Targeting myeloid-derived suppressor cells for cancer immunotherapy.

PubMed

Liu, Yijun; Wei, Guowei; Cheng, Wesley A; Dong, Zhenyuan; Sun, Han; Lee, Vincent Y; Cha, Soung-Chul; Smith, D Lynne; Kwak, Larry W; Qin, Hong

2018-05-31

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with an immune suppressive phenotype. They represent a critical component of the immune suppressive niche described in cancer, where they support immune escape and tumor progression through direct effects on both the innate and adaptive immune responses, largely by contributing to maintenance of a high oxidative stress environment. The number of MDSCs positively correlates with protumoral activity, and often diminishes the effectiveness of immunotherapies, which is particularly problematic with the emergence of personalized medicine. Approaches targeting MDSCs showed promising results in preclinical studies and are under active investigation in clinical trials in combination with various immune checkpoint inhibitors. In this review, we discuss MDSC targets and therapeutic approaches targeting MDSC that have the aim of enhancing the existing tumor therapies.

Demyelination as a Target for Cell-Based Therapy of Chronic Blast-Induced Traumatic Brain Injury

DTIC Science & Technology

2015-10-01

AWARD NUMBER: W81XWH-13-1-0389 TITLE: Demyelination as a Target for Cell-Based Therapy of Chronic Blast-Induced Traumatic Brain Injury...2015 4. TITLE AND SUBTITLE Demyelination as a Target for Cell-Based Therapy of Chronic Blast-Induced Traumatic Brain Injury 5a. CONTRACT NUMBER 5b...disabling behavioral and cognitive abnormalities noted in significant number of combat veterans. These clinical phenotypes suggest impairment in

Natural Killer Cell Immunotherapy Targeting Cancer Stem Cells

PubMed Central

Luna, Jesus I; Grossenbacher, Steven K.; Murphy, William J; Canter, Robert J

2017-01-01

Introduction Standard cytoreductive cancer therapy, such as chemotherapy and radiotherapy, are frequently resisted by a small portion of cancer cells with “stem-cell” like properties including quiescence and repopulation. Immunotherapy represents a breakthrough modality for improving oncologic outcomes in cancer patients. Since the success of immunotherapy is not contingent on target cell proliferation, it may also be uniquely suited to address the problem of resistance and repopulation exerted by cancer stem cells (CSCs). Areas covered Natural killer (NK) cells have long been known for their ability to reject allogeneic hematopoietic stem cells, and there are increasing data demonstrating that NK cells can selectively identify and lyse CSCs. In this report, we review the current knowledge of CSCs and NK cells and highlight recent studies that support the concept that NK cells are capable of targeting CSC in solid tumors, especially in the context of combination therapy simultaneously targeting non-CSCs and CSCs. Expert Opinion Unlike cytotoxic cancer treatments, NK cells are able to target and eliminate quiescent/non-proliferating cells such as CSCs, and these enigmatic cells are an important source of relapse and metastasis. NK targeting of CSCs represents a novel and potentially high impact method to capitalize on the intrinsic therapeutic potential of NK cells. PMID:27960589

N-acetylgalactosamine-functionalized dendrimers as hepatic cancer cell-targeted carriers.

PubMed

Medina, Scott H; Tekumalla, Venkatesh; Chevliakov, Maxim V; Shewach, Donna S; Ensminger, William D; El-Sayed, Mohamed E H

2011-06-01

There is an urgent need for novel polymeric carriers that can selectively deliver a large dose of chemotherapeutic agents into hepatic cancer cells to achieve high therapeutic activity with minimal systemic side effects. PAMAM dendrimers are characterized by a unique branching architecture and a large number of chemical surface groups suitable for coupling of chemotherapeutic agents. In this article, we report the coupling of N-acetylgalactosamine (NAcGal) to generation 5 (G5) of poly(amidoamine) (PAMAM-NH₂) dendrimers via peptide and thiourea linkages to prepare NAcGal-targeted carriers used for targeted delivery of chemotherapeutic agents into hepatic cancer cells. We describe the uptake of NAcGal-targeted and non-targeted G5 dendrimers into hepatic cancer cells (HepG2) as a function of G5 concentration and incubation time. We examine the contribution of the asialoglycoprotein receptor (ASGPR) to the internalization of NAcGal-targeted dendrimers into hepatic cancer cells through a competitive inhibition assay. Our results show that uptake of NAcGal-targeted G5 dendrimers into hepatic cancer cells occurs via ASGPR-mediated endocytosis. Internalization of these targeted carriers increased with the increase in G5 concentration and incubation time following Michaelis-Menten kinetics characteristic of receptor-mediated endocytosis. These results collectively indicate that G5-NAcGal conjugates function as targeted carriers for selective delivery of chemotherapeutic agents into hepatic cancer cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

Highly efficient magnetic targeting of mesenchymal stem cells in spinal cord injury

PubMed Central

Vaněček, Václav; Zablotskii, Vitalii; Forostyak, Serhiy; Růřička, Jiří; Herynek, Vít; Babič, Michal; Jendelová, Pavla; Kubinová, Šárka; Dejneka, Alexandr; Syková, Eva

2012-01-01

The transplantation of mesenchymal stem cells (MSC) is currently under study as a therapeutic approach for spinal cord injury, and the number of transplanted cells that reach the lesioned tissue is one of the critical parameters. In this study, intrathecally transplanted cells labeled with superparamagnetic iron oxide nanoparticles were guided by a magnetic field and successfully targeted near the lesion site in the rat spinal cord. Magnetic resonance imaging and histological analysis revealed significant differences in cell numbers and cell distribution near the lesion site under the magnet in comparison to control groups. The cell distribution correlated well with the calculated distribution of magnetic forces exerted on the transplanted cells in the subarachnoid space and lesion site. The kinetics of the cells’ accumulation near the lesion site is described within the framework of a mathematical model that reveals those parameters critical for cell targeting and suggests ways to enhance the efficiency of magnetic cell delivery. In particular, we show that the targeting efficiency can be increased by using magnets that produce spatially modulated stray fields. Such magnetic systems with tunable geometric parameters may provide the additional level of control needed to enhance the efficiency of stem cell delivery in spinal cord injury. PMID:22888231

MPN estimation of qPCR target sequence recoveries from whole cell calibrator samples.

PubMed

Sivaganesan, Mano; Siefring, Shawn; Varma, Manju; Haugland, Richard A

2011-12-01

DNA extracts from enumerated target organism cells (calibrator samples) have been used for estimating Enterococcus cell equivalent densities in surface waters by a comparative cycle threshold (Ct) qPCR analysis method. To compare surface water Enterococcus density estimates from different studies by this approach, either a consistent source of calibrator cells must be used or the estimates must account for any differences in target sequence recoveries from different sources of calibrator cells. In this report we describe two methods for estimating target sequence recoveries from whole cell calibrator samples based on qPCR analyses of their serially diluted DNA extracts and most probable number (MPN) calculation. The first method employed a traditional MPN calculation approach. The second method employed a Bayesian hierarchical statistical modeling approach and a Monte Carlo Markov Chain (MCMC) simulation method to account for the uncertainty in these estimates associated with different individual samples of the cell preparations, different dilutions of the DNA extracts and different qPCR analytical runs. The two methods were applied to estimate mean target sequence recoveries per cell from two different lots of a commercially available source of enumerated Enterococcus cell preparations. The mean target sequence recovery estimates (and standard errors) per cell from Lot A and B cell preparations by the Bayesian method were 22.73 (3.4) and 11.76 (2.4), respectively, when the data were adjusted for potential false positive results. Means were similar for the traditional MPN approach which cannot comparably assess uncertainty in the estimates. Cell numbers and estimates of recoverable target sequences in calibrator samples prepared from the two cell sources were also used to estimate cell equivalent and target sequence quantities recovered from surface water samples in a comparative Ct method. Our results illustrate the utility of the Bayesian method in accounting for

Copy number determination of genetically-modified hematopoietic stem cells.

PubMed

Schuesler, Todd; Reeves, Lilith; Kalle, Christof von; Grassman, Elke

2009-01-01

Human gene transfer with gammaretroviral, murine leukemia virus (MLV) based vectors has been shown to effectively insert and express transgene sequences at a level of therapeutic benefit. However, there are numerous reports of disruption of the normal cellular processes caused by the viral insertion, even of replication deficient gammaretroviral vectors. Current gammaretroviral and lentiviral vectors do not control the site of insertion into the genome, hence, the possibility of disruption of the target cell genome. Risk related to viral insertions is linked to the number of insertions of the transgene into the cellular DNA, as has been demonstrated for replication competent and replication deficient retroviruses in experiments. At high number of insertions per cell, cell transformation due to vector induced activation of proto-oncogenes is more likely to occur, in particular since more than one transforming event is needed for oncogenesis. Thus, determination of the vector copy number in bulk transduced populations, individual colony forming units, and tissue from the recipient of the transduced cells is an increasingly important safety assay and has become a standard, though not straightforward assay, since the inception of quantitative PCR.

Cooperative tumour cell membrane targeted phototherapy

NASA Astrophysics Data System (ADS)

Kim, Heegon; Lee, Junsung; Oh, Chanhee; Park, Ji-Ho

2017-06-01

The targeted delivery of therapeutics using antibodies or nanomaterials has improved the precision and safety of cancer therapy. However, the paucity and heterogeneity of identified molecular targets within tumours have resulted in poor and uneven distribution of targeted agents, thus compromising treatment outcomes. Here, we construct a cooperative targeting system in which synthetic and biological nanocomponents participate together in the tumour cell membrane-selective localization of synthetic receptor-lipid conjugates (SR-lipids) to amplify the subsequent targeting of therapeutics. The SR-lipids are first delivered selectively to tumour cell membranes in the perivascular region using fusogenic liposomes. By hitchhiking with extracellular vesicles secreted by the cells, the SR-lipids are transferred to neighbouring cells and further spread throughout the tumour tissues where the molecular targets are limited. We show that this tumour cell membrane-targeted delivery of SR-lipids leads to uniform distribution and enhanced phototherapeutic efficacy of the targeted photosensitizer.

Engineered bifunctional proteins and stem cells: next generation of targeted cancer therapeutics.

PubMed

Choi, Sung Hugh; Shah, Khalid

2016-09-01

Redundant survival signaling pathways and their crosstalk within tumor and/or between tumor and their microenvironment are key impediments to developing effective targeted therapies for cancer. Therefore developing therapeutics that target multiple receptor signaling pathways in tumors and utilizing efficient platforms to deliver such therapeutics are critical to the success of future targeted therapies. During the past two decades, a number of bifunctional multi-targeting antibodies, fusion proteins, and oncolytic viruses have been developed and various stem cell types have been engineered to efficiently deliver them to tumors. In this review, we discuss the design and efficacy of therapeutics targeting multiple pathways in tumors and the therapeutic potential of therapeutic stem cells engineered with bifunctional agents.

Inhibitors targeting on cell wall biosynthesis pathway of MRSA.

PubMed

Hao, Haihong; Cheng, Guyue; Dai, Menghong; Wu, Qinghua; Yuan, Zonghui

2012-11-01

Methicillin resistant Staphylococcus aureus (MRSA), widely known as a type of new superbug, has aroused world-wide concern. Cell wall biosynthesis pathway is an old but good target for the development of antibacterial agents. Peptidoglycan and wall teichoic acids (WTAs) biosynthesis are two main processes of the cell wall biosynthesis pathway (CWBP). Other than penicillin-binding proteins (PBPs), some key factors (Mur enzymes, lipid I or II precursor, etc.) in CWBP are becoming attractive molecule targets for the discovery of anti-MRSA compounds. A number of new compounds, with higher affinity for PBPs or with inhibitory activity on such molecule targets in CWBP of MRSA, have been in the pipeline recently. This review concludes recent research achievements and provides a complete picture of CWBP of MRSA, including the peptidoglycan and wall teichoic acids synthesis pathway. The potential inhibitors targeting on CWBP are subsequently presented to improve development of novel therapeutic strategies for MRSA.

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

Tumor-targeting delivery of herb-based drugs with cell-penetrating/tumor-targeting peptide-modified nanocarriers

PubMed Central

Kebebe, Dereje; Liu, Yuanyuan; Wu, Yumei; Vilakhamxay, Maikhone; Liu, Zhidong; Li, Jiawei

2018-01-01

Cancer has become one of the leading causes of mortality globally. The major challenges of conventional cancer therapy are the failure of most chemotherapeutic agents to accumulate selectively in tumor cells and their severe systemic side effects. In the past three decades, a number of drug delivery approaches have been discovered to overwhelm the obstacles. Among these, nanocarriers have gained much attention for their excellent and efficient drug delivery systems to improve specific tissue/organ/cell targeting. In order to enhance targeting efficiency further and reduce limitations of nanocarriers, nanoparticle surfaces are functionalized with different ligands. Several kinds of ligand-modified nanomedicines have been reported. Cell-penetrating peptides (CPPs) are promising ligands, attracting the attention of researchers due to their efficiency to transport bioactive molecules intracellularly. However, their lack of specificity and in vivo degradation led to the development of newer types of CPP. Currently, activable CPP and tumor-targeting peptide (TTP)-modified nanocarriers have shown dramatically superior cellular specific uptake, cytotoxicity, and tumor growth inhibition. In this review, we discuss recent advances in tumor-targeting strategies using CPPs and their limitations in tumor delivery systems. Special emphasis is given to activable CPPs and TTPs. Finally, we address the application of CPPs and/or TTPs in the delivery of plant-derived chemotherapeutic agents. PMID:29563797

Targeting B Cells and Plasma Cells in Autoimmune Diseases

PubMed Central

Hofmann, Katharina; Clauder, Ann-Katrin; Manz, Rudolf Armin

2018-01-01

Success with B cell depletion using rituximab has proven the concept that B lineage cells represent a valid target for the treatment of autoimmune diseases, and has promoted the development of other B cell targeting agents. Present data confirm that B cell depletion is beneficial in various autoimmune disorders and also show that it can worsen the disease course in some patients. These findings suggest that B lineage cells not only produce pathogenic autoantibodies, but also significantly contribute to the regulation of inflammation. In this review, we will discuss the multiple pro- and anti-inflammatory roles of B lineage cells play in autoimmune diseases, in the context of recent findings using B lineage targeting therapies. PMID:29740441

Integrative Analysis Reveals an Outcome-associated and Targetable Pattern of p53 and Cell Cycle Deregulation in Diffuse Large B-cell Lymphoma

PubMed Central

Monti, Stefano; Chapuy, Bjoern; Takeyama, Kunihiko; Rodig, Scott J; Hao, Yangsheng; Yeda, Kelly T.; Inguilizian, Haig; Mermel, Craig; Curie, Treeve; Dogan, Ahmed; Kutok, Jeffery L; Beroukim, Rameen; Neuberg, Donna; Habermann, Thomas; Getz, Gad; Kung, Andrew L; Golub, Todd R; Shipp, Margaret A

2013-01-01

Summary Diffuse large B-cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease with a high proliferation rate. By integrating copy number data with transcriptional profiles and performing pathway analysis in primary DLBCLs, we identified a comprehensive set of copy number alterations (CNAs) that decreased p53 activity and perturbed cell cycle regulation. Primary tumors either had multiple complementary alterations of p53 and cell cycle components or largely lacked these lesions. DLBCLs with p53 and cell cycle pathway CNAs had decreased abundance of p53 target transcripts and increased expression of E2F target genes and the Ki67 proliferation marker. CNAs of the CDKN2A-TP53-RB-E2F axis provide a structural basis for increased proliferation in DLBCL, predict outcome with current therapy and suggest targeted treatment approaches. PMID:22975378

Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

PubMed

Alvarez-Vallina, L; Yañez, R; Blanco, B; Gil, M; Russell, S J

2000-04-01

Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.

Targeting Alpha5 Beta1 Integrin to Prevent Metastatic Breast Cancer Cell Invasion: PhScN Target Site Definition and Plasma Stability

DTIC Science & Technology

2015-11-01

systemic therapy to prevent breast cancer bone colony progression. Figure 6. Colocalization of Ac-PhscNGGK-Bio with DiI in lung– extravasated SUM149PT cells...breast cancer progression that are ultimately fatal. Hence, prevention of extravasation which leads to colony formation would increase life...1 Award Number: W81XWH-12-1-0097 TITLE: “Targeting Alpha5 Beta1 Integrin to Prevent Metastatic Breast Cancer Cell Invasion: PhScN Target Site

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

Mechanisms of epigenetic and cell-type specific regulation of Hey target genes in ES cells and cardiomyocytes.

PubMed

Weber, David; Heisig, Julia; Kneitz, Susanne; Wolf, Elmar; Eilers, Martin; Gessler, Manfred

2015-02-01

Hey bHLH transcription factors are critical effectors of Notch signaling. During mammalian heart development they are expressed in atrial and ventricular cardiomyocytes and in the developing endocardium. Hey knockout mice suffer from lethal cardiac defects, such as ventricular septum defects, valve defects and cardiomyopathy. Despite this functional relevance, little is known about the regulation of downstream targets in relevant cell types. The objective of this study was to elucidate the regulatory mechanisms by which Hey proteins affect gene expression in a cell type specific manner. We used an in vitro cardiomyocyte differentiation system with inducible Hey1 or Hey2 expression to study target gene regulation in cardiomyocytes (CM) generated from murine embryonic stem cells (ESC). The effects of Hey1 and Hey2 are largely redundant, but cell type specific. The number of regulated genes is comparable between ESC and CM, but the total number of binding sites is much higher, especially in ESC, targeting mainly genes involved in transcriptional regulation and developmental processes. Repression by Hey proteins generally correlates with the extent of Hey-binding to target promoters, Hdac recruitment and lower histone acetylation. Functionally, treatment with the Hdac inhibitor TSA abolished Hey target gene regulation. However, in CM the repressive effect of Hey-binding is lost for a subset of genes. These also lack Hey-dependent histone deacetylation in CM and are enriched for binding sites of cardiac specific activators like Srf, Nkx2-5, and Gata4. Ectopic Nkx2-5 overexpression in ESC blocks Hey-mediated repression of these genes. Thus, Hey proteins mechanistically repress target genes via Hdac recruitment and histone deacetylation. In CM Hey-repression is counteracted by cardiac activators, which recruit histone acetylases and prevent Hey mediated deacetylation and subsequent repression for a subset of genes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Human immune cell targeting of protein nanoparticles - caveospheres

NASA Astrophysics Data System (ADS)

Glass, Joshua J.; Yuen, Daniel; Rae, James; Johnston, Angus P. R.; Parton, Robert G.; Kent, Stephen J.; de Rose, Robert

2016-04-01

Nanotechnology has the power to transform vaccine and drug delivery through protection of payloads from both metabolism and off-target effects, while facilitating specific delivery of cargo to immune cells. However, evaluation of immune cell nanoparticle targeting is conventionally restricted to monocultured cell line models. We generated human caveolin-1 nanoparticles, termed caveospheres, which were efficiently functionalized with monoclonal antibodies. Using this platform, we investigated CD4+ T cell and CD20+ B cell targeting within physiological mixtures of primary human blood immune cells using flow cytometry, imaging flow cytometry and confocal microscopy. Antibody-functionalization enhanced caveosphere binding to targeted immune cells (6.6 to 43.9-fold) within mixed populations and in the presence of protein-containing fluids. Moreover, targeting caveospheres to CCR5 enabled caveosphere internalization by non-phagocytic CD4+ T cells--an important therapeutic target for HIV treatment. This efficient and flexible system of immune cell-targeted caveosphere nanoparticles holds promise for the development of advanced immunotherapeutics and vaccines.

Antibody-drug conjugate targeting CD46 eliminates multiple myeloma cells.

