymphoma cells in vitro. By integrating the advantages of sensitive SERS detection with targeted ablation capabilities of immunotherapeutic drug through site specificity, this nanoprobe can be applied as outstanding tools in living imaging, cancer diagnosis and treatment.

Visualization of Endoplasmic Reticulum and Mitochondria in Aurantiochytrium limacinum by the Expression of EGFP with Cell Organelle-Specific Targeting/Retaining Signals.

PubMed

Okino, Nozomu; Wakisaka, Hiroyoshi; Ishibashi, Yohei; Ito, Makoto

2018-04-01

Thraustochytrids are single cell marine eukaryotes that produce large amounts of polyunsaturated fatty acids such as docosahexaenoic acid. In the present study, we report the visualization of endoplasmic reticulum (ER) and mitochondria in a type strain of the thraustochytrid, Aurantiochytrium limacinum ATCC MYA-1381, using the enhanced green fluorescent protein (EGFP) with specific targeting/retaining signals. We expressed the egfp gene with ER targeting/retaining signals from A. limacinum calreticulin or BiP/GRP78 in the thraustochytrid, resulting in the distribution of EGFP signals at the perinuclear region and near lipid droplets. ER-Tracker™ Red, an authentic fluorescent probe for the visualization of ER in mammalian cells, also stained the same region. We observed small lipid droplets generated from the visualized ER in the early growth phase of cell culture. Expression of the egfp gene with the mitochondria targeting signal from A. limacinum cytochrome c oxidase resulted in the localization of EGFP near the plasma membrane. The distribution of EGFP signals coincided with that of MitoTracker® Red CMXRos, which is used to visualize mitochondria in eukaryotes. The ER and mitochondria of A. limacinum were visualized for the first time by EGFP with thraustochytrid cell organelle-specific targeting/retaining signals. These results will contribute to classification of the intracellular localization of proteins expressed in ER and mitochondria as well as analyses of these cell organelles in thraustochytrids.

HER2 activating mutations are targets for colorectal cancer treatment.

PubMed

Kavuri, Shyam M; Jain, Naveen; Galimi, Francesco; Cottino, Francesca; Leto, Simonetta M; Migliardi, Giorgia; Searleman, Adam C; Shen, Wei; Monsey, John; Trusolino, Livio; Jacobs, Samuel A; Bertotti, Andrea; Bose, Ron

2015-08-01

The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of patients with colorectal cancer. Introduction of the HER2 mutations S310F, L755S, V777L, V842I, and L866M into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutants are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors neratinib and afatinib. HER2 gene sequencing of 48 cetuximab-resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) wild-type (WT) colorectal cancer patient-derived xenografts (PDX) identified 4 PDXs with HER2 mutations. HER2-targeted therapies were tested on two PDXs. Treatment with a single HER2-targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2-targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2-mutated PDXs. HER2 activating mutations cause EGFR antibody resistance in colorectal cell lines, and PDXs with HER2 mutations show durable tumor regression when treated with dual HER2-targeted therapy. These data provide a strong preclinical rationale for clinical trials targeting HER2 activating mutations in metastatic colorectal cancer. ©2015 American Association for Cancer Research.

Formulation and evaluation of targeted nanoparticles for breast cancer theranostic system.

PubMed

Dadras, Pegah; Atyabi, Fatemeh; Irani, Shiva; Ma'mani, Leila; Foroumadi, Alireza; Mirzaie, Zahra Hadavand; Ebrahimi, Marzieh; Dinarvand, R