PubMed

Sherbenou, Daniel W; Aftab, Blake T; Su, Yang; Behrens, Christopher R; Wiita, Arun; Logan, Aaron C; Acosta-Alvear, Diego; Hann, Byron C; Walter, Peter; Shuman, Marc A; Wu, Xiaobo; Atkinson, John P; Wolf, Jeffrey L; Martin, Thomas G; Liu, Bin

2016-12-01

Multiple myeloma is incurable by standard approaches because of inevitable relapse and development of treatment resistance in all patients. In our prior work, we identified a panel of macropinocytosing human monoclonal antibodies against CD46, a negative regulator of the innate immune system, and constructed antibody-drug conjugates (ADCs). In this report, we show that an anti-CD46 ADC (CD46-ADC) potently inhibited proliferation in myeloma cell lines with little effect on normal cells. CD46-ADC also potently eliminated myeloma growth in orthometastatic xenograft models. In primary myeloma cells derived from bone marrow aspirates, CD46-ADC induced apoptosis and cell death, but did not affect the viability of nontumor mononuclear cells. It is of clinical interest that the CD46 gene resides on chromosome 1q, which undergoes genomic amplification in the majority of relapsed myeloma patients. We found that the cell surface expression level of CD46 was markedly higher in patient myeloma cells with 1q gain than in those with normal 1q copy number. Thus, genomic amplification of CD46 may serve as a surrogate for target amplification that could allow patient stratification for tailored CD46-targeted therapy. Overall, these findings indicate that CD46 is a promising target for antibody-based treatment of multiple myeloma, especially in patients with gain of chromosome 1q.

Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements

PubMed Central

Mumbach, Maxwell R; Satpathy, Ansuman T; Boyle, Evan A; Dai, Chao; Gowen, Benjamin G; Cho, Seung Woo; Nguyen, Michelle L; Rubin, Adam J; Granja, Jeffrey M; Kazane, Katelynn R; Wei, Yuning; Nguyen, Trieu; Greenside, Peyton G; Corces, M Ryan; Tycko, Josh; Simeonov, Dimitre R; Suliman, Nabeela; Li, Rui; Xu, Jin; Flynn, Ryan A; Kundaje, Anshul; Khavari, Paul A; Marson, Alexander; Corn, Jacob E; Quertermous, Thomas; Greenleaf, William J; Chang, Howard Y

2018-01-01

The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer–promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases. PMID:28945252

Impact of genetic targets on therapy in head and neck squamous cell carcinoma.

PubMed

Chaikhoutdinov, Irina; Goldenberg, David

2013-01-01

Despite advances in surgical technique, radiation therapy and chemotherapy, the mortality from head and neck squamous cell carcinoma (HNSCC) has not improved significantly. Squamous cell carcinoma is caused by tobacco use, alcohol consumption and infection with high-risk types of human papillomavirus. It is the 6th most common cancer in the world, with upwards of 45,000 new cases reported yearly in the United States alone.In recent years, there has been a significant increase in the understanding of the molecular and genetic pathogenesis of head and neck cancer, shedding light on the unexpected heterogeneity of the disease. Genetic analysis has led to new classification schemes for HNSCC, with different subgroups exhibiting different prognoses. In addition, multiple targets in aberrant signaling pathways have been identified using increasingly sophisticated bio-informatics tools. Advances in technology have allowed for novel delivery mechanisms to introduce genetic material into cells to produce a therapeutic effect by targeting cancer cells via a number of different approaches.A pressing need to develop novel therapies to augment current treatment modalities has led to a number of translational studies involving gene therapy in the treatment of HNSCC. This article will focus on a review of the most recent developments in molecular biology of head and neck squamous cell carcinoma in regards to possible targets for gene therapy, as well as the array of novel therapeutic strategies directed at these targets.

Nanobiotechnology for the Therapeutic Targeting of Cancer Cells in Blood.

PubMed

Li, Jiahe; Sharkey, Charles C; Huang, Dantong; King, Michael R

During metastasis, circulating tumor cells migrate away from a primary tumor via the blood circulation to form secondary tumors in distant organs. Mounting evidence from clinical observations indicates that the number of circulating tumor cells (CTCs) in the blood correlates with the progression of solid tumors before and during chemotherapy. Beyond the well-established role of CTCs as a fluid biopsy, however, the field of targeting CTCs for the prevention or reduction of metastases has just emerged. Conventional cancer therapeutics have a relatively short circulation time in the blood which may render the killing of CTCs inefficient due to reduced exposure of CTCs to drugs. Nevertheless, over the past few decades, the development of nanoparticles and nanoformulations to improve the half-life and release profile of drugs in circulation has rejuvenated certain traditional medicines in the emerging field of CTC neutralization. This review focuses on how the principles of nanomedicine may be applied to target CTCs. Moreover, inspired by the interactions between CTCs and host cells in the blood circulation, novel biomimetic approaches for targeted drug delivery are presented.

Reduced mtDNA copy number increases the sensitivity of tumor cells to chemotherapeutic drugs.

PubMed

Mei, H; Sun, S; Bai, Y; Chen, Y; Chai, R; Li, H

2015-04-02

Many cancer drugs are toxic to cells by activating apoptotic pathways. Previous studies have shown that mitochondria have key roles in apoptosis in mammalian cells, but the role of mitochondrial DNA (mtDNA) copy number variation in the pathogenesis of tumor cell apoptosis remains largely unknown. We used the HEp-2, HNE2, and A549 tumor cell lines to explore the relationship between mtDNA copy number variation and cell apoptosis. We first induced apoptosis in three tumor cell lines and one normal adult human skin fibroblast cell line (HSF) with cisplatin (DDP) or doxorubicin (DOX) treatment and found that the mtDNA copy number significantly increased in apoptotic tumor cells, but not in HSF cells. We then downregulated the mtDNA copy number by transfection with shRNA-TFAM plasmids or treatment with ethidium bromide and found that the sensitivity of tumor cells to DDP or DOX was significantly increased. Furthermore, we observed that levels of reactive oxygen species (ROS) increased significantly in tumor cells with lower mtDNA copy numbers, and this might be related to a low level of antioxidant gene expression. Finally, we rescued the increase of ROS in tumor cells with lipoic acid or N-acetyl-L-cysteine and found that the apoptosis rate decreased. Our studies suggest that the increase of mtDNA copy number is a self-protective mechanism of tumor cells to prevent apoptosis and that reduced mtDNA copy number increases ROS levels in tumor cells, increases the tumor cells' sensitivity to chemotherapeutic drugs, and increases the rate of apoptosis. This research provides evidence that mtDNA copy number variation might be a promising new therapeutic target for the clinical treatment of tumors.

Ku protein as a potential human T-cell leukemia virus type 1 (HTLV-1) Tax target in clastogenic chromosomal instability of mammalian cells

PubMed Central

Majone, Franca; Luisetto, Roberto; Zamboni, Daniela; Iwanaga, Yoichi; Jeang, Kuan-Teh

2005-01-01

The HTLV-1 Tax oncoprotein rapidly induces cytogenetic damage which can be measured by a significant increase in the number of micronuclei (MN) in cells. Tax is thought to have both aneuploidogenic and clastogenic effects. To examine the cellular target for Tax which might mechanistically explain the clastogenic phenomenon, we tested the ability of Tax to induce MN in rodents cells genetically defective for either the Ku80 protein or the catalytic subunit of DNA protein kinase (DNAPKcs). We found that cells genetically mutated in Ku80 were refractory to Tax's induction of MN while cells knocked-out for DNAPKcs showed increased number of Tax-induced MN. Using a cytogenetic method termed FISHI (Fluorescent In Situ Hybridization and Incorporation) which measures the number of DNA-breaks in cells that contained unprotected 3'-OH ends, we observed that Tax increased the prevalence of unprotected DNA breaks in Ku80-intact cells, but not in Ku80-mutated cells. Taken together, our findings suggest Ku80 as a cellular factor targeted by Tax in engendering clastogenic DNA damage. PMID:16014171

Cell-specific targeting by heterobivalent ligands.

PubMed

Josan, Jatinder S; Handl, Heather L; Sankaranarayanan, Rajesh; Xu, Liping; Lynch, Ronald M; Vagner, Josef; Mash, Eugene A; Hruby, Victor J; Gillies, Robert J

2011-07-20

Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach--to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle(4), dPhe(7)]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20-50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH(2). Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging.

Cell-Specific Targeting by Heterobivalent Ligands

PubMed Central

Josan, Jatinder S.; Handl, Heather L.; Sankaranarayanan, Rajesh; Xu, Liping; Lynch, Ronald M.; Vagner, Josef; Mash, Eugene A.; Hruby, Victor J.; Gillies, Robert J.

2012-01-01

Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach—to specifically target combinations of cell-surface receptors using heteromultivalent ligands (“receptor combination approach”). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle4, DPhe7]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20–50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH2. Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging. PMID:21639139

Measuring mRNA copy-number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization (smFISH)

PubMed Central

Skinner, Samuel O.; Sepúlveda, Leonardo A.; Xu, Heng; Golding, Ido

2014-01-01

We present a method for measuring the absolute number of mRNA molecules from a gene of interest in individual, chemically fixed Escherichia coli cells. A set of fluorescently-labeled oligonucleotide probes are hybridized to the target mRNA, so that each mRNA molecule is decorated by a known number of fluorescent dyes. Cells are then imaged using fluorescence microscopy. The number of target mRNA is estimated from the total intensity of fluorescent foci in the cell, rather than from counting discrete “spots” as in other currently available protocols. Image analysis is performed using an automated algorithm. The measured mRNA copy-number distribution obtained from many individual cells can be used to extract the parameters of stochastic gene activity, namely the frequency and size of transcription bursts from the gene of interest. The experimental procedure takes 2 days, with another 2-3 days typically required for image and data analysis. PMID:23680982

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.

A hypothesis of target cell formation in sickle cell disease.

PubMed

Wong, P

2016-08-01

A fraction of erythrocytes appear as target cells in stained blood smears in sickle cell disease, due to a inheritance of the hemoglobin variant Hb S, polymerizing upon deoxygenation. These cells appear in a three dimension as thin cups. A process of their formation in this disease is proposed based on a band 3-based mechanism of the erythrocyte shape control, able to explain the erythrocyte echinocytosis by glucose depletion. It indicates that their formation is due to a stomatocytogenic slow outward transport of the dibasic form of endogenous Pi with an H(+) by band 3, promoted by the decrease of the Donnan ratio, which decreases cell pH and volume, attributed by a decrease of cell KCl concentration by the higher efflux of K(+)Cl(-) cotransport and Ca(2+) activation of the Gardos channel. Its implications are briefly discussed with respect to target cells per se, target cell formation in other hemoglobinopathies, acquired and inherited disorders of the lipid metabolism and dehydrated hereditary stomatocytosis as well as a stomatocyte presence in a double heterozygote of Hb S and Hb C and of an involvement of the process of target cell formation in acanthocytosis in acquired and inherited disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evolving phage vectors for cell targeted gene delivery.

PubMed

Larocca, David; Burg, Michael A; Jensen-Pergakes, Kristen; Ravey, Edward Prenn; Gonzalez, Ana Maria; Baird, Andrew

2002-03-01

We adapted filamentous phage vectors for targeted gene delivery to mammalian cells by inserting a mammalian reporter gene expression cassette (GFP) into the vector backbone and fusing the pIII coat protein to a cell targeting ligand (i.e. FGF2, EGF). Like transfection with animal viral vectors, targeted phage gene delivery is concentration, time, and ligand dependent. Importantly, targeted phage particles are specific for the appropriate target cell surface receptor. Phage have distinct advantages over existing gene therapy vectors because they are simple, economical to produce at high titer, have no intrinsic tropism for mammalian cells, and are relatively simple to genetically modify and evolve. Initially transduction by targeted phage particles was low resulting in foreign gene expression in 1-2% of transfected cells. We increased transduction efficiency by modifying both the transfection protocol and vector design. For example, we stabilized the display of the targeting ligand to create multivalent phagemid-based vectors with transduction efficiencies of up to 45% in certain cell lines when combined with genotoxic treatment. Taken together, these studies establish that the efficiency of phage-mediated gene transfer can be significantly improved through genetic modification. We are currently evolving phage vectors with enhanced cell targeting, increased stability, reduced immunogenicity and other properties suitable for gene therapy.

Modeling activity and target-dependent developmental cell death of mouse retinal ganglion cells ex vivo.

PubMed

Voyatzis, Sylvie; Muzerelle, Aude; Gaspar, Patricia; Nicol, Xavier

2012-01-01

Programmed cell death is widespread during the development of the central nervous system and serves multiple purposes including the establishment of neural connections. In the mouse retina a substantial reduction of retinal ganglion cells (RGCs) occurs during the first postnatal week, coinciding with the formation of retinotopic maps in the superior colliculus (SC). We previously established a retino-collicular culture preparation which recapitulates the progressive topographic ordering of RGC projections during early post-natal life. Here, we questioned whether this model could also be suitable to examine the mechanisms underlying developmental cell death of RGCs. Brn3a was used as a marker of the RGCs. A developmental decline in the number of Brn3a-immunolabelled neurons was found in the retinal explant with a timing that paralleled that observed in vivo. In contrast, the density of photoreceptors or of starburst amacrine cells increased, mimicking the evolution of these cell populations in vivo. Blockade of neural activity with tetrodotoxin increased the number of surviving Brn3a-labelled neurons in the retinal explant, as did the increase in target availability when one retinal explant was confronted with 2 or 4 collicular slices. Thus, this ex vivo model reproduces the developmental reduction of RGCs and recapitulates its regulation by neural activity and target availability. It therefore offers a simple way to analyze developmental cell death in this classic system. Using this model, we show that ephrin-A signaling does not participate to the regulation of the Brn3a population size in the retina, indicating that eprhin-A-mediated elimination of exuberant projections does not involve developmental cell death.

Cancer stem cells (CSCs), cervical CSCs and targeted therapies

PubMed Central

Huang, Ruixia; Rofstad, Einar K.

2017-01-01

Accumulating evidence has shown that cancer stem cells (CSCs) have a tumour-initiating capacity and play crucial roles in tumour metastasis, relapse and chemo/radio-resistance. As tumour propagation initiators, CSCs are considered to be promising targets for obtaining a better therapeutic outcome. Cervical carcinoma is the most common gynaecological malignancy and has a high cancer mortality rate among females. As a result, the investigation of cervical cancer stem cells (CCSCs) is of great value. However, the numbers of cancer cells and corresponding CSCs in malignancy are dynamically balanced, and CSCs may reside in the CSC niche, about which little is known to date. Therefore, due to their complicated molecular phenotypes and biological behaviours, it remains challenging to obtain “purified” CSCs and continuously culture CSCs for further in vitro studies without the cells losing their stem properties. At present, CSC-related markers and functional assays are used to purify, identify and therapeutically target CSCs both in vitro and in vivo. Nevertheless, CSC-related markers are not universal to all tumour types, although some markers may be valid in multiple tumour types. Additionally, functional identifications based on CSC-specific properties are usually limited in in vivo studies. Furthermore, an optimal method for identifying potential CCSCs in CCSC studies has not been previously published, and these techniques are currently of great importance. This article updates our knowledge on CSCs and CCSCs, reviews potential stem cell markers and functional assays for identifying CCSCs, and describes the potential of targeting CCSCs in the treatment of cervical carcinoma. PMID:27343550

Cancer stem cells (CSCs), cervical CSCs and targeted therapies.

PubMed

Huang, Ruixia; Rofstad, Einar K

2017-05-23

Accumulating evidence has shown that cancer stem cells (CSCs) have a tumour-initiating capacity and play crucial roles in tumour metastasis, relapse and chemo/radio-resistance. As tumour propagation initiators, CSCs are considered to be promising targets for obtaining a better therapeutic outcome. Cervical carcinoma is the most common gynaecological malignancy and has a high cancer mortality rate among females. As a result, the investigation of cervical cancer stem cells (CCSCs) is of great value. However, the numbers of cancer cells and corresponding CSCs in malignancy are dynamically balanced, and CSCs may reside in the CSC niche, about which little is known to date. Therefore, due to their complicated molecular phenotypes and biological behaviours, it remains challenging to obtain "purified" CSCs and continuously culture CSCs for further in vitro studies without the cells losing their stem properties. At present, CSC-related markers and functional assays are used to purify, identify and therapeutically target CSCs both in vitro and in vivo. Nevertheless, CSC-related markers are not universal to all tumour types, although some markers may be valid in multiple tumour types. Additionally, functional identifications based on CSC-specific properties are usually limited in in vivo studies. Furthermore, an optimal method for identifying potential CCSCs in CCSC studies has not been previously published, and these techniques are currently of great importance. This article updates our knowledge on CSCs and CCSCs, reviews potential stem cell markers and functional assays for identifying CCSCs, and describes the potential of targeting CCSCs in the treatment of cervical carcinoma.

Targeting Breast Cancer Recurrence via Hedgehog-mediated Sensitization of Breast Cancer Stem Cells

DTIC Science & Technology

2011-07-01

Hedgehog -mediated Sensitization of Breast Cancer Stem Cells PRINCIPAL INVESTIGATOR: David J. Robbins, Ph.D...June 2010 – 14 June 2011 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Targeting Breast Cancer Recurrence via Hedgehog -mediated Sensitization of...this award. Introduction The purpose of the research supported by this award is to determine if targeting the hedgehog signaling pathway in

Targeting Common but Complex Proteoglycans on Breast Cancer Cells and Stem Cells Using Evolutionary Refined Malaria Proteins

DTIC Science & Technology

2014-09-01

chondroitin sulfate A proteoglycans present on all tested breast cancer cells and the vast majority of tested tissue biopsies. Using pull down assays...Invited, Daugaard. C) 2014. Gordon research conference, July 6-11; Targeting of cancer-specific chondroitin sulfate on circulating tumor cells using...now successfully identified a number of proteoglycans that interact with the recombinant malaria protein VAR2CSA when sulfated on carbon 4 of the CS

Statistical Modeling of Single Target Cell Encapsulation

PubMed Central

Moon, SangJun; Ceyhan, Elvan; Gurkan, Umut Atakan; Demirci, Utkan

2011-01-01

High throughput drop-on-demand systems for separation and encapsulation of individual target cells from heterogeneous mixtures of multiple cell types is an emerging method in biotechnology that has broad applications in tissue engineering and regenerative medicine, genomics, and cryobiology. However, cell encapsulation in droplets is a random process that is hard to control. Statistical models can provide an understanding of the underlying processes and estimation of the relevant parameters, and enable reliable and repeatable control over the encapsulation of cells in droplets during the isolation process with high confidence level. We have modeled and experimentally verified a microdroplet-based cell encapsulation process for various combinations of cell loading and target cell concentrations. Here, we explain theoretically and validate experimentally a model to isolate and pattern single target cells from heterogeneous mixtures without using complex peripheral systems. PMID:21814548

Magselectofection: an integrated method of nanomagnetic separation and genetic modification of target cells.

PubMed

Sanchez-Antequera, Yolanda; Mykhaylyk, Olga; van Til, Niek P; Cengizeroglu, Arzu; de Jong, J Henk; Huston, Marshall W; Anton, Martina; Johnston, Ian C D; Pojda, Zygmunt; Wagemaker, Gerard; Plank, Christian

2011-04-21

Research applications and cell therapies involving genetically modified cells require reliable, standardized, and cost-effective methods for cell manipulation. We report a novel nanomagnetic method for integrated cell separation and gene delivery. Gene vectors associated with magnetic nanoparticles are used to transfect/transduce target cells while being passaged and separated through a high gradient magnetic field cell separation column. The integrated method yields excellent target cell purity and recovery. Nonviral and lentiviral magselectofection is efficient and highly specific for the target cell population as demonstrated with a K562/Jurkat T-cell mixture. Both mouse and human enriched hematopoietic stem cell pools were effectively transduced by lentiviral magselectofection, which did not affect the hematopoietic progenitor cell number determined by in vitro colony assays. Highly effective reconstitution of T and B lymphocytes was achieved by magselectofected murine wild-type lineage-negative Sca-1(+) cells transplanted into Il2rg(-/-) mice, stably expressing GFP in erythroid, myeloid, T-, and B-cell lineages. Furthermore, nonviral, lentiviral, and adenoviral magselectofection yielded high transfection/transduction efficiency in human umbilical cord mesenchymal stem cells and was fully compatible with their differentiation potential. Upscaling to a clinically approved automated cell separation device was feasible. Hence, once optimized, validated, and approved, the method may greatly facilitate the generation of genetically engineered cells for cell therapies.

Oxidant-induced DNA damage of target cells.

PubMed Central

Schraufstätter, I; Hyslop, P A; Jackson, J H; Cochrane, C G

1988-01-01

In this study we examined the leukocytic oxidant species that induce oxidant damage of DNA in whole cells. H2O2 added extracellularly in micromolar concentrations (10-100 microM) induced DNA strand breaks in various target cells. The sensitivity of a specific target cell was inversely correlated to its catalase content and the rate of removal of H2O2 by the target cell. Oxidant species produced by xanthine oxidase/purine or phorbol myristate acetate-stimulated monocytes induced DNA breakage of target cells in proportion to the amount of H2O2 generated. These DNA strand breaks were prevented by extracellular catalase, but not by superoxide dismutase. Cytotoxic doses of HOCl, added to target cells, did not induce DNA strand breakage, and myeloperoxidase added extracellularly in the presence of an H2O2-generating system, prevented the formation of DNA strand breaks in proportion to its H2O2 degrading capacity. The studies also indicated that H2O2 formed hydroxyl radical (.OH) intracellularly, which appeared to be the most likely free radical responsible for DNA damage: .OH was detected in cells exposed to H2O2; the DNA base, deoxyguanosine, was hydroxylated in cells exposed to H2O2; and intracellular iron was essential for induction of DNA strand breaks. PMID:2843565

Targeting Peripheral-Derived Regulatory T Cells as a Means of Enhancing Immune Responses Directed against Prostate Cancer

DTIC Science & Technology

2017-08-01

Award Number: W81XWH-15-1-0328 TITLE: Targeting Peripheral-Derived Regulatory T Cells as a Means of Enhancing Immune Responses Directed against...1 August 2016 - 31 July 2017 4. TITLE AND SUBTITLE Targeting Peripheral-Derived Regulatory T Cells as a Means of Enhancing Immune Responses Directed...discovered that a subset of regulatory T cells (Tregs), termed peripheral-derived Tregs (pTregs), impair immune responses directed against tumor

Neocortical glial cell numbers in human brains.

PubMed

Pelvig, D P; Pakkenberg, H; Stark, A K; Pakkenberg, B

2008-11-01

Stereological cell counting was applied to post-mortem neocortices of human brains from 31 normal individuals, age 18-93 years, 18 females (average age 65 years, range 18-93) and 13 males (average age 57 years, range 19-87). The cells were differentiated in astrocytes, oligodendrocytes, microglia and neurons and counting were done in each of the four lobes. The study showed that the different subpopulations of glial cells behave differently as a function of age; the number of oligodendrocytes showed a significant 27% decrease over adult life and a strong correlation to the total number of neurons while the total astrocyte number is constant through life; finally males have a 28% higher number of neocortical glial cells and a 19% higher neocortical neuron number than females. The overall total number of neocortical neurons and glial cells was 49.3 billion in females and 65.2 billion in males, a difference of 24% with a high biological variance. These numbers can serve as reference values in quantitative studies of the human neocortex.

Designing oral vaccines targeting intestinal dendritic cells.

PubMed

Devriendt, Bert; De Geest, Bruno G; Cox, Eric

2011-04-01

Most pathogens colonize and invade the host at mucosal surfaces, such as the lung and the intestine. To combat intestinal pathogens the induction of local adaptive immune responses is required, which is mainly achieved through oral vaccination. However, most vaccines are ineffective when given orally owing to the hostile environment in the gastrointestinal tract. The encapsulation of antigens in biodegradable microparticulate delivery systems enhances their immunogenicity; however, the uptake of these delivery systems by intestinal immune cells is rather poor. Surface decoration of the particulates with targeting ligands could increase the uptake and mediate the selective targeting of the vaccine to intestinal antigen-presenting cells, including dendritic cells. In this review, current knowledge on dendritic cell subsets is discussed, along with progress in the development of selective antigen targeting to these cells, in addition to focusing on data obtained in mice and, where possible, the pig, as a non-rodent animal model for humans. Moreover, the potential use and benefits of Fcγ receptor-mediated targeting of antigen delivery systems are highlighted. In conclusion, dendritic cell targeting ligands grafted on antigen carrier systems should preferably bind to a conserved endocytotic receptor, facilitating the design of a multispecies vaccine platform, which could elicit robust protective immune responses against enteric pathogens.

Shared target antigens on cancer cells and tissue stem cells: go or no-go for CAR T cells?

PubMed

Hombach, Andreas A; Abken, Hinrich

2017-02-01

Adoptive therapy with chimeric antigen receptor (CAR) T cells redirected towards CD19 produces remissions of B cell malignancies, however, it also eradicates healthy B cells sharing the target antigen. Such 'on-target off-tumor' toxicity raises serious safety concerns when the target antigen is also expressed by tissue stem cells, with the risk of lasting tissue destruction. Areas covered: We discuss CAR T cell targeting of activation antigens versus lineage associated antigens on the basis of recent experimental and animal data and the literature in the field. Expert commentary: Targeting an activation associated antigen which is transiently expressed by stem cells seems to be safe, like CAR T cells targeting CD30 spare CD30 + hematopoietic stem and progenitor cells while eliminating CD30 + lymphoma cells, whereas targeting lineage associated antigens which increase in expression during cell maturation, like folate receptor-β and CD123, is of risk to destruct tissue stem cells.

Targeting tumor cell motility to prevent metastasis

PubMed Central

Palmer, Trenis D.; Ashby, William J.; Lewis, John D.; Zijlstra, Andries

2011-01-01

Mortality and morbidity in patients with solid tumors invariably results from the disruption of normal biological function caused by disseminating tumor cells. Tumor cell migration is under intense investigation as the underlying cause of cancer metastasis. The need for tumor cell motility in the progression of metastasis has been established experimentally and is supported empirically by basic and clinical research implicating a large collection of migration-related genes. However, there are few clinical interventions designed to specifically target the motility of tumor cells and adjuvant therapy to specifically prevent cancer cell dissemination is severely limited. In an attempt to define motility targets suitable for treating metastasis, we have parsed the molecular determinants of tumor cell motility into five underlying principles including cell autonomous ability, soluble communication, cell-cell adhesion, cell-matrix adhesion, and integrating these determinants of migration on molecular scaffolds. The current challenge is to implement meaningful and sustainable inhibition of metastasis by developing clinically viable disruption of molecular targets that control these fundamental capabilities. PMID:21664937

Cytotoxic T cells use mechanical force to potentiate target cell killing

PubMed Central

Basu, Roshni; Whitlock, Benjamin M.; Husson, Julien; Le Floc’h, Audrey; Jin, Weiyang; Oyler-Yaniv, Alon; Dotiwala, Farokh; Giannone, Gregory; Hivroz, Claire; Biais, Nicolas; Lieberman, Judy; Kam, Lance C.; Huse, Morgan

2016-01-01

SUMMARY The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals. PMID:26924577

Cytotoxic T Cells Use Mechanical Force to Potentiate Target Cell Killing.

PubMed

Basu, Roshni; Whitlock, Benjamin M; Husson, Julien; Le Floc'h, Audrey; Jin, Weiyang; Oyler-Yaniv, Alon; Dotiwala, Farokh; Giannone, Gregory; Hivroz, Claire; Biais, Nicolas; Lieberman, Judy; Kam, Lance C; Huse, Morgan

2016-03-24

The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals. Copyright © 2016 Elsevier Inc. All rights reserved.

Cell-to-Cell Transmission Can Overcome Multiple Donor and Target Cell Barriers Imposed on Cell-Free HIV

PubMed Central

Ilinskaya, Anna; Dorjbal, Batsukh; Truong, Rosaline; Derse, David; Uchil, Pradeep D.; Heidecker, Gisela; Mothes, Walther

2013-01-01

Virus transmission can occur either by a cell-free mode through the extracellular space or by cell-to-cell transmission involving direct cell-to-cell contact. The factors that determine whether a virus spreads by either pathway are poorly understood. Here, we assessed the relative contribution of cell-free and cell-to-cell transmission to the spreading of the human immunodeficiency virus (HIV). We demonstrate that HIV can spread by a cell-free pathway if all the steps of the viral replication cycle are efficiently supported in highly permissive cells. However, when the cell-free path was systematically hindered at various steps, HIV transmission became contact-dependent. Cell-to-cell transmission overcame barriers introduced in the donor cell at the level of gene expression and surface retention by the restriction factor tetherin. Moreover, neutralizing antibodies that efficiently inhibit cell-free HIV were less effective against cell-to-cell transmitted virus. HIV cell-to-cell transmission also efficiently infected target T cells that were relatively poorly susceptible to cell-free HIV. Importantly, we demonstrate that the donor and target cell types influence critically the extent by which cell-to-cell transmission can overcome each barrier. Mechanistically, cell-to-cell transmission promoted HIV spread to more cells and infected target cells with a higher proviral content than observed for cell-free virus. Our data demonstrate that the frequently observed contact-dependent spread of HIV is the result of specific features in donor and target cell types, thus offering an explanation for conflicting reports on the extent of cell-to-cell transmission of HIV. PMID:23308151

CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level

PubMed Central

Yuen, Garmen; Khan, Fehad J.; Gao, Shaojian; Stommel, Jayne M.; Batchelor, Eric; Wu, Xiaolin

2017-01-01

Abstract CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform. PMID:29036671

CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level.

PubMed

Yuen, Garmen; Khan, Fehad J; Gao, Shaojian; Stommel, Jayne M; Batchelor, Eric; Wu, Xiaolin; Luo, Ji

2017-11-16

CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

MicroRNA-184 inhibits neuroblastoma cell survival through targeting the serine/threonine kinase AKT2

PubMed Central

2010-01-01

Background Neuroblastoma is a paediatric cancer of the sympathetic nervous system. The single most important genetic indicator of poor clinical outcome is amplification of the MYCN transcription factor. One of many down-stream MYCN targets is miR-184, which is either directly or indirectly repressed by this transcription factor, possibly due to its pro-apoptotic effects when ectopically over-expressed in neuroblastoma cells. The purpose of this study was to elucidate the molecular mechanism by which miR-184 conveys pro-apoptotic effects. Results We demonstrate that the knock-down of endogenous miR-184 has the opposite effect of ectopic up-regulation, leading to enhanced neuroblastoma cell numbers. As a mechanism of how miR-184 causes apoptosis when over-expressed, and increased cell numbers when inhibited, we demonstrate direct targeting and degradation of AKT2, a major downstream effector of the phosphatidylinositol 3-kinase (PI3K) pathway, one of the most potent pro-survival pathways in cancer. The pro-apoptotic effects of miR-184 ectopic over-expression in neuroblastoma cell lines is reproduced by siRNA inhibition of AKT2, while a positive effect on cell numbers similar to that obtained by the knock-down of endogenous miR-184 can be achieved by ectopic up-regulation of AKT2. Moreover, co-transfection of miR-184 with an AKT2 expression vector lacking the miR-184 target site in the 3'UTR rescues cells from the pro-apoptotic effects of miR-184. Conclusions MYCN contributes to tumorigenesis, in part, by repressing miR-184, leading to increased levels of AKT2, a direct target of miR-184. Thus, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through the actions of miR-184. As an inhibitor of AKT2, miR-184 could be of potential benefit in miRNA mediated therapeutics of MYCN amplified neuroblastoma and other forms of cancer. PMID:20409325

Single-Cell Droplet Microfluidic Screening for Antibodies Specifically Binding to Target Cells.

PubMed

Shembekar, Nachiket; Hu, Hongxing; Eustace, David; Merten, Christoph A

2018-02-20

Monoclonal antibodies are a main player in modern drug discovery. Many antibody screening formats exist, each with specific advantages and limitations. Nonetheless, it remains challenging to screen antibodies for the binding of cell-surface receptors (the most important class of all drug targets) or for the binding to target cells rather than purified proteins. Here, we present a high-throughput droplet microfluidics approach employing dual-color normalized fluorescence readout to detect antibody binding. This enables us to obtain quantitative data on target cell recognition, using as little as 33 fg of IgG per assay. Starting with an excess of hybridoma cells releasing unspecific antibodies, individual clones secreting specific binders (of target cells co-encapsulated into droplets) could be enriched 220-fold after sorting 80,000 clones in a single experiment. This opens the way for therapeutic antibody discovery, especially since the single-cell approach is in principle also applicable to primary human plasma cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Targeting Alpha5 Beta1 Integrin to Prevent Metastatic Breast Cancer Cell Invasion: PhScN Target Site Definition and Plasma Stability

DTIC Science & Technology

2015-11-01

increased PhScN potency as a result of preventing endoproteolytic degradation. Finally, the in vivo lung extravasation and colonization data, as well as...successful colonization are late stages in breast cancer progression that are ultimately fatal. Hence, prevention of extravasation which leads to colony...Award Number: TITLE: “Targeting Alpha5 Beta1 Integrin to Prevent Metastatic Breast Cancer Cell Invasion: PhScN Target Site Definition and Plasma

Epratuzumab modulates B-cell signaling without affecting B-cell numbers or B-cell functions in a mouse model with humanized CD22.

PubMed

Özgör, Lamia; Brandl, Carolin; Shock, Anthony; Nitschke, Lars

2016-09-01

Treatment of systemic lupus erythematosus patients with epratuzumab (Emab), a humanized monoclonal antibody targeting CD22, leads to moderately reduced B-cell numbers but does not completely deplete B cells. Emab appears to induce immunomodulation of B cells, but the exact mode of action has not been defined. In the present study, we aimed to understand the effects of Emab on B cells using a humanized mouse model (Huki CD22), in which the B cells express human instead of murine CD22. Emab administration to Huki CD22 mice results in rapid and long-lasting CD22 internalization. There was no influence on B-cell turnover, but B-cell apoptosis ex vivo was increased. Emab administration to Huki CD22 mice had no effect on B-cell numbers in several lymphatic organs, nor in blood. In vitro exposure of B cells from Huki CD22 mice to Emab resulted in decreased B-cell receptor (BCR) induced Ca(2+) mobilization, whereas B-cell proliferation after Toll-like receptor (TLR) stimulation was not affected. In addition, IL-10 production was slightly increased after TLR and anti-CD40 stimulation, whereas IL-6 production was unchanged. In conclusion, Emab appears to inhibit BCR signaling in a CD22-dependent fashion without strong influence on B-cell development and B-cell populations. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photochemical internalization (PCI) of immunotoxins targeting CD133 is specific and highly potent at femtomolar levels in cells with cancer stem cell properties.

PubMed

Bostad, Monica; Berg, Kristian; Høgset, Anders; Skarpen, Ellen; Stenmark, Harald; Selbo, Pål K

2013-06-28

CD133 is a putative cancer stem cell (CSC) marker for a number of different cancers and is suggested to be a therapeutic target. Since also normal stem cells express CD133 it is of paramount importance that targeting strategies provide a specific and efficient delivery of cytotoxic drugs in only CD133-positive CSCs. In this study, we have employed photochemical internalization (PCI), a minimally invasive method for light-controlled, specific delivery of membrane-impermeable macromolecules from endocytic vesicles to the cytosol, to specifically target CD133-positive cancer cells. We demonstrate that PCI increases the cytotoxic effect of an immunotoxin (IT) targeting CD133-expressing cancer cells of colon (WiDr and HCT116) and pancreas (BxPC-3) origin. The IT consisted of the mAb CD133/1 (AC133) bound to the ribosome inactivating plant toxin saporin (anti-CD133/1-sap). We show that TPCS2a-PCI of anti-CD133/1-sap is specific, and highly cytotoxic at femto-molar concentrations. Specific binding and uptake of CD133/1, was shown by fluorescence microscopy and co-localization with TPCS2a in endosomes/lysosomes was determined by confocal microscopy. CD133(high) WiDr cells, isolated by fluorescence activated cell sorting, had a 7-fold higher capacity to initiate spheroids than CD133(low) cells (P<0.001) and were resistant to photodynamic therapy (PDT). However, PDT-resistance was bypassed by the PCI strategy. Tumor initiation and aggressive growth in athymic nude mice was obtained with only 10 CD133(high) cells in contrast to CD133(low) cells where substantially higher cell numbers were needed. The excellent high efficacy and selectivity of eliminating CD133-expressing cells by PCI warrant further pre-clinical evaluations of this novel therapeutic approach. Copyright © 2013 Elsevier B.V. All rights reserved.

Controversies in targeted therapy of adult T cell leukemia/lymphoma: ON target or OFF target effects?

PubMed

Nasr, Rihab; El Hajj, Hiba; Kfoury, Youmna; de Thé, Hugues; Hermine, Olivier; Bazarbachi, Ali

2011-06-01

Adult T cell leukemia/lymphoma (ATL) represents an ideal model for targeted therapy because of intrinsic chemo-resistance of ATL cells and the presence of two well identified targets: the HTLV-I retrovirus and the viral oncoprotein Tax. The combination of zidovudine (AZT) and interferon-alpha (IFN) has a dramatic impact on survival of ATL patients. Although the mechanism of action remains unclear, arguments in favor or against a direct antiviral effect will be discussed. Yet, most patients relapse and alternative therapies are mandatory. IFN and arsenic trioxide induce Tax proteolysis, synergize to induce apoptosis in ATL cells and cure Tax-driven ATL in mice through specific targeting of leukemia initiating cell activity. These results provide a biological basis for the clinical success of arsenic/IFN/AZT therapy in ATL patients and suggest that both extinction of viral replication (AZT) and Tax degradation (arsenic/IFN) are needed to cure ATL.

Cell lines derived from feline fibrosarcoma display unstable chromosomal aneuploidy and additionally centrosome number aberrations.

PubMed

von Erichsen, J; Hecht, W; Löhberg-Gruene, C; Reinacher, M

2012-07-01

The purpose of the study was to evaluate clonality and presence of numerical chromosomal and centrosomal aberrations in 5 established feline fibrosarcoma cell lines and in a fetal dermal fibroblast cell line as a control. The clonality of all cell lines was examined using limited-dilution cloning. The number of chromosomes was counted in metaphase spreads. The immunocytochemical analysis of centrosome numbers was performed by indirect immunofluorescence using a monoclonal antibody that targets γ-tubulin, a well-characterized component of centrosomes. Monoclonal cell populations could be established from all cell lines. In all feline fibrosarcoma cell lines, the number of chromosomes deviated abnormally from the normal feline chromosome number of 2n = 38, ranging from 19 to 155 chromosomes per cell. Centrosome hyperamplification was observed in all 5 feline fibrosarcoma cell lines with a proportion of cells (5.7 to 15.2%) having more than 2 centrosomes. In the control cell line, only 0.6% of the cells had more than 2 centrosomes. In conclusion, the examinations revealed that centrosome hyperamplification occurs in feline fibrosarcoma cell lines. The feline fibrosarcoma cell lines possessed 10 to 25 times as many cells with centrosome hyperamplification as the control cell line. These observations suggest an association of numerical centrosome aberrations with karyotype instability by increasing the frequency of chromosome missegregation. The results of this study may be helpful for further characterization of feline fibrosarcomas and may contribute to the knowledge of cytogenetic factors that may be important for the pathogenesis of feline fibrosarcomas.

Antibody-targeted interleukin 2 stimulates T-cell killing of autologous tumor cells.

PubMed Central

Gillies, S D; Reilly, E B; Lo, K M; Reisfeld, R A

1992-01-01

A genetically engineered fusion protein consisting of a chimeric anti-ganglioside GD2 antibody (ch14.18) and interleukin 2 (IL2) was tested for its ability to enhance the killing of autologous GD2-expressing melanoma target cells by a tumor-infiltrating lymphocyte line (660 TIL). The fusion of IL2 to the carboxyl terminus of the immunoglobulin heavy chain did not reduce IL2 activity as measured in a standard proliferation assay using either mouse or human T-cell lines. Antigen-binding activity was greater than that of the native chimeric antibody. The ability of resting 660 TIL cells to kill their autologous GD2-positive target cells was enhanced if the target cells were first coated with the fusion protein. This stimulation of killing was greater than that of uncoated cells in the presence of equivalent or higher concentrations of free IL2. Such antibody-cytokine fusion proteins may prove useful in targeting the biological effect of IL2 and other cytokines to tumor cells and in this way stimulate their immune destruction. Images PMID:1741398

Targeting Host Cell Surface Nucleolin for RSV Therapy: Challenges and Opportunities.