2017-01-15

Theranostic polymeric NPs developed for both cancer diagnosis and cancer therapy. This multifunctional polymeric vehicle was prepared by a single emulsion evaporation method, using carboxyl-terminated PLGA. LHRH as a targeting moiety, was conjugated to the surface of polymeric carrier by applying polyethylene glycol. The results indicated that the diameter of NPs was ~185.4±4.6nm as defined by DLS. The entrapment efficacy of docetaxel, silibinin, and SPIONs was 84.6±4.1%, 80.6±2.7%, and 77.9±4.3%, respectively. The NPs showed a triphasic in-vitro drug release pattern. MTT assay was done on two cell lines, MCF-7 and SKOV-3. Enhanced cellular uptake ability of the targeted NPs to MCF-7 was evaluated in-vitro by confocal laser scanning microscopy. The results indicated that compared to non-targeted NPs, the LHRH targeted NPs had significant efficacy at IC50 concentration. The effect of the NPs on VEGF expression in MCF-7 and SKOV-3 cells was investigated by Real-Time PCR method. VEGF mRNA level expression in MCF-7 cell line reduced by 83% in comparison to control cell line. The designed NPs can be used as promising multifunctional platform for detection and targeted drug delivery in breast cancer. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of verteporfin-PDT on epithelial growth factor receptor (EGFR) signaling pathway in cholangiocarcinoma cell lines

NASA Astrophysics Data System (ADS)

Andreola, Fausto; Cerec, Virginie; Pereira, Stephen P.

2009-06-01

EGFR, a member of the ERBB family, plays a pivotal role in carcinogenesis. EGFR overexpression is implicated in DNA repair and synergistic interactions between EGFR-targeting drugs and conventional chemo/radiotherapy have been reported in preclinical studies for different cancers but not cholangiocarcinoma (CCA). To date there are no in vitro data available on the cellular response and effect of either photodynamic therapy (PDT) or EGFR-targeting drugs on CCA. Therefore, we aimed to study the: (i) response to Verteporfin PDT and to EGFR-targeting drugs, as single agents; (ii) effect of PDT on ERBBs expression, phosporylation status and activation of its signaling pathways; (iii) response to combination of PDT and EGFR-targeting agents. We showed that two cholangiocarcinoma cell lines (HuCCT1 and TFK1 cells, intra- and extrahepatic, respectively) differentially respond to verteporfin-PDT treatment and are resistant to EGFR-targeting agents. A constitutive activation of EGFR in both cell lines was also observed, which could partly account for the observed resistance to EGFR-targeting drugs. In addition, verteporfin-PDT induced further phosphorylation of both EGFR and other Receptor Tyrosine Kinases. Mitochondria-independent apoptosis was induced by PDT in both CCA cell lines; in particular, PDT modulated the expression of members of the Inhibitor of Apoptosis (IAP) family of proteins. Interestingly, there was a PDT-induced EGFR nuclear translocation in both cell lines; co-treatment with either an EGFR-inhibitor (Cetuximab) or a nuclear import blocking agent (Wheat Germ Agglutinin) had an additive effect on PDT cell killing, thus implying a role of EGFR in repairing the potential PDT-induced DNA damage.

Toward intracellular targeted delivery of cancer therapeutics: progress and clinical outlook for brain tumor therapy.

PubMed

Pandya, Hetal; Debinski, Waldemar

2012-08-01

A number of anti-cancer drugs have their targets localized to particular intracellular compartments. These drugs reach the targets mainly through diffusion, dependent on biophysical and biochemical forces that allow cell penetration. This means that both cancer cells and normal cells will be subjected to such diffusion; hence many of these drugs, like chemotherapeutics, are potentially toxic and the concentration achieved at the site of their action is often suboptimal. The same relates to radiation that indiscriminately affects normal and diseased cells. However, nature-designed systems enable compounds present in the extracellular environment to end up inside the cell and even travel to more specific intracellular compartments. For example, viruses and bacterial toxins can more or less specifically recognize eukaryotic cells, enter these cells, and direct some protein portions to designated intracellular areas. These phenomena have led to creative thinking, such as employing viruses or bacterial toxins for cargo delivery to cells and, more specifically, to cancer cells. Proteins can be genetically engineered in order to not only mimic what viruses and bacterial toxins can do, but also to add new functions, extending or changing the intracellular routes. It is possible to make conjugates or, more preferably, single-chain proteins that recognize cancer cells and deliver cargo inside the cells, even to the desired subcellular compartment. These findings offer new opportunities to deliver drugs/labels only to cancer cells and only to their site of action within the cells. The development of such dual-specificity vectors for targeting cancer cells is an attractive and potentially safer and more efficacious way of delivering drugs. We provide examples of this approach for delivering brain cancer therapeutics, using a specific biomarker on glioblastoma tumor cells.