PubMed

Mastrangelo, Peter; Norris, Michael J; Duan, Wenming; Barrett, Edward G; Moraes, Theo J; Hegele, Richard G

2017-09-19

Nucleolin (NCL) has been reported as a cellular receptor for the human respiratory syncytial virus (RSV). We studied the effects of re-purposing AS1411, an anti-cancer compound that binds cell surface NCL, as a possible novel strategy for RSV therapy in vitro and in vivo. AS1411 was administered to RSV-infected cultures of non-polarized (HEp-2) and polarized (MDCK) epithelial cells and to virus-infected mice and cotton rats. Results of in vitro experiments showed that AS1411, used in micromolar concentrations, was associated with decreases in the number of virus-positive cells. Intranasal administration of AS1411 (50 mg/kg) to RSV-infected mice and cotton rats was associated with partial reductions in lung viral titers, decreased virus-associated airway inflammation, and decreased IL-4/IFN-γ ratios when compared to untreated, infected animals. In conclusion, our findings indicate that therapeutic use of AS1411 has modest effects on RSV replication and host response. While the results underscore the challenges of targeting cell surface NCL as a potential novel strategy for RSV therapy, they also highlight the potential of cell surface NCL as a therapeutic target.

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.

Recognition of glioma stem cells by genetically modified T cells targeting EGFRvIII and development of adoptive cell therapy for glioma.

PubMed

Morgan, Richard A; Johnson, Laura A; Davis, Jeremy L; Zheng, Zhili; Woolard, Kevin D; Reap, Elizabeth A; Feldman, Steven A; Chinnasamy, Nachimuthu; Kuan, Chien-Tsun; Song, Hua; Zhang, Wei; Fine, Howard A; Rosenberg, Steven A

2012-10-01

No curative treatment exists for glioblastoma, with median survival times of less than 2 years from diagnosis. As an approach to develop immune-based therapies for glioblastoma, we sought to target antigens expressed in glioma stem cells (GSCs). GSCs have multiple properties that make them significantly more representative of glioma tumors than established glioma cell lines. Epidermal growth factor receptor variant III (EGFRvIII) is the result of a novel tumor-specific gene rearrangement that produces a unique protein expressed in approximately 30% of gliomas, and is an ideal target for immunotherapy. Using PCR primers spanning the EGFRvIII-specific deletion, we found that this tumor-specific gene is expressed in three of three GCS lines. Based on the sequence information of seven EGFRvIII-specific monoclonal antibodies (mAbs), we assembled chimeric antigen receptors (CARs) and evaluated the ability of CAR-engineered T cells to recognize EGFRvIII. Three of these anti-EGFRvIII CAR-engineered T cells produced the effector cytokine, interferon-γ, and lysed antigen-expressing target cells. We concentrated development on a CAR produced from human mAb 139, which specifically recognized GSC lines and glioma cell lines expressing mutant EGFRvIII, but not wild-type EGFR and did not recognize any normal human cell tested. Using the 139-based CAR, T cells from glioblastoma patients could be genetically engineered to recognize EGFRvIII-expressing tumors and could be expanded ex vivo to large numbers, and maintained their antitumor activity. Based on these observations, a γ-retroviral vector expressing this EGFRvIII CAR was produced for clinical application.

Controversies in Targeted Therapy of Adult T Cell Leukemia/Lymphoma: ON Target or OFF Target Effects?

PubMed Central

Nasr, Rihab; Hajj, Hiba El; Kfoury, Youmna; de Thé, Hugues; Hermine, Olivier; Bazarbachi, Ali

2011-01-01

Adult T cell leukemia/lymphoma (ATL) represents an ideal model for targeted therapy because of intrinsic chemo-resistance of ATL cells and the presence of two well identified targets: the HTLV-I retrovirus and the viral oncoprotein Tax. The combination of zidovudine (AZT) and interferon-alpha (IFN) has a dramatic impact on survival of ATL patients. Although the mechanism of action remains unclear, arguments in favor or against a direct antiviral effect will be discussed. Yet, most patients relapse and alternative therapies are mandatory. IFN and arsenic trioxide induce Tax proteolysis, synergize to induce apoptosis in ATL cells and cure Tax-driven ATL in mice through specific targeting of leukemia initiating cell activity. These results provide a biological basis for the clinical success of arsenic/IFN/AZT therapy in ATL patients and suggest that both extinction of viral replication (AZT) and Tax degradation (arsenic/IFN) are needed to cure ATL. PMID:21994752

Targeting dysfunctional beta-cell signaling for the potential treatment of type 1 diabetes mellitus.

PubMed

Fenske, Rachel J; Kimple, Michelle E

2018-03-01

Since its discovery and purification by Frederick Banting in 1921, exogenous insulin has remained almost the sole therapy for type 1 diabetes mellitus. While insulin alleviates the primary dysfunction of the disease, many other aspects of the pathophysiology of type 1 diabetes mellitus are unaffected. Research aimed towards the discovery of novel type 1 diabetes mellitus therapeutics targeting different cell signaling pathways is gaining momentum. The focus of these efforts has been almost entirely on the impact of immunomodulatory drugs, particularly those that have already received FDA-approval for other autoimmune diseases. However, these drugs can often have severe side effects, while also putting already immunocompromised individuals at an increased risk for other infections. Potential therapeutic targets in the insulin-producing beta-cell have been largely ignored by the type 1 diabetes mellitus field, save the glucagon-like peptide 1 receptor. While there is preliminary evidence to support the clinical exploration of glucagon-like peptide 1 receptor-based drugs as type 1 diabetes mellitus adjuvant therapeutics, there is a vast space for other putative therapeutic targets to be explored. The alpha subunit of the heterotrimeric G z protein (Gα z ) has been shown to promote beta-cell inflammation, dysfunction, death, and failure to replicate in the context of diabetes in a number of mouse models. Genetic loss of Gα z or inhibition of the Gα z signaling pathway through dietary interventions is protective against the development of insulitis and hyperglycemia. The multifaceted effects of Gα z in regards to beta-cell health in the context of diabetes make it an ideal therapeutic target for further study. It is our belief that a low-risk, effective therapy for type 1 diabetes mellitus will involve a multidimensional approach targeting a number of regulatory systems, not the least of which is the insulin-producing beta-cell. Impact statement The expanding

Driver genes in non-small cell lung cancer: Characteristics, detection methods, and targeted therapies

PubMed Central

He, Bing; Zhang, Hu-Qin

2017-01-01

Lung cancer is one of the most common causes of cancer-related death in the world. The large number of lung cancer cases is non-small cell lung cancer (NSCLC), which approximately accounting for 75% of lung cancer. Over the past years, our comprehensive knowledge about the molecular biology of NSCLC has been rapidly enriching, which has promoted the discovery of driver genes in NSCLC and directed FDA-approved targeted therapies. Of course, the targeted therapies based on driver genes provide a more exact option for advanced non-small cell lung cancer, improving the survival rate of patients. Now, we will review the landscape of driver genes in NSCLC including the characteristics, detection methods, the application of target therapy and challenges. PMID:28915704

Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy.

PubMed

Lin, Huan-Ting; Okumura, Takashi; Yatsuda, Yukinori; Ito, Satoru; Nakauchi, Hiromitsu; Otsu, Makoto

2016-10-01

Stable gene transfer into target cell populations via integrating viral vectors is widely used in stem cell gene therapy (SCGT). Accurate vector copy number (VCN) estimation has become increasingly important. However, existing methods of estimation such as real-time quantitative PCR are more restricted in practicality, especially during clinical trials, given the limited availability of sample materials from patients. This study demonstrates the application of an emerging technology called droplet digital PCR (ddPCR) in estimating VCN states in the context of SCGT. Induced pluripotent stem cells (iPSCs) derived from a patient with X-linked chronic granulomatous disease were used as clonable target cells for transduction with alpharetroviral vectors harboring codon-optimized CYBB cDNA. Precise primer-probe design followed by multiplex analysis conferred assay specificity. Accurate estimation of per-cell VCN values was possible without reliance on a reference standard curve. Sensitivity was high and the dynamic range of detection was wide. Assay reliability was validated by observation of consistent, reproducible, and distinct VCN clustering patterns for clones of transduced iPSCs with varying numbers of transgene copies. Taken together, use of ddPCR appears to offer a practical and robust approach to VCN estimation with a wide range of clinical and research applications.

Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy

PubMed Central

Lin, Huan-Ting; Okumura, Takashi; Yatsuda, Yukinori; Ito, Satoru; Nakauchi, Hiromitsu; Otsu, Makoto

2016-01-01

Stable gene transfer into target cell populations via integrating viral vectors is widely used in stem cell gene therapy (SCGT). Accurate vector copy number (VCN) estimation has become increasingly important. However, existing methods of estimation such as real-time quantitative PCR are more restricted in practicality, especially during clinical trials, given the limited availability of sample materials from patients. This study demonstrates the application of an emerging technology called droplet digital PCR (ddPCR) in estimating VCN states in the context of SCGT. Induced pluripotent stem cells (iPSCs) derived from a patient with X-linked chronic granulomatous disease were used as clonable target cells for transduction with alpharetroviral vectors harboring codon-optimized CYBB cDNA. Precise primer–probe design followed by multiplex analysis conferred assay specificity. Accurate estimation of per-cell VCN values was possible without reliance on a reference standard curve. Sensitivity was high and the dynamic range of detection was wide. Assay reliability was validated by observation of consistent, reproducible, and distinct VCN clustering patterns for clones of transduced iPSCs with varying numbers of transgene copies. Taken together, use of ddPCR appears to offer a practical and robust approach to VCN estimation with a wide range of clinical and research applications. PMID:27763786

Coexpression of the Follicle Stimulating Hormone Receptor and Stem Cell Markers: A Novel Approach to Target Ovarian Cancer Stem Cells

DTIC Science & Technology

2016-03-01

1 AD_____________ Award Number: W81XWH-11-1-0623 TITLE: Coexpression of the Follicle Stimulating Hormone Receptor and Stem Cell Markers: A...Novel Approach to Target Ovarian Cancer Stem Cells PRINCIPAL INVESTIGATOR: David W. Schomberg, PhD CONTRACTING ORGANIZATION: Duke University...Durham, NC 27705 REPORT DATE: March 2016 TYPE OF REPORT: Final PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick

The number and distribution of AMPA receptor channels containing fast kinetic GluA3 and GluA4 subunits at auditory nerve synapses depend on the target cells.

PubMed

Rubio, María E; Matsui, Ko; Fukazawa, Yugo; Kamasawa, Naomi; Harada, Harumi; Itakura, Makoto; Molnár, Elek; Abe, Manabu; Sakimura, Kenji; Shigemoto, Ryuichi

2017-11-01

The neurotransmitter receptor subtype, number, density, and distribution relative to the location of transmitter release sites are key determinants of signal transmission. AMPA-type ionotropic glutamate receptors (AMPARs) containing GluA3 and GluA4 subunits are prominently expressed in subsets of neurons capable of firing action potentials at high frequencies, such as auditory relay neurons. The auditory nerve (AN) forms glutamatergic synapses on two types of relay neurons, bushy cells (BCs) and fusiform cells (FCs) of the cochlear nucleus. AN-BC and AN-FC synapses have distinct kinetics; thus, we investigated whether the number, density, and localization of GluA3 and GluA4 subunits in these synapses are differentially organized using quantitative freeze-fracture replica immunogold labeling. We identify a positive correlation between the number of AMPARs and the size of AN-BC and AN-FC synapses. Both types of AN synapses have similar numbers of AMPARs; however, the AN-BC have a higher density of AMPARs than AN-FC synapses, because the AN-BC synapses are smaller. A higher number and density of GluA3 subunits are observed at AN-BC synapses, whereas a higher number and density of GluA4 subunits are observed at AN-FC synapses. The intrasynaptic distribution of immunogold labeling revealed that AMPAR subunits, particularly GluA3, are concentrated at the center of the AN-BC synapses. The central distribution of AMPARs is absent in GluA3-knockout mice, and gold particles are evenly distributed along the postsynaptic density. GluA4 gold labeling was homogenously distributed along both synapse types. Thus, GluA3 and GluA4 subunits are distributed at AN synapses in a target-cell-dependent manner.

Medulloblastoma stem cells: Promising targets in medulloblastoma therapy.

PubMed

Huang, Guo-Hao; Xu, Qing-Fu; Cui, You-Hong; Li, Ningning; Bian, Xiu-Wu; Lv, Sheng-Qing

2016-05-01

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Despite great improvements in the therapeutic regimen, relapse and leptomeningeal dissemination still pose great challenges to the long-term survival of MB patients. Developing more effective strategies has become extremely urgent. In recent years, a number of malignancies, including MB, have been found to contain a subpopulation of cancer cells known as cancer stem cells (CSCs), or tumor initiating/propagating cells. The CSCs are thought to be largely responsible for tumor initiation, maintenance, dissemination, and relapse; therefore, their pivotal roles have revealed them to be promising targets in MB therapy. Our growing understanding of the major medulloblastoma molecular subgroups and the derivation of some of these groups from specific stem or progenitor cells adds additional layers to the CSC knowledge base. Herein we review the current knowledge of MB stem cells, highlight the molecular mechanisms relating to MB relapse and leptomeningeal dissemination, and incorporate these with the need to develop more effective and accurate therapies for MB patients. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Targeting hepatic cancer cells with pegylated dendrimers displaying N-acetylgalactosamine and SP94 peptide ligands.

PubMed

Medina, Scott H; Tiruchinapally, Gopinath; Chevliakov, Maxim V; Durmaz, Yasemin Yuksel; Stender, Rachell N; Ensminger, William D; Shewach, Donna S; Elsayed, Mohamed E H

2013-10-01

Poly(amidoamine) (PAMAM) dendrimers are branched water-soluble polymers defined by consecutive generation numbers (Gn) indicating a parallel increase in size, molecular weight, and number of surface groups available for conjugation of bioactive agents. In this article, we compare the biodistribution of N-acetylgalactosamine (NAcGal)-targeted [(14) C]1 -G5-(NH2 )5 -(Ac)108 -(NAcGal)14 particles to non-targeted [(14) C]1 -G5-(NH2 )127 and PEGylated [(14) C]1 -G5-(NH2 )44 -(Ac)73 -(PEG)10 particles in a mouse hepatic cancer model. Results show that both NAcGal-targeted and non-targeted particles are rapidly cleared from the systemic circulation with high distribution to the liver. However, NAcGal-targeted particles exhibited 2.5-fold higher accumulation in tumor tissue compared to non-targeted ones. In comparison, PEGylated particles showed a 16-fold increase in plasma residence time and a 5-fold reduction in liver accumulation. These results motivated us to engineer new PEGylated G5 particles with PEG chains anchored to the G5 surface via acid-labile cis-aconityl linkages where the free PEG tips are functionalized with NAcGal or SP94 peptide to investigate their potential as targeting ligands for hepatic cancer cells as a function of sugar conformation (α versus β), ligand concentration (100-4000 nM), and incubation time (2 and 24 hours) compared to fluorescently (Fl)-labeled and non-targeted G5-(Fl)6 -(NH2 )122 and G5-(Fl)6 -(Ac)107 -(cPEG)15 particles. Results show G5-(Fl)6 -(Ac)107 -(cPEG[NAcGalβ ])14 particles achieve faster uptake and higher intracellular concentrations in HepG2 cancer cells compared to other G5 particles while escaping the non-specific adsorption of serum protein and phagocytosis by Kupffer cells, which make these particles the ideal carrier for selective drug delivery into hepatic cancer cells. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Wilms Tumor NCAM-Expressing Cancer Stem Cells as Potential Therapeutic Target for Polymeric Nanomedicine.

PubMed

Markovsky, Ela; Vax, Einav; Ben-Shushan, Dikla; Eldar-Boock, Anat; Shukrun, Rachel; Yeini, Eilam; Barshack, Iris; Caspi, Revital; Harari-Steinberg, Orit; Pode-Shakked, Naomi; Dekel, Benjamin; Satchi-Fainaro, Ronit

2017-11-01

Cancer stem cells (CSC) form a specific population within the tumor that has been shown to have self-renewal and differentiation properties, increased ability to migrate and form metastases, and increased resistance to chemotherapy. Consequently, even a small number of cells remaining after therapy can repopulate the tumor and cause recurrence of the disease. CSCs in Wilms tumor, a pediatric renal cancer, were previously shown to be characterized by neural cell adhesion molecule (NCAM) expression. Therefore, NCAM provides a specific biomarker through which the CSC population in this tumor can be targeted. We have recently developed an NCAM-targeted nanosized conjugate of paclitaxel bound to a biodegradable polyglutamic acid polymer. In this work, we examined the ability of the conjugate to inhibit Wilms tumor by targeting the NCAM-expressing CSCs. Results show that the conjugate selectively depleted the CSC population of the tumors and effectively inhibited tumor growth without causing toxicity. We propose that the NCAM-targeted conjugate could be an effective therapeutic for Wilms tumor. Mol Cancer Ther; 16(11); 2462-72. ©2017 AACR . ©2017 American Association for Cancer Research.

New targeted therapies for indolent B-cell malignancies in older patients.

PubMed

Krem, Maxwell M; Gopal, Ajay K

2015-01-01

Molecularly targeted agents have become an established component of the treatment of indolent B-cell malignancies (iNHL). iNHL disproportionately affects older adults, so treatments that have excellent tolerability and efficacy across multiple lines of therapy are in demand. The numbers and classes of targeted therapies for iNHL have proliferated rapidly in recent years; classes of agents that show promise for older patients with iNHL include anti-CD20 antibodies, phosphatidyl-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway inhibitors, immunomodulators, proteasome inhibitors, epigenetic modulators, and immunotherapies. Here, we review the proposed mechanisms of action, efficacy, and tolerability of novel agents for iNHL, with an emphasis on their applicability to older patients.

Merkel cell polyomavirus small T antigen induces genome instability by E3 ubiquitin ligase targeting.

PubMed

Kwun, H J; Wendzicki, J A; Shuda, Y; Moore, P S; Chang, Y

2017-12-07

The formation of a bipolar mitotic spindle is an essential process for the equal segregation of duplicated DNA into two daughter cells during mitosis. As a result of deregulated cellular signaling pathways, cancer cells often suffer a loss of genome integrity that might etiologically contribute to carcinogenesis. Merkel cell polyomavirus (MCV) small T (sT) oncoprotein induces centrosome overduplication, aneuploidy, chromosome breakage and the formation of micronuclei by targeting cellular ligases through a sT domain that also inhibits MCV large T oncoprotein turnover. These results provide important insight as to how centrosome number and chromosomal stability can be affected by the E3 ligase targeting capacity of viral oncoproteins such as MCV sT, which may contribute to Merkel cell carcinogenesis.

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.

Changes in mast cell number and stem cell factor expression in human skin after radiotherapy for breast cancer.

PubMed

Westbury, Charlotte B; Freeman, Alex; Rashid, Mohammed; Pearson, Ann; Yarnold, John R; Short, Susan C

2014-05-01

Mast cells are involved in the pathogenesis of radiation fibrosis and may be a therapeutic target. The mechanism of increased mast cell number in relation to acute and late tissue responses in human skin was investigated. Punch biopsies of skin 1 and 15-18 months after breast radiotherapy and a contralateral control biopsy were collected. Mast cells were quantified by immunohistochemistry using the markers c-Kit and tryptase. Stem cell factor (SCF) and collagen-1 expression was analysed by qRT-PCR. Clinical photographic scores were performed at post-surgical baseline and 18 months and 5 years post-radiotherapy. Primary human dermal microvascular endothelial cell (HDMEC) cultures were exposed to 2Gy ionising radiation and p53 and SCF expression was analysed by Western blotting and ELISA. Dermal mast cell numbers were increased at 1 (p=0.047) and 18 months (p=0.040) using c-Kit, and at 18 months (p=0.024) using tryptase immunostaining. Collagen-1 mRNA in skin was increased at 1 month (p=0.047) and 18 months (p=0.032) and SCF mRNA increased at 1 month (p=0.003). None of 16 cases scored had a change in photographic appearance at 5 years, compared to baseline. SCF expression was not increased in HDMECs irradiated in vitro. Increased mast cell number was associated with up-regulated collagen-1 expression in human skin at early and late time points. This increase could be secondary to elevated SCF expression at 1 month after radiotherapy. Although mast cells accumulate around blood vessels, no endothelial cell secretion of SCF was seen after in vitro irradiation. Modification of mast cell number and collagen-1 expression may be observed in skin at 1 and 18 months after radiotherapy in breast cancer patients with no change in photographic breast appearance at 5 years. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

In chronic spontaneous urticaria, high numbers of dermal endothelial cells, but not mast cells, are linked to recurrent angio-oedema.

PubMed

Terhorst, D; Koti, I; Krause, K; Metz, M; Maurer, M

2018-03-01

Chronic spontaneous urticaria (CSU) is an inflammatory skin disorder characterized by recurrent weals, angio-oedema or both. Recent studies have shown that the number of endothelial cells is increased in the skin of patients with CSU, but the underlying mechanisms and clinical implications of this are unclear. To evaluate whether mast cell (MC) or endothelial cell (EC) numbers correlate with CSU and whether they are relevant for disease duration, disease activity or the presence of clinical features. We determined the numbers of CD31+ ECs and MCs in nonlesional skin of 30 patients with CSU using quantitative histomorphometry, and assessed their correlation with each other and with clinical features such as disease duration, disease activity and occurrence of angio-oedema. The numbers of MCs and ECs were high in the nonlesional skin of patients with CSU, but did not correlate with each other. Neither MC number nor EC number correlated with disease duration or disease activity. Interestingly, patients with high numbers of cutaneous CD31+ ECs had higher rates of recurrent angio-oedema and vice versa. Based on these findings, we speculate that vascular remodelling and MC hyperplasia in patients with CSU occurs independently and via different mechanisms. Targeting of the mechanisms that drive neoangiogenesis in CSU may result in novel therapeutic strategies for the management of patients with angio-oedema. © 2017 British Association of Dermatologists.

In vivo stem cell transplantation using reduced cell numbers.

PubMed

Tsutsui, Takeo W

2015-01-01

Dental pulp stem cell (DPSC) characterization is essential for regeneration of a dentin/pulp like complex in vivo. This is especially important for identifying the potential of DPSCs to function as stem cells. Previously reported DPSC transplantation methods have used with huge numbers of cells, along with hydroxyapatite/tricalcium phosphate (HA/TCP), gelatin and fibrin, and collagen scaffolds. This protocol describe a transplantation protocol that uses fewer cells and a temperature-responsive cell culture dish.

Prediction of intracellular exposure bridges the gap between target- and cell-based drug discovery

PubMed Central

Gordon, Laurie J.; Wayne, Gareth J.; Almqvist, Helena; Axelsson, Hanna; Seashore-Ludlow, Brinton; Treyer, Andrea; Lundbäck, Thomas; West, Andy; Hann, Michael M.; Artursson, Per

2017-01-01

Inadequate target exposure is a major cause of high attrition in drug discovery. Here, we show that a label-free method for quantifying the intracellular bioavailability (Fic) of drug molecules predicts drug access to intracellular targets and hence, pharmacological effect. We determined Fic in multiple cellular assays and cell types representing different targets from a number of therapeutic areas, including cancer, inflammation, and dementia. Both cytosolic targets and targets localized in subcellular compartments were investigated. Fic gives insights on membrane-permeable compounds in terms of cellular potency and intracellular target engagement, compared with biochemical potency measurements alone. Knowledge of the amount of drug that is locally available to bind intracellular targets provides a powerful tool for compound selection in early drug discovery. PMID:28701380

Recognition of Glioma Stem Cells by Genetically Modified T Cells Targeting EGFRvIII and Development of Adoptive Cell Therapy for Glioma

PubMed Central

Johnson, Laura A.; Davis, Jeremy L.; Zheng, Zhili; Woolard, Kevin D.; Reap, Elizabeth A.; Feldman, Steven A.; Chinnasamy, Nachimuthu; Kuan, Chien-Tsun; Song, Hua; Zhang, Wei; Fine, Howard A.; Rosenberg, Steven A.

2012-01-01

Abstract No curative treatment exists for glioblastoma, with median survival times of less than 2 years from diagnosis. As an approach to develop immune-based therapies for glioblastoma, we sought to target antigens expressed in glioma stem cells (GSCs). GSCs have multiple properties that make them significantly more representative of glioma tumors than established glioma cell lines. Epidermal growth factor receptor variant III (EGFRvIII) is the result of a novel tumor-specific gene rearrangement that produces a unique protein expressed in approximately 30% of gliomas, and is an ideal target for immunotherapy. Using PCR primers spanning the EGFRvIII-specific deletion, we found that this tumor-specific gene is expressed in three of three GCS lines. Based on the sequence information of seven EGFRvIII-specific monoclonal antibodies (mAbs), we assembled chimeric antigen receptors (CARs) and evaluated the ability of CAR-engineered T cells to recognize EGFRvIII. Three of these anti-EGFRvIII CAR-engineered T cells produced the effector cytokine, interferon-γ, and lysed antigen-expressing target cells. We concentrated development on a CAR produced from human mAb 139, which specifically recognized GSC lines and glioma cell lines expressing mutant EGFRvIII, but not wild-type EGFR and did not recognize any normal human cell tested. Using the 139-based CAR, T cells from glioblastoma patients could be genetically engineered to recognize EGFRvIII-expressing tumors and could be expanded ex vivo to large numbers, and maintained their antitumor activity. Based on these observations, a γ-retroviral vector expressing this EGFRvIII CAR was produced for clinical application. PMID:22780919

Do bacterial cell numbers follow a theoretical Poisson distribution? Comparison of experimentally obtained numbers of single cells with random number generation via computer simulation.

PubMed

Koyama, Kento; Hokunan, Hidekazu; Hasegawa, Mayumi; Kawamura, Shuso; Koseki, Shigenobu

2016-12-01

We investigated a bacterial sample preparation procedure for single-cell studies. In the present study, we examined whether single bacterial cells obtained via 10-fold dilution followed a theoretical Poisson distribution. Four serotypes of Salmonella enterica, three serotypes of enterohaemorrhagic Escherichia coli and one serotype of Listeria monocytogenes were used as sample bacteria. An inoculum of each serotype was prepared via a 10-fold dilution series to obtain bacterial cell counts with mean values of one or two. To determine whether the experimentally obtained bacterial cell counts follow a theoretical Poisson distribution, a likelihood ratio test between the experimentally obtained cell counts and Poisson distribution which parameter estimated by maximum likelihood estimation (MLE) was conducted. The bacterial cell counts of each serotype sufficiently followed a Poisson distribution. Furthermore, to examine the validity of the parameters of Poisson distribution from experimentally obtained bacterial cell counts, we compared these with the parameters of a Poisson distribution that were estimated using random number generation via computer simulation. The Poisson distribution parameters experimentally obtained from bacterial cell counts were within the range of the parameters estimated using a computer simulation. These results demonstrate that the bacterial cell counts of each serotype obtained via 10-fold dilution followed a Poisson distribution. The fact that the frequency of bacterial cell counts follows a Poisson distribution at low number would be applied to some single-cell studies with a few bacterial cells. In particular, the procedure presented in this study enables us to develop an inactivation model at the single-cell level that can estimate the variability of survival bacterial numbers during the bacterial death process. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

PubMed

Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

2016-01-01

The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling.

The Quest for Targets Executing MYC-Dependent Cell Transformation.

PubMed

Hartl, Markus

2016-01-01

MYC represents a transcription factor with oncogenic potential converting multiple cellular signals into a broad transcriptional response, thereby controlling the expression of numerous protein-coding and non-coding RNAs important for cell proliferation, metabolism, differentiation, and apoptosis. Constitutive activation of MYC leads to neoplastic cell transformation, and deregulated MYC alleles are frequently observed in many human cancer cell types. Multiple approaches have been performed to isolate genes differentially expressed in cells containing aberrantly activated MYC proteins leading to the identification of thousands of putative targets. Functional analyses of genes differentially expressed in MYC-transformed cells had revealed that so far more than 40 upregulated or downregulated MYC targets are actively involved in cell transformation or tumorigenesis. However, further systematic and selective approaches are required for determination of the known or yet unidentified targets responsible for processing the oncogenic MYC program. The search for critical targets in MYC-dependent tumor cells is exacerbated by the fact that during tumor development, cancer cells progressively evolve in a multistep process, thereby acquiring their characteristic features in an additive manner. Functional expression cloning, combinatorial gene expression, and appropriate in vivo tests could represent adequate tools for dissecting the complex scenario of MYC-specified cell transformation. In this context, the central goal is to identify a minimal set of targets that suffices to phenocopy oncogenic MYC. Recently developed genomic editing tools could be employed to confirm the requirement of crucial transformation-associated targets. Knowledge about essential MYC-regulated genes is beneficial to expedite the development of specific inhibitors to interfere with growth and viability of human tumor cells in which MYC is aberrantly activated. Approaches based on the principle of

The Quest for Targets Executing MYC-Dependent Cell Transformation

PubMed Central

Hartl, Markus

2016-01-01

MYC represents a transcription factor with oncogenic potential converting multiple cellular signals into a broad transcriptional response, thereby controlling the expression of numerous protein-coding and non-coding RNAs important for cell proliferation, metabolism, differentiation, and apoptosis. Constitutive activation of MYC leads to neoplastic cell transformation, and deregulated MYC alleles are frequently observed in many human cancer cell types. Multiple approaches have been performed to isolate genes differentially expressed in cells containing aberrantly activated MYC proteins leading to the identification of thousands of putative targets. Functional analyses of genes differentially expressed in MYC-transformed cells had revealed that so far more than 40 upregulated or downregulated MYC targets are actively involved in cell transformation or tumorigenesis. However, further systematic and selective approaches are required for determination of the known or yet unidentified targets responsible for processing the oncogenic MYC program. The search for critical targets in MYC-dependent tumor cells is exacerbated by the fact that during tumor development, cancer cells progressively evolve in a multistep process, thereby acquiring their characteristic features in an additive manner. Functional expression cloning, combinatorial gene expression, and appropriate in vivo tests could represent adequate tools for dissecting the complex scenario of MYC-specified cell transformation. In this context, the central goal is to identify a minimal set of targets that suffices to phenocopy oncogenic MYC. Recently developed genomic editing tools could be employed to confirm the requirement of crucial transformation-associated targets. Knowledge about essential MYC-regulated genes is beneficial to expedite the development of specific inhibitors to interfere with growth and viability of human tumor cells in which MYC is aberrantly activated. Approaches based on the principle of

Imatinib and Nilotinib Off-Target Effects on Human NK Cells, Monocytes, and M2 Macrophages.

PubMed

Bellora, Francesca; Dondero, Alessandra; Corrias, Maria Valeria; Casu, Beatrice; Regis, Stefano; Caliendo, Fabio; Moretta, Alessandro; Cazzola, Mario; Elena, Chiara; Vinti, Luciana; Locatelli, Franco; Bottino, Cristina; Castriconi, Roberta

2017-08-15

Tyrosine kinase inhibitors (TKIs) are used in the clinical management of hematological neoplasms. Moreover, in solid tumors such as stage 4 neuroblastomas (NB), imatinib showed benefits that might depend on both on-target and immunological off-target effects. We investigated the effects of imatinib and nilotinib on human NK cells, monocytes, and macrophages. High numbers of monocytes died upon exposure to TKI concentrations similar to those achieved in patients. Conversely, NK cells were highly resistant to the TKI cytotoxic effect, were properly activated by immunostimulatory cytokines, and degranulated in the presence of NB cells. In NB, neither drug reduced the expression of ligands for activating NK receptors or upregulated that of HLA class I, B7-H3, PD-L1, and PD-L2, molecules that might limit NK cell function. Interestingly, TKIs modulated the chemokine receptor repertoire of immune cells. Acting at the transcriptional level, they increased the surface expression of CXCR4, an effect observed also in NK cells and monocytes of patients receiving imatinib for chronic myeloid leukemia. Moreover, TKIs reduced the expression of CXCR3 (in NK cells) and CCR1 (in monocytes). Monocytes also decreased the expression of M-CSFR, and low numbers of cells underwent differentiation toward macrophages. M0 and M2 macrophages were highly resistant to TKIs and maintained their phenotypic and functional characteristics. Importantly, also in the presence of TKIs, the M2 immunosuppressive polarization was reverted by TLR engagement, and M1-oriented macrophages fully activated autologous NK cells. Our results contribute to better interpreting the off-target efficacy of TKIs in tumors and to envisaging strategies aimed at facilitating antitumor immune responses. Copyright © 2017 by The American Association of Immunologists, Inc.

Cell cycle-tailored targeting of metastatic melanoma: Challenges and opportunities.

PubMed

Haass, Nikolas K; Gabrielli, Brian

2017-07-01

The advent of targeted therapies of metastatic melanoma, such as MAPK pathway inhibitors and immune checkpoint antagonists, has turned dermato-oncology from the "bad guy" to the "poster child" in oncology. Current targeted therapies are effective, although here is a clear need to develop combination therapies to delay the onset of resistance. Many antimelanoma drugs impact on the cell cycle but are also dependent on certain cell cycle phases resulting in cell cycle phase-specific drug insensitivity. Here, we raise the question: Have combination trials been abandoned prematurely as ineffective possibly only because drug scheduling was not optimized? Firstly, if both drugs of a combination hit targets in the same melanoma cell, cell cycle-mediated drug insensitivity should be taken into account when planning combination therapies, timing of dosing schedules and choice of drug therapies in solid tumors. Secondly, if the combination is designed to target different tumor cell subpopulations of a heterogeneous tumor, one drug effective in a particular subpopulation should not negatively impact on the other drug targeting another subpopulation. In addition to the role of cell cycle stage and progression on standard chemotherapeutics and targeted drugs, we discuss the utilization of cell cycle checkpoint control defects to enhance chemotherapeutic responses or as targets themselves. We propose that cell cycle-tailored targeting of metastatic melanoma could further improve therapy outcomes and that our real-time cell cycle imaging 3D melanoma spheroid model could be utilized as a tool to measure and design drug scheduling approaches. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Sonoporation of endothelial cells by vibrating targeted microbubbles.

PubMed

Kooiman, Klazina; Foppen-Harteveld, Miranda; van der Steen, Antonius F W; de Jong, Nico

2011-08-25

Molecular imaging using ultrasound makes use of targeted microbubbles. In this study we investigated whether these microbubbles could also be used to induce sonoporation in endothelial cells. Lipid-coated microbubbles were targeted to CD31 and insonified at 1 MHz at low peak negative acoustic pressures at six sequences of 10 cycle sine-wave bursts. Vibration of the targeted microbubbles was recorded with the Brandaris-128 high-speed camera (~13 million frames per second). In total, 31 cells were studied that all had one microbubble (1.2-4.2 micron in diameter) attached per cell. After insonification at 80 kPa, 30% of the cells (n=6) had taken up propidium iodide, while this was 20% (n=1) at 120 kPa and 83% (n=5) at 200 kPa. Irrespective of the peak negative acoustic pressure, uptake of propidium iodide was observed when the relative vibration amplitude of targeted microbubbles was greater than 0.5. No relationship was found between the position of the microbubble on the cell and induction of sonoporation. This study shows that targeted microbubbles can also be used to induce sonoporation, thus making it possible to combine molecular imaging and drug delivery. Copyright © 2011 Elsevier B.V. All rights reserved.

Obtaining control of cell surface functionalizations via Pre-targeting and Supramolecular host guest interactions

NASA Astrophysics Data System (ADS)

Rood, Mark T. M.; Spa, Silvia J.; Welling, Mick M.; Ten Hove, Jan Bart; van Willigen, Danny M.; Buckle, Tessa; Velders, Aldrik H.; van Leeuwen, Fijs W. B.

2017-01-01

The use of mammalian cells for therapeutic applications is finding its way into modern medicine. However, modification or “training” of cells to make them suitable for a specific application remains complex. By envisioning a chemical toolbox that enables specific, but straight-forward and generic cellular functionalization, we investigated how membrane-receptor (pre)targeting could be combined with supramolecular host-guest interactions based on β-cyclodextrin (CD) and adamantane (Ad). The feasibility of this approach was studied in cells with membranous overexpression of the chemokine receptor 4 (CXCR4). By combining specific targeting of CXCR4, using an adamantane (Ad)-functionalized Ac-TZ14011 peptide (guest; KD = 56 nM), with multivalent host molecules that entailed fluorescent β-CD-Poly(isobutylene-alt-maleic-anhydride)-polymers with different fluorescent colors and number of functionalities, host-guest cell-surface modifications could be studied in detail. A second set of Ad-functionalized entities enabled introduction of additional surface functionalities. In addition, the attraction between CD and Ad could be used to drive cell-cell interactions. Combined we have shown that supramolecular interactions, that are based on specific targeting of an overexpressed membrane-receptor, allow specific and stable, yet reversible, surface functionalization of viable cells and how this approach can be used to influence the interaction between cells and their surroundings.

Targeted silver nanoparticles for ratiometric cell phenotyping

NASA Astrophysics Data System (ADS)

Willmore, Anne-Mari A.; Simón-Gracia, Lorena; Toome, Kadri; Paiste, Päärn; Kotamraju, Venkata Ramana; Mölder, Tarmo; Sugahara, Kazuki N.; Ruoslahti, Erkki; Braun, Gary B.; Teesalu, Tambet

2016-04-01

Affinity targeting is used to deliver nanoparticles to cells and tissues. For efficient targeting, it is critical to consider the expression and accessibility of the relevant receptors in the target cells. Here, we describe isotopically barcoded silver nanoparticles (AgNPs) as a tool for auditing affinity ligand receptors in cells. Tumor penetrating peptide RPARPAR (receptor: NRP-1) and tumor homing peptide GKRK (receptor: p32) were used as affinity ligands on the AgNPs. The binding and uptake of the peptide-functionalized AgNPs by cultured PPC-1 prostate cancer and M21 melanoma cells was dependent on the cell surface expression of the cognate peptide receptors. Barcoded peptide-functionalized AgNPs were synthesized from silver and palladium isotopes. The cells were incubated with a cocktail of the barcoded nanoparticles [RPARPAR (R), GKRK (K), and control], and cellular binding and internalization of each type of nanoparticle was assessed by inductively coupled plasma mass spectrometry. The results of isotopic analysis were in agreement with data obtained using optical methods. Using ratiometric measurements, we were able to classify the PPC-1 cell line as mainly NRP-1-positive, with 75 +/- 5% R-AgNP uptake, and the M21 cell line as only p32-positive, with 89 +/- 9% K-AgNP uptake. The isotopically barcoded multiplexed AgNPs are useful as an in vitro ratiometric phenotyping tool and have potential uses in functional evaluation of the expression of accessible homing peptide receptors in vivo.Affinity targeting is used to deliver nanoparticles to cells and tissues. For efficient targeting, it is critical to consider the expression and accessibility of the relevant receptors in the target cells. Here, we describe isotopically barcoded silver nanoparticles (AgNPs) as a tool for auditing affinity ligand receptors in cells. Tumor penetrating peptide RPARPAR (receptor: NRP-1) and tumor homing peptide GKRK (receptor: p32) were used as affinity ligands on the AgNPs. The

Hesr1 and Hesr3 are essential to generate undifferentiated quiescent satellite cells and to maintain satellite cell numbers

PubMed Central

Fukada, So-ichiro; Yamaguchi, Masahiko; Kokubo, Hiroki; Ogawa, Ryo; Uezumi, Akiyoshi; Yoneda, Tomohiro; Matev, Miroslav M.; Motohashi, Norio; Ito, Takahito; Zolkiewska, Anna; Johnson, Randy L.; Saga, Yumiko; Miyagoe-Suzuki, Yuko; Tsujikawa, Kazutake; Takeda, Shin’ichi; Yamamoto, Hiroshi

2011-01-01

Satellite cells, which are skeletal muscle stem cells, divide to provide new myonuclei to growing muscle fibers during postnatal development, and then are maintained in an undifferentiated quiescent state in adult skeletal muscle. This state is considered to be essential for the maintenance of satellite cells, but their molecular regulation is unknown. We show that Hesr1 (Hey1) and Hesr3 (Heyl) (which are known Notch target genes) are expressed simultaneously in skeletal muscle only in satellite cells. In Hesr1 and Hesr3 single-knockout mice, no obvious abnormalities of satellite cells or muscle regenerative potentials are observed. However, the generation of undifferentiated quiescent satellite cells is impaired during postnatal development in Hesr1/3 double-knockout mice. As a result, myogenic (MyoD and myogenin) and proliferative (Ki67) proteins are expressed in adult satellite cells. Consistent with the in vivo results, Hesr1/3-null myoblasts generate very few Pax7+ MyoD– undifferentiated cells in vitro. Furthermore, the satellite cell number gradually decreases in Hesr1/3 double-knockout mice even after it has stabilized in control mice, and an age-dependent regeneration defect is observed. In vivo results suggest that premature differentiation, but not cell death, is the reason for the reduced number of satellite cells in Hesr1/3 double-knockout mice. These results indicate that Hesr1 and Hesr3 are essential for the generation of adult satellite cells and for the maintenance of skeletal muscle homeostasis. PMID:21989910

What's your number? The effects of trial order on the one-target advantage.