Toward Intracellular Targeted Delivery of Cancer Therapeutics

PubMed Central

Pandya, Hetal; Debinski, Waldemar

2013-01-01

A number of anti-cancer drugs have their targets localized to particular intracellular compartments. These drugs reach the targets mainly through diffusion, dependent on biophysical and biochemical forces that allow cell penetration. This means that both cancer cells and normal cells will be subjected to such diffusion; hence many of these drugs, like chemotherapeutics, are potentially toxic and the concentration achieved at the site of their action is often suboptimal. The same relates to radiation that indiscriminately affects normal and diseased cells. However, nature-designed systems enable compounds present in the extracellular environment to end up inside the cell and even travel to more specific intracellular compartments. For example, viruses and bacterial toxins can more or less specifically recognize eukaryotic cells, enter these cells, and direct some protein portions to designated intracellular areas. These phenomena have led to creative thinking, such as employing viruses or bacterial toxins for cargo delivery to cells and, more specifically, to cancer cells. Proteins can be genetically engineered in order to not only mimic what viruses and bacterial toxins can do, but also to add new functions, extending or changing the intracellular routes. It is possible to make conjugates or, more preferably, single-chain proteins that recognize cancer cells and deliver cargo inside the cells, even to the desired subcellular compartment. These findings offer new opportunities to deliver drugs/labels only to cancer cells and only to their site of action within the cells. The development of such dual-specificity vectors for targeting cancer cells is an attractive and potentially safer and more efficacious way of delivering drugs. We provide examples of this approach for delivering brain cancer therapeutics, using a specific biomarker on glioblastoma tumor cells. PMID:22671766

Tetraspecific scFv construct provides NK cell mediated ADCC and self-sustaining stimuli via insertion of IL-15 as a cross-linker

PubMed Central

Schmohl, Joerg U.; Felices, Martin; Todhunter, Deborah; Taras, Elizabeth; Miller, Jeffrey S.; Vallera, Daniel A.

2016-01-01

Background The design of a highly effective anti-cancer immune-engager would include targeting of highly drug refractory cancer stem cells (CSC). The design would promote effective antibody-dependent cell-mediated cytotoxicity (ADCC) and simultaneously promote costimulation to expand and self-sustain the effector NK cell population. Based on our bispecific NK cell engager platform we constructed a tetraspecific killer engager (TetraKE) comprising single-chain variable fragments (scFvs) binding FcγRIII (CD16) on NK cells, EpCAM on carcinoma cells and CD133 on cancer stem cells in order to promote ADCC. Furthermore, an Interleukin (IL)-15-crosslinker enhanced NK cell related proliferation resulting in a highly active drug termed 1615EpCAM133. Results Proliferation assays showed TetraKE promoted proliferation and enhanced NK cell survival. Drug-target binding, NK cell related degranulation, and IFN-γ production was specific for both tumor related antigens in EpCAM and CD133 bearing cancer cell lines. The TetraKE showed higher killing activity and superior dose dependent degranulation. Cytokine profiling showed a moderately enhanced IFN-γ production, enhanced GM-CSF production, but no evidence of induction of excessive cytokine release. Methods Assembly and synthesis of hybrid genes encoding the TetraKE were performed using DNA shuffling and ligation. The TetraKE was tested for efficacy, specificity, proliferation, survival, and cytokine production using carcinoma cell lines and functional assays measuring NK cell activity. Conclusion 1615EpCAM133 combines improved induction of ADCC with enhanced proliferation, limited cytokine response, and prolonged survival and proliferation of NK cells. By linking scFv-related targeting of carcinoma and CSCs with a sustaining IL-15 signal, our new construct shows great promise to target cancer and CSCs. PMID:27650544