PubMed

Bested, Stephen R; Khan, Michael A; Lawrence, Gavin P; Tremblay, Luc

2018-05-01

When moving our upper-limb towards a single target, movement times are typically shorter than when movement to a second target is required. This is known as the one-target advantage. Most studies that have demonstrated the one-target advantage have employed separate trial blocks for the one- and two-segment movements. To test if the presence of the one-target advantage depends on advance knowledge of the number of segments, the present study investigated whether the one-target advantage would emerge under different trial orders/sequences. One- and two-segment responses were organized in blocked (i.e., 1-1-1, 2-2-2), alternating (i.e., 1-2-1-2-1-2), and random (i.e., 1-1-2-1-2-2) trial sequences. Similar to previous studies, where only blocked schedules have typically been utilized, the one-target advantage emerged during the blocked and alternate conditions, but not in the random condition. This finding indicates that the one-target advantage is contingent on participants knowing the number of movement segments prior to stimulus onset. Copyright © 2018 Elsevier B.V. All rights reserved.

A novel double-targeted nondrug delivery system for targeting cancer stem cells

PubMed Central

Qiao, Shupei; Zhao, Yufang; Geng, Shuai; Li, Yong; Hou, Xiaolu; Liu, Yi; Lin, Feng-Huei; Yao, Lifen; Tian, Weiming

2016-01-01

Instead of killing cancer stem cells (CSCs), the conventional chemotherapy used for cancer treatment promotes the enrichment of CSCs, which are responsible for tumor growth, metastasis, and recurrence. However, most therapeutic agents are only able to kill a small proportion of CSCs by targeting one or two cell surface markers or dysregulated CSC pathways, which are usually shared with normal stem cells (NSCs). In this study, we developed a novel nondrug delivery system for the dual targeting of CSCs by conjugating hyaluronic acid (HA) and grafting the doublecortin-like kinase 1 (DCLK1) monoclonal antibody to the surface of poly(ethylene glycol) (PEG)–poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs), which can specifically target CD44 receptors and the DCLK1 surface marker – the latter was shown to possess the capacity to distinguish between CSCSs and NSCs. The size and morphology of these NPs were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). This was followed by studies of NP encapsulation efficiency and in vitro drug release properties. Then, the cytotoxicity of the NPs was tested via Cell Counting Kit-8 assay. Finally, the 4T1 CSCs were obtained from the alginate-based platform, which we developed as an in vitro tumor model. Tumor-bearing nude mice were used as in vivo models to systematically detect the ability of NPs to target CSCs. Our results showed that the DCLK1–HA–PEG–PLGA NPs exhibited a targeting effect toward CSCs both in vitro and in vivo. These findings have important implications for the rational design of drug delivery systems that target CSCs with high efficacy. PMID:27994463

Increased mast cell numbers in a calcaneal tendon overuse model.

PubMed

Pingel, J; Wienecke, J; Kongsgaard, M; Behzad, H; Abraham, T; Langberg, H; Scott, A

2013-12-01

Tendinopathy is often discovered late because the initial development of tendon pathology is asymptomatic. The aim of this study was to examine the potential role of mast cell involvement in early tendinopathy using a high-intensity uphill running (HIUR) exercise model. Twenty-four male Wistar rats were divided in two groups: running group (n = 12); sedentary control group (n = 12). The running-group was exposed to the HIUR exercise protocol for 7 weeks. The calcaneal tendons of both hind limbs were dissected. The right tendon was used for histologic analysis using Bonar score, immunohistochemistry, and second harmonic generation microscopy (SHGM). The left tendon was used for quantitative polymerase chain reaction (qPCR) analysis. An increased tendon cell density in the runners were observed compared to the controls (P = 0.05). Further, the intensity of immunostaining of protein kinase B, P = 0.03; 2.75 ± 0.54 vs 1.17 ± 0.53, was increased in the runners. The Bonar score (P = 0.05), and the number of mast cells (P = 0.02) were significantly higher in the runners compared to the controls. Furthermore, SHGM showed focal collagen disorganization in the runners, and reduced collagen density (P = 0.03). IL-3 mRNA levels were correlated with mast cell number in sedentary animals. The qPCR analysis showed no significant differences between the groups in the other analyzed targets. The current study demonstrates that 7-week HIUR causes structural changes in the calcaneal tendon, and further that these changes are associated with an increased mast cell density. © 2013 The Authors. Scand J Med Sci Sports published by John Wiley & Sons Ltd.

Targeted gene therapy and cell reprogramming in Fanconi anemia

PubMed Central

Rio, Paula; Baños, Rocio; Lombardo, Angelo; Quintana-Bustamante, Oscar; Alvarez, Lara; Garate, Zita; Genovese, Pietro; Almarza, Elena; Valeri, Antonio; Díez, Begoña; Navarro, Susana; Torres, Yaima; Trujillo, Juan P; Murillas, Rodolfo; Segovia, Jose C; Samper, Enrique; Surralles, Jordi; Gregory, Philip D; Holmes, Michael C; Naldini, Luigi; Bueren, Juan A

2014-01-01

Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies. PMID:24859981

Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein

PubMed Central

Viktorova, Ekaterina G.; Nchoutmboube, Jules; Ford-Siltz, Lauren A.

2015-01-01

ABSTRACT It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses. IMPORTANCE Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can

Advanced cell therapies: targeting, tracking and actuation of cells with magnetic particles.

PubMed

Connell, John J; Patrick, P Stephen; Yu, Yichao; Lythgoe, Mark F; Kalber, Tammy L

2015-01-01

Regenerative medicine would greatly benefit from a new platform technology that enabled measurable, controllable and targeting of stem cells to a site of disease or injury in the body. Superparamagnetic iron-oxide nanoparticles offer attractive possibilities in biomedicine and can be incorporated into cells, affording a safe and reliable means of tagging. This review describes three current and emerging methods to enhance regenerative medicine using magnetic particles to guide therapeutic cells to a target organ; track the cells using MRI and assess their spatial localization with high precision and influence the behavior of the cell using magnetic actuation. This approach is complementary to the systemic injection of cell therapies, thus expanding the horizon of stem cell therapeutics.

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.

Targeting NCK-Mediated Endothelial Cell Front-Rear Polarity Inhibits Neovascularization.

PubMed

Dubrac, Alexandre; Genet, Gael; Ola, Roxana; Zhang, Feng; Pibouin-Fragner, Laurence; Han, Jinah; Zhang, Jiasheng; Thomas, Jean-Léon; Chedotal, Alain; Schwartz, Martin A; Eichmann, Anne

2016-01-26

Sprouting angiogenesis is a key process driving blood vessel growth in ischemic tissues and an important drug target in a number of diseases, including wet macular degeneration and wound healing. Endothelial cells forming the sprout must develop front-rear polarity to allow sprout extension. The adaptor proteins Nck1 and 2 are known regulators of cytoskeletal dynamics and polarity, but their function in angiogenesis is poorly understood. Here, we show that the Nck adaptors are required for endothelial cell front-rear polarity and migration downstream of the angiogenic growth factors VEGF-A and Slit2. Mice carrying inducible, endothelial-specific Nck1/2 deletions fail to develop front-rear polarized vessel sprouts and exhibit severe angiogenesis defects in the postnatal retina and during embryonic development. Inactivation of NCK1 and 2 inhibits polarity by preventing Cdc42 and Pak2 activation by VEGF-A and Slit2. Mechanistically, NCK binding to ROBO1 is required for both Slit2- and VEGF-induced front-rear polarity. Selective inhibition of polarized endothelial cell migration by targeting Nck1/2 prevents hypersprouting induced by Notch or Bmp signaling inhibition, and pathological ocular neovascularization and wound healing, as well. These data reveal a novel signal integration mechanism involving NCK1/2, ROBO1/2, and VEGFR2 that controls endothelial cell front-rear polarity during sprouting angiogenesis. © 2015 American Heart Association, Inc.

An innovative pre-targeting strategy for tumor cell specific imaging and therapy

NASA Astrophysics Data System (ADS)

Qin, Si-Yong; Peng, Meng-Yun; Rong, Lei; Jia, Hui-Zhen; Chen, Si; Cheng, Si-Xue; Feng, Jun; Zhang, Xian-Zheng

2015-08-01

A programmed pre-targeting system for tumor cell imaging and targeting therapy was established based on the ``biotin-avidin'' interaction. In this programmed functional system, transferrin-biotin can be actively captured by tumor cells with the overexpression of transferrin receptors, thus achieving the pre-targeting modality. Depending upon avidin-biotin recognition, the attachment of multivalent FITC-avidin to biotinylated tumor cells not only offered the rapid fluorescence labelling, but also endowed the pre-targeted cells with targeting sites for the specifically designed biotinylated peptide nano-drug. Owing to the successful pre-targeting, tumorous HepG2 and HeLa cells were effectively distinguished from the normal 3T3 cells via fluorescence imaging. In addition, the self-assembled peptide nano-drug resulted in enhanced cell apoptosis in the observed HepG2 cells. The tumor cell specific pre-targeting strategy is applicable for a variety of different imaging and therapeutic agents for tumor treatments.A programmed pre-targeting system for tumor cell imaging and targeting therapy was established based on the ``biotin-avidin'' interaction. In this programmed functional system, transferrin-biotin can be actively captured by tumor cells with the overexpression of transferrin receptors, thus achieving the pre-targeting modality. Depending upon avidin-biotin recognition, the attachment of multivalent FITC-avidin to biotinylated tumor cells not only offered the rapid fluorescence labelling, but also endowed the pre-targeted cells with targeting sites for the specifically designed biotinylated peptide nano-drug. Owing to the successful pre-targeting, tumorous HepG2 and HeLa cells were effectively distinguished from the normal 3T3 cells via fluorescence imaging. In addition, the self-assembled peptide nano-drug resulted in enhanced cell apoptosis in the observed HepG2 cells. The tumor cell specific pre-targeting strategy is applicable for a variety of different imaging

Stromal cells in breast cancer as a potential therapeutic target

PubMed Central

Dykes, Samantha S.; Hughes, Veronica S.; Wiggins, Jennifer M.; Fasanya, Henrietta O.; Tanaka, Mai; Siemann, Dietmar

2018-01-01

Breast cancer in the United States is the second most commonly diagnosed cancer in women. About 1 in 8 women will develop invasive breast cancer over the course of her lifetime and breast cancer remains the second leading cause of cancer-related death. In pursuit of novel therapeutic strategies, researchers have examined the tumor microenvironment as a potential anti-cancer target. In addition to neoplastic cells, the tumor microenvironment is composed of several critical normal cell types, including fibroblasts, vascular and lymph endothelial cells, osteoclasts, adipocytes, and immune cells. These cells have important roles in healthy tissue stasis, which frequently are altered in tumors. Indeed, tumor-associated stromal cells often contribute to tumorigenesis, tumor progression, and metastasis. Consequently, these host cells may serve as a possible target in anti-tumor and anti-metastatic therapeutic strategies. Targeting the tumor associated host cells offers the benefit that such cells do not mutate and develop resistance in response to treatment, a major cause of failure in cancer therapeutics targeting neoplastic cells. This review discusses the role of host cells in the tumor microenvironment during tumorigenesis, progression, and metastasis, and provides an overview of recent developments in targeting these cell populations to enhance cancer therapy efficacy.

Improved and targeted delivery of bioactive molecules to cells with magnetic layer-by-layer assembled microcapsules

NASA Astrophysics Data System (ADS)

Pavlov, Anton M.; Gabriel, Samantha A.; Sukhorukov, Gleb B.; Gould, David J.

2015-05-01

Despite our increasing knowledge of cell biology and the recognition of an increasing repertoire of druggable intracellular therapeutic targets, there remain a limited number of approaches to deliver bioactive molecules to cells and even fewer that enable targeted delivery. Layer-by-layer (LbL) microcapsules are assembled using alternate layers of oppositely charged molecules and are potential cell delivery vehicles for applications in nanomedicine. There are a wide variety of charged molecules that can be included in the microcapsule structure including metal nanoparticles that introduce physical attributes. Delivery of bioactive molecules to cells with LbL microcapsules has recently been demonstrated, so in this study we explore the delivery of bioactive molecules (luciferase enzyme and plasmid DNA) to cells using biodegradable microcapsules containing a layer of magnetite nanoparticles. Interestingly, significantly improved intracellular luciferase enzyme activity (25 fold) and increased transfection efficiency with plasmid DNA (3.4 fold) was observed with magnetic microcapsules. The use of a neodymium magnet enabled efficient targeting of magnetic microcapsules which further improved the delivery efficiency of the cargoes as a consequence of increased microcapsule concentration at the magnetic site. Microcapsules were well tolerated by cells in these experiments and only displayed signs of toxicity at a capsule : cell ratio of 100 : 1 and with extended exposure. These studies illustrate how multi-functionalization of LbL microcapsules can improve and target delivery of bioactive molecules to cells.

Mast cell proteases as pharmacological targets

PubMed Central

Caughey, George H.

2015-01-01

Mast cells are rich in proteases, which are the major proteins of intracellular granules and are released with histamine and heparin by activated cells. Most of these proteases are active in the granule as well outside of the mast cell when secreted, and can cleave targets near degranulating mast cells and in adjoining tissue compartments. Some proteases released from mast cells reach the bloodstream and may have far-reaching actions. In terms of relative amounts, the major mast cell proteases include the tryptases, chymases, cathepsin G, carboxypeptidase A3, dipeptidylpeptidase I/cathepsin C, and cathepsins L and S. Some mast cells also produce granzyme B, plasminogen activators, and matrix metalloproteinases. Tryptases and chymases are almost entirely mast cell-specific, whereas other proteases, such as cathepsins G, C, and L are expressed by a variety of inflammatory cells. Carboxypeptidase A3 expression is a property shared by basophils and mast cells. Other proteases, such as mastins, are largely basophil-specific, although human basophils are protease-deficient compared with their murine counterparts. The major classes of mast cell proteases have been targeted for development of therapeutic inhibitors. Also, a human β-tryptase has been proposed as a potential drug itself, to inactivate of snake venins. Diseases linked to mast cell proteases include allergic diseases, such as asthma, eczema, and anaphylaxis, but also include non-allergic diseases such inflammatory bowel disease, autoimmune arthritis, atherosclerosis, aortic aneurysms, hypertension, myocardial infarction, heart failure, pulmonary hypertension and scarring diseases of lungs and other organs. In some cases, studies performed in mouse models suggest protective or homeostatic roles for specific proteases (or groups of proteases) in infections by bacteria, worms and other parasites, and even in allergic inflammation. At the same time, a clearer picture has emerged of differences in the properties

Stem cell signaling as a target for novel drug discovery: recent progress in the WNT and Hedgehog pathways.

PubMed

An, Songzhu Michael; Ding, Qiang Peter; Li, Ling-song

2013-06-01

One of the most exciting fields in biomedical research over the past few years is stem cell biology, and therapeutic application of stem cells to replace the diseased or damaged tissues is also an active area in development. Although stem cell therapy has a number of technical challenges and regulatory hurdles to overcome, the use of stem cells as tools in drug discovery supported by mature technologies and established regulatory paths is expected to generate more immediate returns. In particular, the targeting of stem cell signaling pathways is opening up a new avenue for drug discovery. Aberrations in these pathways result in various diseases, including cancer, fibrosis and degenerative diseases. A number of drug targets in stem cell signaling pathways have been identified. Among them, WNT and Hedgehog are two most important signaling pathways, which are the focus of this review. A hedgehog pathway inhibitor, vismodegib (Erivedge), has recently been approved by the US FDA for the treatment of skin cancer, while several drug candidates for the WNT pathway are entering clinical trials. We have discovered that the stem cell signaling pathways respond to traditional Chinese medicines. Substances isolated from herbal medicine may act specifically on components of stem cell signaling pathways with high affinities. As many of these events can be explained through molecular interactions, these phenomena suggest that discovery of stem cell-targeting drugs from natural products may prove to be highly successful.

Prostate Stem Cell Antigen: A Prospective Therapeutic and Diagnostic Target

PubMed Central

Raff, Adam B.; Gray, Andrew; Kast, W. Martin

2009-01-01

The development of novel clinical tools to combat cancer is an intense field of research and recent efforts have been directed at the identification of proteins that may provide diagnostic, prognostic and/or therapeutic applications due to their restricted expression. To date, a number of protein candidates have emerged as potential clinical tools in the treatment of prostate cancer. Discovered over ten year ago, prostate stem cell antigen (PSCA) is a cell surface antigen that belongs to the Ly-6/Thy-1 family of glycosylphosphatidylinositol-anchored proteins. PSCA is highly overexpressed in human prostate cancer, with limited expression in normal tissues, making it an ideal target for both diagnosis and therapy. Several studies have now clearly correlated the expression of PSCA with relevant clinical benchmarks, such as Gleason score and metastasis, while others have demonstrated the efficacy of PSCA targeting in treatment through various modalities. The purpose of this review is to present the current body of knowledge about PSCA and its potential role in the treatment of human prostate cancer. PMID:18838214

Limitations and possibilities of low cell number ChIP-seq

PubMed Central

2012-01-01

Background Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) offers high resolution, genome-wide analysis of DNA-protein interactions. However, current standard methods require abundant starting material in the range of 1–20 million cells per immunoprecipitation, and remain a bottleneck to the acquisition of biologically relevant epigenetic data. Using a ChIP-seq protocol optimised for low cell numbers (down to 100,000 cells / IP), we examined the performance of the ChIP-seq technique on a series of decreasing cell numbers. Results We present an enhanced native ChIP-seq method tailored to low cell numbers that represents a 200-fold reduction in input requirements over existing protocols. The protocol was tested over a range of starting cell numbers covering three orders of magnitude, enabling determination of the lower limit of the technique. At low input cell numbers, increased levels of unmapped and duplicate reads reduce the number of unique reads generated, and can drive up sequencing costs and affect sensitivity if ChIP is attempted from too few cells. Conclusions The optimised method presented here considerably reduces the input requirements for performing native ChIP-seq. It extends the applicability of the technique to isolated primary cells and rare cell populations (e.g. biobank samples, stem cells), and in many cases will alleviate the need for cell culture and any associated alteration of epigenetic marks. However, this study highlights a challenge inherent to ChIP-seq from low cell numbers: as cell input numbers fall, levels of unmapped sequence reads and PCR-generated duplicate reads rise. We discuss a number of solutions to overcome the effects of reducing cell number that may aid further improvements to ChIP performance. PMID:23171294

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

Targeting survivin with prodigiosin isolated from cell wall of Serratia marcescens induces apoptosis in hepatocellular carcinoma cells.

PubMed

Yenkejeh, R A; Sam, M R; Esmaeillou, M

2017-04-01

Abnormal activation of the Wnt/β-catenin signaling pathway increases survivin expression that is involved in hepatocarcinogenesis. Therefore, downregulation of survivin may provide an attractive strategy for treatment of hepatocellular carcinoma. In this regard, little is known about the anticancer effects of prodigiosin isolated from cell wall of Serratia marcescens on the survivin expression and induction of apoptosis in hepatocellular carcinoma cells. Human hepatocellular carcinoma (HepG2) cells were treated with 100-, 200-, 400-, and 600-nM prodigiosin after which morphology of cells, cell number, growth inhibition, survivin expression, caspase-3 activation, and apoptotic rate were evaluated by inverted microscope, hemocytometer, MTT assay, RT-PCR, fluorometric immunosorbent enzyme assay, and flow cytometric analysis, respectively. Prodigiosin changed morphology of cells to apoptotic forms and disrupted cell connections. This compound significantly increased growth inhibition rate and decreased metabolic activity of HepG2 cells in a dose- and time-dependent manner. After 24-, 48-, and 72-h treatments with prodigiosin at concentrations ranging from 100 nM to 600 nM, growth inhibition rates were measured to be 1.5-10%, 24-47.5%, and 55.5-72.5%, respectively, compared to untreated cells. At the same conditions, metabolic activities were measured to be 91-83%, 74-53%, and 47-31% for indicated concentrations of prodigiosin, respectively, compared to untreated cells. We also found that treatment of HepG2 cells for 48 h decreased significantly cell number and survivin expression and increased caspase-3 activation in a dose-dependent manner. Specifically, treatment with 600-nM prodigiosin resulted in 77% decrease in cell number, 88.5% decrease in survivin messenger RNA level, and 330% increase in caspase-3 activation level compared to untreated cells. An increase in the number of apoptotic cells (late apoptosis) ranging from 36.9% to 97.4% was observed with increasing

Surface-modified gold nanorods for specific cell targeting

NASA Astrophysics Data System (ADS)

Wang, Chan-Ung; Arai, Yoshie; Kim, Insun; Jang, Wonhee; Lee, Seonghyun; Hafner, Jason H.; Jeoung, Eunhee; Jung, Deokho; Kwon, Youngeun

2012-05-01

Gold nanoparticles (GNPs) have unique properties that make them highly attractive materials for developing functional reagents for various biomedical applications including photothermal therapy, targeted drug delivery, and molecular imaging. For in vivo applications, GNPs need to be prepared with very little or negligible cytotoxicitiy. Most GNPs are, however, prepared using growth-directing surfactants such as cetyl trimethylammonium bromide (CTAB), which are known to have considerable cytotoxicity. In this paper, we describe an approach to remove CTAB to a non-toxic concentration. We optimized the conditions for surface modification with methoxypolyethylene glycol thiol (mPEG), which replaced CTAB and formed a protective layer on the surface of gold nanorods (GNRs). The cytotoxicities of pristine and surface-modified GNRs were measured in primary human umbilical vein endothelial cells and human cell lines derived from hepatic carcinoma cells, embryonic kidney cells, and thyroid papillary carcinoma cells. Cytotoxicity assays revealed that treating cells with GNRs did not significantly affect cell viability except for thyroid papillary carcinoma cells. Thyroid cancer cells were more susceptible to residual CTAB, so CTAB had to be further removed by dialysis in order to use GNRs for thyroid cell targeting. PEGylated GNRs are further modified to present monoclonal antibodies that recognize a specific surface marker, Na-I symporter, for thyroid cells. Antibody-conjugated GNRs specifically targeted human thyroid cells in vitro.

The effects of variations in the number and sequence of targeting signals on nuclear uptake

PubMed Central

1988-01-01

To determine if the number of targeting signals affects the transport of proteins into the nucleus, Xenopus oocytes were injected with colloidal gold particles, ranging in diameter from 20 to 280 A, that were coated with BSA cross-linked with synthetic peptides containing the SV-40 large T-antigen nuclear transport signal. Three BSA conjugate preparations were used; they had an average of 5, 8, and 11 signals per molecule of carrier protein. In addition, large T-antigen, which contains one signal per monomer, was used as a coating agent. The cells were fixed at various times after injection and subsequently analyzed by electron microscopy. Gold particles coated with proteins containing the SV-40 signal entered the nucleus through central channels located within the nuclear pores. Analysis of the intracellular distribution and size of the tracers that entered the nucleus indicated that the number of signals per molecule affect both the relative uptake of particles and the functional size of the channels available for translocation. In control experiments, gold particles coated with BSA or BSA conjugated with inactive peptides similar to the SV-40 transport signal were virtually excluded from the nucleus. Gold particles coated with nucleoplasmin, an endogenous karyophilic protein that contains five targeting signals per molecule, was transported through the nuclear pores more effectively than any of the BSA-peptide conjugates. Based on a correlation between the peri-envelope density of gold particles and their relative uptake, it is suggested that the differences in the activity of the two targeting signals is related to their binding affinity for envelope receptors. It was also determined, by performing coinjection experiments, that individual pores are capable of recognizing and transporting proteins that contain different nuclear targeting signals. PMID:3170630

Curcumin targets fibroblast–tumor cell interactions in oral squamous cell carcinoma

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

Dudás, József, E-mail: jozsef.dudas@i-med.ac.at; Fullár, Alexandra, E-mail: fullarsz@gmail.com; 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, 1085 Budapest

Co-culture of periodontal ligament fibroblasts (PDLs) and SCC-25 oral squamous carcinoma cells (OSCC) results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs) and induces epithelial-to mesenchymal transition (EMT) of OSCC tumor cells. We hypothesized that Curcumin targets this dynamic mutual interaction between CAFs and tumor cells. Normal and 2 μM Curcumin-treated co-culture were performed for 4 days, followed by analysis of tumor cell invasivity, mRNA/protein expression of EMT-markers and mediators, activity measure of matrix metalloproteinase 9 (MMP-9), and western blot analysis of signal transduction in tumor cells and fibroblasts. In Curcumin-treated co-culture, in tumor cells, the levels of nuclear factormore » κB (NFκBα) and early response kinase (ERK)—decreased, in fibroblasts, integrin αv protein synthesis decreased compared to corresponding cells in normal co-culture. The signal modulatory changes induced by Curcumin caused decreased release of EMT-mediators in CAFs and reversal of EMT in tumor cells, which was associated with decreased invasion. These data confirm the palliative potential of Curcumin in clinical application. - Graphical abstract: Co-culture of periodontal ligament fibroblasts (PDLs) and SCC-25 oral squamous carcinoma cells (OSCC) results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs) and induces epithelial-to mesenchymal transition (EMT) of tumor cells. Curcumin targets this dynamic mutual interaction between CAFs and tumor cells by inhibiting the production of EMT mediators in CAFs and by modification of intracellular signaling in tumor cells. This causes less invasivity and reversal of EMT in tumor cells. Highlights: ► Curcumin targets tumor–fibroblast interaction in head and neck cancer. ► Curcumin suppresses mediators of epithelial–mesenchymal transition. ► Curcumin decreases the invasivity of tumor cells.« less

Single-cell copy number variation detection

PubMed Central

2011-01-01

Detection of chromosomal aberrations from a single cell by array comparative genomic hybridization (single-cell array CGH), instead of from a population of cells, is an emerging technique. However, such detection is challenging because of the genome artifacts and the DNA amplification process inherent to the single cell approach. Current normalization algorithms result in inaccurate aberration detection for single-cell data. We propose a normalization method based on channel, genome composition and recurrent genome artifact corrections. We demonstrate that the proposed channel clone normalization significantly improves the copy number variation detection in both simulated and real single-cell array CGH data. PMID:21854607

Therapeutic potential of the metabolic modulator phenformin in targeting the stem cell compartment in melanoma.

PubMed

Petrachi, Tiziana; Romagnani, Alessandra; Albini, Adriana; Longo, Caterina; Argenziano, Giuseppe; Grisendi, Giulia; Dominici, Massimo; Ciarrocchi, Alessia; Dallaglio, Katiuscia

2017-01-24

Melanoma is the most dangerous and treatment-resistant skin cancer. Tumor resistance and recurrence are due to the persistence in the patient of aggressive cells with stem cell features, the cancer stem cells (CSC). Recent evidences have shown that CSC display a distinct metabolic profile as compared to tumor bulk population: a promising anti-tumor strategy is therefore to target specific metabolic pathways driving CSC behavior. Biguanides (metformin and phenformin) are anti-diabetic drugs able to perturb cellular metabolism and displaying anti-cancer activity. However, their ability to target the CSC compartment in melanoma is not known. Here we show that phenformin, but not metformin, strongly reduces melanoma cell viability, growth and invasion in both 2D and 3D (spheroids) models. While phenformin decreases melanoma CSC markers expression and the levels of the pro-survival factor MITF, MITF overexpression fails to prevent phenformin effects. Phenformin significantly reduces cell viability in melanoma by targeting both CSC (ALDHhigh) and non-CSC cells and by significantly reducing the number of viable cells in ALDHhigh and ALDHlow-derived spheroids. Consistently, phenformin reduces melanoma cell viability and growth independently from SOX2 levels. Our results show that phenformin is able to affect both CSC and non-CSC melanoma cell viability and growth and suggests its potential use as anti-cancer therapy in melanoma.

Therapeutic potential of the metabolic modulator phenformin in targeting the stem cell compartment in melanoma

PubMed Central

Albini, Adriana; Longo, Caterina; Argenziano, Giuseppe; Grisendi, Giulia; Dominici, Massimo; Ciarrocchi, Alessia; Dallaglio, Katiuscia

2017-01-01

Melanoma is the most dangerous and treatment-resistant skin cancer. Tumor resistance and recurrence are due to the persistence in the patient of aggressive cells with stem cell features, the cancer stem cells (CSC). Recent evidences have shown that CSC display a distinct metabolic profile as compared to tumor bulk population: a promising anti-tumor strategy is therefore to target specific metabolic pathways driving CSC behavior. Biguanides (metformin and phenformin) are anti-diabetic drugs able to perturb cellular metabolism and displaying anti-cancer activity. However, their ability to target the CSC compartment in melanoma is not known. Here we show that phenformin, but not metformin, strongly reduces melanoma cell viability, growth and invasion in both 2D and 3D (spheroids) models. While phenformin decreases melanoma CSC markers expression and the levels of the pro-survival factor MITF, MITF overexpression fails to prevent phenformin effects. Phenformin significantly reduces cell viability in melanoma by targeting both CSC (ALDHhigh) and non-CSC cells and by significantly reducing the number of viable cells in ALDHhigh and ALDHlow-derived spheroids. Consistently, phenformin reduces melanoma cell viability and growth independently from SOX2 levels. Our results show that phenformin is able to affect both CSC and non-CSC melanoma cell viability and growth and suggests its potential use as anti-cancer therapy in melanoma. PMID:28036292

Estimation of the radiation-induced DNA double-strand breaks number by considering cell cycle and absorbed dose per cell nucleus

PubMed Central

Mori, Ryosuke; Matsuya, Yusuke; Yoshii, Yuji; Date, Hiroyuki

2018-01-01

Abstract DNA double-strand breaks (DSBs) are thought to be the main cause of cell death after irradiation. In this study, we estimated the probability distribution of the number of DSBs per cell nucleus by considering the DNA amount in a cell nucleus (which depends on the cell cycle) and the statistical variation in the energy imparted to the cell nucleus by X-ray irradiation. The probability estimation of DSB induction was made following these procedures: (i) making use of the Chinese Hamster Ovary (CHO)-K1 cell line as the target example, the amounts of DNA per nucleus in the logarithmic and the plateau phases of the growth curve were measured by flow cytometry with propidium iodide (PI) dyeing; (ii) the probability distribution of the DSB number per cell nucleus for each phase after irradiation with 1.0 Gy of 200 kVp X-rays was measured by means of γ-H2AX immunofluorescent staining; (iii) the distribution of the cell-specific energy deposition via secondary electrons produced by the incident X-rays was calculated by WLTrack (in-house Monte Carlo code); (iv) according to a mathematical model for estimating the DSB number per nucleus, we deduced the induction probability density of DSBs based on the measured DNA amount (depending on the cell cycle) and the calculated dose per nucleus. The model exhibited DSB induction probabilities in good agreement with the experimental results for the two phases, suggesting that the DNA amount (depending on the cell cycle) and the statistical variation in the local energy deposition are essential for estimating the DSB induction probability after X-ray exposure. PMID:29800455

Estimation of the radiation-induced DNA double-strand breaks number by considering cell cycle and absorbed dose per cell nucleus.

PubMed

Mori, Ryosuke; Matsuya, Yusuke; Yoshii, Yuji; Date, Hiroyuki

2018-05-01

DNA double-strand breaks (DSBs) are thought to be the main cause of cell death after irradiation. In this study, we estimated the probability distribution of the number of DSBs per cell nucleus by considering the DNA amount in a cell nucleus (which depends on the cell cycle) and the statistical variation in the energy imparted to the cell nucleus by X-ray irradiation. The probability estimation of DSB induction was made following these procedures: (i) making use of the Chinese Hamster Ovary (CHO)-K1 cell line as the target example, the amounts of DNA per nucleus in the logarithmic and the plateau phases of the growth curve were measured by flow cytometry with propidium iodide (PI) dyeing; (ii) the probability distribution of the DSB number per cell nucleus for each phase after irradiation with 1.0 Gy of 200 kVp X-rays was measured by means of γ-H2AX immunofluorescent staining; (iii) the distribution of the cell-specific energy deposition via secondary electrons produced by the incident X-rays was calculated by WLTrack (in-house Monte Carlo code); (iv) according to a mathematical model for estimating the DSB number per nucleus, we deduced the induction probability density of DSBs based on the measured DNA amount (depending on the cell cycle) and the calculated dose per nucleus. The model exhibited DSB induction probabilities in good agreement with the experimental results for the two phases, suggesting that the DNA amount (depending on the cell cycle) and the statistical variation in the local energy deposition are essential for estimating the DSB induction probability after X-ray exposure.

Cell-type-specific, Aptamer-functionalized Agents for Targeted Disease Therapy

PubMed Central

Zhou, Jiehua; Rossi, John J.

2014-01-01

One hundred years ago, Dr. Paul Ehrlich popularized the “magic bullet” concept for cancer therapy in which an ideal therapeutic agent would only kill the specific tumor cells it targeted. Since then, “targeted therapy” that specifically targets the molecular defects responsible for a patient's condition has become a long-standing goal for treating human disease. However, safe and efficient drug delivery during the treatment of cancer and infectious disease remains a major challenge for clinical translation and the development of new therapies. The advent of SELEX technology has inspired many groundbreaking studies that successfully adapted cell-specific aptamers for targeted delivery of active drug substances in both in vitro and in vivo models. By covalently linking or physically functionalizing the cell-specific aptamers with therapeutic agents, such as siRNA, microRNA, chemotherapeutics or toxins, or delivery vehicles, such as organic or inorganic nanocarriers, the targeted cells and tissues can be specifically recognized and the therapeutic compounds internalized, thereby improving the local concentration of the drug and its therapeutic efficacy. Currently, many cell-type-specific aptamers have been developed that can target distinct diseases or tissues in a cell-type-specific manner. In this review, we discuss recent advances in the use of cell-specific aptamers for targeted disease therapy, as well as conjugation strategies and challenges. PMID:24936916

Trispecific antibodies for CD16A-directed NK cell engagement and dual-targeting of tumor cells.

PubMed

Gantke, Thorsten; Weichel, Michael; Herbrecht, Carmen; Reusch, Uwe; Ellwanger, Kristina; Fucek, Ivica; Eser, Markus; Müller, Thomas; Griep, Remko; Molkenthin, Vera; Zhukovsky, Eugene A; Treder, Martin

2017-09-01

Bispecific antibodies that redirect the lytic activity of cytotoxic immune effector cells, such as T- and NK cells, onto tumor cells have emerged as a highly attractive and clinically validated treatment modality for hematological malignancies. Advancement of this therapeutic concept into solid tumor indications, however, is hampered by the scarcity of targetable antigens that are surface-expressed on tumor cells but demonstrate only limited expression on healthy tissues. To overcome this limitation, the concept of dual-targeting, i.e. the simultaneous targeting of two tumor-expressed surface antigens with limited co-expression on non-malignant cells, with multispecific antibodies has been proposed to increase tumor selectivity of antibody-induced effector cell cytotoxicity. Here, a novel CD16A (FcγRIIIa)-directed trispecific, tetravalent antibody format, termed aTriFlex, is described, that is capable of redirecting NK cell cytotoxicity to two surface-expressed antigens. Using a BCMA/CD200-based in vitro model system, the potential use of aTriFlex antibodies for dual-targeting and selective induction of NK cell-mediated target cell lysis was investigated. Bivalent bispecific target cell binding was found to result in significant avidity gains and up to 17-fold increased in vitro potency. These data suggest trispecific aTriFlex antibodies may support dual-targeting strategies to redirect NK cell cytotoxicity with increased selectivity to enable targeting of solid tumor antigens. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Dendritic cell targeted vaccines: Recent progresses and challenges

PubMed Central

Chen, Pengfei; Liu, Xinsheng; Sun, Yuefeng; Zhou, Peng; Wang, Yonglu; Zhang, Yongguang

2016-01-01

ABSTRACT Dendritic cells (DCs) are known to be a set of morphology, structure and function of heterogeneous professional antigen presenting cells (APCs), as well as the strongest functional antigen presenting cells, which can absorb, process and present antigens. As the key regulators of innate and adaptive immune responses, DCs are at the center of the immune system and capable of interacting with both B cells and T cells, thereby manipulating the humoral and cellular immune responses. DCs provide an essential link between the innate and adaptive immunity, and the strong immune activation function of DCs and their properties of natural adjuvants, make them a valuable target for antigen delivery. Targeting antigens to DC-specific endocytic receptors in combination with the relevant antibodies or ligands along with immunostimulatory adjuvants has been recently recognized as a promising strategy for designing an effective vaccine that elicits a strong and durable T cell response against intracellular pathogens and cancer. This opinion article provides a brief summary of the rationales, superiorities and challenges of existing DC-targeting approaches. PMID:26513200

miR-26a suppresses autophagy in swine Sertoli cells by targeting ULK2.

PubMed

Ran, M; Li, Z; Cao, R; Weng, B; Peng, F; He, C; Chen, B

2018-05-14

A large number of microRNAs (miRNAs) have been detected from porcine testicular tissues thanks to the development of high-throughput sequencing technology. However, the regulatory roles of most identified miRNAs in swine testicular development or spermatogenesis are poorly understood. In our previous study, ULK2 (uncoordinated-51-like kinase 2) was predicted as a target gene of miR-26a. In this study, we aimed to investigate the role of miR-26a in swine Sertoli cell autophagy. The relative expression of miR-26a and ULK2 levels has a significant negative correlation (R 2  = .5964, p ≤ .01) in nine developmental stages of swine testicular tissue. Dual-luciferase reporter assay results show that miR-26a directly targets the 3'UTR of the ULK2 gene (position 618-624). In addition, both the mRNA and protein expression of ULK2 were downregulated by miR-26a in swine Sertoli cells. These results indicate that miR-26a targets the ULK2 gene and downregulates its expression in swine Sertoli cells. Based on the expression of marker genes (LC3, p62 and Beclin-1), overexpression of miR-26a or knock-down of ULK2 inhibits swine Sertoli cell autophagy. Taken together, these findings demonstrate that miR-26a suppresses autophagy in swine Sertoli cells by targeting ULK2. © 2018 Blackwell Verlag GmbH.

Selective tumor cell targeting by the disaccharide moiety of bleomycin.

PubMed

Yu, Zhiqiang; Schmaltz, Ryan M; Bozeman, Trevor C; Paul, Rakesh; Rishel, Michael J; Tsosie, Krystal S; Hecht, Sidney M

2013-02-27

In a recent study, the well-documented tumor targeting properties of the antitumor agent bleomycin (BLM) were studied in cell culture using microbubbles that had been derivatized with multiple copies of BLM. It was shown that BLM selectively targeted MCF-7 human breast carcinoma cells but not the "normal" breast cell line MCF-10A. Furthermore, it was found that the BLM analogue deglycobleomycin, which lacks the disaccharide moiety of BLM, did not target either cell line, indicating that the BLM disaccharide moiety is necessary for tumor selectivity. Not resolved in the earlier study were the issues of whether the BLM disaccharide moiety alone is sufficient for tumor cell targeting and the possible cellular uptake of the disaccharide. In the present study, we conjugated BLM, deglycoBLM, and BLM disaccharide to the cyanine dye Cy5**. It was found that the BLM and BLM disaccharide conjugates, but not the deglycoBLM conjugate, bound selectively to MCF-7 cells and were internalized. The same was also true for the prostate cancer cell line DU-145 (but not for normal PZ-HPV-7 prostate cells) and for the pancreatic cancer cell line BxPC-3 (but not for normal SVR A221a pancreas cells). The targeting efficiency of the disaccharide was only slightly less than that of BLM in MCF-7 and DU-145 cells and comparable to that of BLM in BxPC-3 cells. These results establish that the BLM disaccharide is both necessary and sufficient for tumor cell targeting, a finding with obvious implications for the design of novel tumor imaging and therapeutic agents.

Targeted Therapies in Non-Small Cell Lung Cancer—Beyond EGFR and ALK

PubMed Central

Rothschild, Sacha I.

2015-01-01

Systemic therapy for non-small cell lung cancer (NSCLC) has undergone a dramatic paradigm shift over the past decade. Advances in our understanding of the underlying biology of NSCLC have revealed distinct molecular subtypes. A substantial proportion of NSCLC depends on oncogenic molecular aberrations (so-called “driver mutations”) for their malignant phenotype. Personalized therapy encompasses the strategy of matching these subtypes with effective targeted therapies. EGFR mutations and ALK translocation are the most effectively targeted oncogenes in NSCLC. EGFR mutations and ALK gene rearrangements are successfully being targeted with specific tyrosine kinase inhibitors. The number of molecular subgroups of NSCLC continues to grow. The scope of this review is to discuss recent data on novel molecular targets as ROS1, BRAF, KRAS, HER2, c-MET, RET, PIK3CA, FGFR1 and DDR2. Thereby the review will focus on therapeutic strategies targeting these aberrations. Moreover, the emerging challenge of acquired resistance to initially effective therapies will be discussed. PMID:26018876

Targeted Therapies in Non-Small Cell Lung Cancer-Beyond EGFR and ALK.

PubMed

Rothschild, Sacha I

2015-05-26

Systemic therapy for non-small cell lung cancer (NSCLC) has undergone a dramatic paradigm shift over the past decade. Advances in our understanding of the underlying biology of NSCLC have revealed distinct molecular subtypes. A substantial proportion of NSCLC depends on oncogenic molecular aberrations (so-called "driver mutations") for their malignant phenotype. Personalized therapy encompasses the strategy of matching these subtypes with effective targeted therapies. EGFR mutations and ALK translocation are the most effectively targeted oncogenes in NSCLC. EGFR mutations and ALK gene rearrangements are successfully being targeted with specific tyrosine kinase inhibitors. The number of molecular subgroups of NSCLC continues to grow. The scope of this review is to discuss recent data on novel molecular targets as ROS1, BRAF, KRAS, HER2, c-MET, RET, PIK3CA, FGFR1 and DDR2. Thereby the review will focus on therapeutic strategies targeting these aberrations. Moreover, the emerging challenge of acquired resistance to initially effective therapies will be discussed.

miR-24-2 controls H2AFX expression regardless of gene copy number alteration and induces apoptosis by targeting antiapoptotic gene BCL-2: a potential for therapeutic intervention.

PubMed

Srivastava, Niloo; Manvati, Siddharth; Srivastava, Archita; Pal, Ranjana; Kalaiarasan, Ponnusamy; Chattopadhyay, Shilpi; Gochhait, Sailesh; Dua, Raina; Bamezai, Rameshwar N K

2011-04-04

New levels of gene regulation with microRNA (miR) and gene copy number alterations (CNAs) have been identified as playing a role in various cancers. We have previously reported that sporadic breast cancer tissues exhibit significant alteration in H2AX gene copy number. However, how CNA affects gene expression and what is the role of miR, miR-24-2, known to regulate H2AX expression, in the background of the change in copy number, are not known. Further, many miRs, including miR-24-2, are implicated as playing a role in cell proliferation and apoptosis, but their specific target genes and the pathways contributing to them remain unexplored. Changes in gene copy number and mRNA/miR expression were estimated using real-time polymerase chain reaction assays in two mammalian cell lines, MCF-7 and HeLa, and in a set of sporadic breast cancer tissues. In silico analysis was performed to find the putative target for miR-24-2. MCF-7 cells were transfected with precursor miR-24-2 oligonucleotides, and the gene expression levels of BRCA1, BRCA2, ATM, MDM2, TP53, CHEK2, CYT-C, BCL-2, H2AFX and P21 were examined using TaqMan gene expression assays. Apoptosis was measured by flow cytometric detection using annexin V dye. A luciferase assay was performed to confirm BCL-2 as a valid cellular target of miR-24-2. It was observed that H2AX gene expression was negatively correlated with miR-24-2 expression and not in accordance with the gene copy number status, both in cell lines and in sporadic breast tumor tissues. Further, the cells overexpressing miR-24-2 were observed to be hypersensitive to DNA damaging drugs, undergoing apoptotic cell death, suggesting the potentiating effect of mir-24-2-mediated apoptotic induction in human cancer cell lines treated with anticancer drugs. BCL-2 was identified as a novel cellular target of miR-24-2. mir-24-2 is capable of inducing apoptosis by modulating different apoptotic pathways and targeting BCL-2, an antiapoptotic gene. The study suggests

An innovative pre-targeting strategy for tumor cell specific imaging and therapy.

PubMed

Qin, Si-Yong; Peng, Meng-Yun; Rong, Lei; Jia, Hui-Zhen; Chen, Si; Cheng, Si-Xue; Feng, Jun; Zhang, Xian-Zheng

2015-09-21

A programmed pre-targeting system for tumor cell imaging and targeting therapy was established based on the "biotin-avidin" interaction. In this programmed functional system, transferrin-biotin can be actively captured by tumor cells with the overexpression of transferrin receptors, thus achieving the pre-targeting modality. Depending upon avidin-biotin recognition, the attachment of multivalent FITC-avidin to biotinylated tumor cells not only offered the rapid fluorescence labelling, but also endowed the pre-targeted cells with targeting sites for the specifically designed biotinylated peptide nano-drug. Owing to the successful pre-targeting, tumorous HepG2 and HeLa cells were effectively distinguished from the normal 3T3 cells via fluorescence imaging. In addition, the self-assembled peptide nano-drug resulted in enhanced cell apoptosis in the observed HepG2 cells. The tumor cell specific pre-targeting strategy is applicable for a variety of different imaging and therapeutic agents for tumor treatments.

Estimation of lactic acid bacterial cell number by DNA quantification.

PubMed

Ishii, Masaki; Matsumoto, Yasuhiko; Sekimizu, Kazuhisa

2018-01-01

Lactic acid bacteria are provided by fermented foods, beverages, medicines, and supplements. Because the beneficial effects of medicines and supplements containing functional lactic acid bacteria are related to the bacterial cell number, it is important to establish a simple method for estimating the total number of lactic acid bacterial cells in the products for quality control. Almost all of the lactic acid bacteria in the products are dead, however, making it difficult to estimate the total number of lactic acid bacterial cells in the products using a standard colony-counting method. Here we estimated the total lactic acid bacterial cell number in samples containing dead bacteria by quantifying the DNA. The number of viable Enterococcus faecalis 0831-07 cells decreased to less than 1 × 10 -8 by 15 min of heat treatment at 80°C. The amount of extracted DNA from heat-treated cells was 78% that of non-heated cells. The number of viable Lactobacillus paraplantarum 11-1 cells decreased to 1 × 10 -4 after 4 days culture. The amount of extracted DNA of the long-cultured cells, however, was maintained at 97%. These results suggest that cell number of lactic acid bacteria killed by heat-treatment or long-term culture can be estimated by DNA quantification.

Targeting cancer stem cell-specific markers and/or associated signaling pathways for overcoming cancer drug resistance.

PubMed

Ranji, Peyman; Salmani Kesejini, Tayyebali; Saeedikhoo, Sara; Alizadeh, Ali Mohammad

2016-10-01

Cancer stem cells (CSCs) are a small subpopulation of tumor cells with capabilities of self-renewal, dedifferentiation, tumorigenicity, and inherent chemo-and-radio therapy resistance. Tumor resistance is believed to be caused by CSCs that are intrinsically challenging to common treatments. A number of CSC markers including CD44, CD133, receptor tyrosine kinase, aldehyde dehydrogenases, epithelial cell adhesion molecule/epithelial specific antigen, and ATP-binding cassette subfamily G member 2 have been proved as the useful targets for defining CSC population in solid tumors. Furthermore, targeting CSC markers through new therapeutic strategies will ultimately improve treatments and overcome cancer drug resistance. Therefore, the identification of novel strategies to increase sensitivity of CSC markers has major clinical implications. This review will focus on the innovative treatment methods such as nano-, immuno-, gene-, and chemotherapy approaches for targeting CSC-specific markers and/or their associated signaling pathways.

Strategies to target non-T-cell HIV reservoirs.

PubMed

Sacha, Jonah B; Ndhlovu, Lishomwa C

2016-07-01

A central question for the HIV cure field is to determine new ways to target clinically relevant, latently and actively replicating HIV-infected cells beyond resting memory CD4 T cells, particularly in anatomical areas of low drug penetrability. HIV eradication strategies being positioned for targeting HIV for extinction in the CD4 T-cell compartment may also show promise in non-CD4 T-cells reservoirs. Furthermore, several exciting novel therapeutic approaches specifically focused on HIV clearance from non-CD4 T-cell populations are being developed. Although reservoir validity in these non-CD4 T cells continues to remain debated, this review will highlight recent advances and make an argument as to their clinical relevancy as we progress towards an HIV cure.

Gene Targets in Prostate Tumor Cells that Mediate Aberrant Growth and Invasiveness

DTIC Science & Technology

2005-02-01

Craig A. Hauser , Ph.D. Gabriele Foos, Ph.D. CONTRACTING ORGANIZATION: The Burnham Institute La Jolla, California 92037 REPORT DATE: February 2005 TYPE...NUMBERS Gene Targets in Prostate Tumor Cells that Mediate DAMD17-02-1-0019 Aberrant Growth and Invasiveness 6. AUTHOR(S) Craig A. Hauser , Ph.D. Gabriele...REPORTABLE OUTCOMES Foos G, Hauser CA (2004) The role of Ets transcription factors in mediating cellular transformation. In: Handbook of Experimental

MOLECULARLY TARGETED THERAPIES IN NON-SMALL CELL LUNG CANCER ANNUAL UPDATE 2014

PubMed Central

Morgensztern, Daniel; Campo, Meghan J.; Dahlberg, Suzanne E.; Doebele, Robert C.; Garon, Edward; Gerber, David E.; Goldberg, Sarah B.; Hammerman, Peter S.; Heist, Rebecca; Hensing, Thomas; Horn, Leora; Ramalingam, Suresh S.; Rudin, Charles M.; Salgia, Ravi; Sequist, Lecia; Shaw, Alice T.; Simon, George R.; Somaiah, Neeta; Spigel, David R.; Wrangle, John; Johnson, David; Herbst, Roy S.; Bunn, Paul; Govindan, Ramaswamy

2015-01-01

There have been significant advances in the understanding of the biology and treatment of non-small cell lung cancer (NSCLC) over the past few years. A number of molecularly targeted agents are in the clinic or in development for patients with advanced NSCLC (Table 1). We are beginning to understand the mechanisms of acquired resistance following exposure to tyrosine kinase inhibitors in patients with oncogene addicted NSCLC. The advent of next generation sequencing has enabled to study comprehensively genomic alterations in lung cancer. Finally, early results from immune checkpoint inhibitors are very encouraging. This review summarizes recent advances in the area of cancer genomics, targeted therapies and immunotherapy. PMID:25535693

Effectors of root sedentary nematodes target diverse plant cell compartments to manipulate plant functions and promote infection.

PubMed

Jaouannet, Maëlle; Rosso, Marie-Noëlle

2013-09-01

Sedentary plant-parasitic nematodes maintain a biotrophic relationship with their hosts over a period of several weeks and induce the differentiation of root cells into specialized feeding cells. Nematode effectors, which are synthesized in the esophageal glands and injected into the plant tissue through the syringe-like stylet, play a central role in these processes. Previous work on nematode effectors has shown that the apoplasm is targeted during invasion of the host while the cytoplasm is targeted during the induction and the maintenance of the feeding site. A large number of candidate effectors potentially secreted by the nematode into the plant tissues to promote infection have now been identified. This work has shown that the targeting and the role of effectors are more complex than previously thought. This review will not cover the prolific recent findings in nematode effector function but will instead focus on recent selected examples that illustrate the variety of plant cell compartments that effectors are addressed to in order reach their plant targets.

Targeted gene therapy and cell reprogramming in Fanconi anemia.

PubMed

Rio, Paula; Baños, Rocio; Lombardo, Angelo; Quintana-Bustamante, Oscar; Alvarez, Lara; Garate, Zita; Genovese, Pietro; Almarza, Elena; Valeri, Antonio; Díez, Begoña; Navarro, Susana; Torres, Yaima; Trujillo, Juan P; Murillas, Rodolfo; Segovia, Jose C; Samper, Enrique; Surralles, Jordi; Gregory, Philip D; Holmes, Michael C; Naldini, Luigi; Bueren, Juan A

2014-06-01

Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

Potential targets for lung squamous cell carcinoma

Cancer.gov

Researchers have identified potential therapeutic targets in lung squamous cell carcinoma, the second most common form of lung cancer. The Cancer Genome Atlas (TCGA) Research Network study comprehensively characterized the lung squamous cell carcinoma gen

The Clinical Potential of Targeted Nanomedicine: Delivering to Cancer Stem-like Cells

PubMed Central

Kim, Sang-Soo; Rait, Antonina; Rubab, Farwah; Rao, Abhi K; Kiritsy, Michael C; Pirollo, Kathleen F; Wang, Shangzi; Weiner, Louis M; Chang, Esther H

2014-01-01

Cancer stem-like cells (CSCs) have been implicated in recurrence and treatment resistance in many human cancers. Thus, a CSC-targeted drug delivery strategy to eliminate CSCs is a desirable approach for developing a more effective anticancer therapy. We have developed a tumor-targeting nanodelivery platform (scL) for systemic administration of molecular medicines. Following treatment with the scL nanocomplex carrying various payloads, we have observed exquisite tumor-targeting specificity and significant antitumor response with long-term survival benefit in numerous animal models. We hypothesized that this observed efficacy might be attributed, at least in part, to elimination of CSCs. Here, we demonstrate the ability of scL to target both CSCs and differentiated nonstem cancer cells (non-CSCs) in various mouse models including subcutaneous and intracranial xenografts, syngeneic, and chemically induced tumors. We also show that systemic administration of scL carrying the wtp53 gene was able to induce tumor growth inhibition and the death of both CSCs and non-CSCs in subcutaneous colorectal cancer xenografts suggesting that this could be an effective method to reduce cancer recurrence and treatment resistance. This scL nanocomplex is being evaluated in a number of clinical trials where it has been shown to be well tolerated with indications of anticancer activity. PMID:24113515

Cell cycle proteins as promising targets in cancer therapy.

PubMed

Otto, Tobias; Sicinski, Piotr

2017-01-27

Cancer is characterized by uncontrolled tumour cell proliferation resulting from aberrant activity of various cell cycle proteins. Therefore, cell cycle regulators are considered attractive targets in cancer therapy. Intriguingly, animal models demonstrate that some of these proteins are not essential for proliferation of non-transformed cells and development of most tissues. By contrast, many cancers are uniquely dependent on these proteins and hence are selectively sensitive to their inhibition. After decades of research on the physiological functions of cell cycle proteins and their relevance for cancer, this knowledge recently translated into the first approved cancer therapeutic targeting of a direct regulator of the cell cycle. In this Review, we focus on proteins that directly regulate cell cycle progression (such as cyclin-dependent kinases (CDKs)), as well as checkpoint kinases, Aurora kinases and Polo-like kinases (PLKs). We discuss the role of cell cycle proteins in cancer, the rationale for targeting them in cancer treatment and results of clinical trials, as well as the future therapeutic potential of various cell cycle inhibitors.

Identification of Wnt Pathway Target Genes Regulating the Division and Differentiation of Larval Seam Cells and Vulval Precursor Cells in Caenorhabditis elegans.

PubMed

Gorrepati, Lakshmi; Krause, Michael W; Chen, Weiping; Brodigan, Thomas M; Correa-Mendez, Margarita; Eisenmann, David M

2015-06-05

The evolutionarily conserved Wnt/β-catenin signaling pathway plays a fundamental role during metazoan development, regulating numerous processes including cell fate specification, cell migration, and stem cell renewal. Wnt ligand binding leads to stabilization of the transcriptional effector β-catenin and upregulation of target gene expression to mediate a cellular response. During larval development of the nematode Caenorhabditis elegans, Wnt/β-catenin pathways act in fate specification of two hypodermal cell types, the ventral vulval precursor cells (VPCs) and the lateral seam cells. Because little is known about targets of the Wnt signaling pathways acting during larval VPC and seam cell differentiation, we sought to identify genes regulated by Wnt signaling in these two hypodermal cell types. We conditionally activated Wnt signaling in larval animals and performed cell type-specific "mRNA tagging" to enrich for VPC and seam cell-specific mRNAs, and then used microarray analysis to examine gene expression compared to control animals. Two hundred thirty-nine genes activated in response to Wnt signaling were identified, and we characterized 50 genes further. The majority of these genes are expressed in seam and/or vulval lineages during normal development, and reduction of function for nine genes caused defects in the proper division, fate specification, fate execution, or differentiation of seam cells and vulval cells. Therefore, the combination of these techniques was successful at identifying potential cell type-specific Wnt pathway target genes from a small number of cells and at increasing our knowledge of the specification and behavior of these C. elegans larval hypodermal cells. Copyright © 2015 Gorrepati et al.

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.

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.

Folate-conjugated immunoglobulin targets melanoma tumor cells for NK cell effector functions

PubMed Central

Skinner, Cassandra C.; McMichael, Elizabeth L.; Jaime-Ramirez, Alena C.; Abrams, Zachary B.; Lee, Robert J.; Carson, William E.

2016-01-01

The folate receptor (FR) is over-expressed on the vascular side of cancerous cells including those of the breast, ovaries, testes, and cervix. We hypothesized that a folate-conjugated immunoglobulin (F-IgG) would bind to the FR that is over-expressed on melanoma tumor cells to target these cells for lysis by natural killer (NK) cells. Folate receptor expression was confirmed in the Mel-39 (human melanoma) cell line by flow cytometry and immunoblot analysis, using KB (human oral epithelial) and F01 (human melanoma) as a positive and negative control, respectively. FR-positive and negative cell lines were treated with F-IgG or control immunoglobulin G (C-IgG) in the presence or absence of cytokines in order to determine NK cell ability to lyse FR-positive cell lines. NK cell activation was significantly upregulated and lysis of Mel 39 tumor cells enhanced following treatment with F-IgG, as compared to C-IgG at all effector:target (E:T) ratios (p<0.01). This trend was further enhanced by NK cell stimulation with the activating cytokine interleukin-12 (IL-12). NK cell production of cytokines such as interferon-gamma (IFN-γ), macrophage inflammatory protein 1 alpha (MIP-1α), and regulated on activation normal T-cell expressed and secreted (RANTES) were also significantly increased in response to co-stimulation with IL-12 stimulation and F-IgG-coated Mel 39 target cells, as compared to controls (p<0.01). In contrast, F-IgG did not bind to the FR-negative cell line F01 and had no significant effect on NK cell lysis or cytokine production. This research indicates the potential use of F-IgG for its ability to induce an immune response from NK cells against FR-positive melanoma tumor cells which can be further enhanced by the addition of cytokines. PMID:27035691

Identification of Wnt Pathway Target Genes Regulating the Division and Differentiation of Larval Seam Cells and Vulval Precursor Cells in Caenorhabditis elegans

PubMed Central

Gorrepati, Lakshmi; Krause, Michael W.; Chen, Weiping; Brodigan, Thomas M.; Correa-Mendez, Margarita; Eisenmann, David M.

2015-01-01

The evolutionarily conserved Wnt/β-catenin signaling pathway plays a fundamental role during metazoan development, regulating numerous processes including cell fate specification, cell migration, and stem cell renewal. Wnt ligand binding leads to stabilization of the transcriptional effector β-catenin and upregulation of target gene expression to mediate a cellular response. During larval development of the nematode Caenorhabditis elegans, Wnt/β-catenin pathways act in fate specification of two hypodermal cell types, the ventral vulval precursor cells (VPCs) and the lateral seam cells. Because little is known about targets of the Wnt signaling pathways acting during larval VPC and seam cell differentiation, we sought to identify genes regulated by Wnt signaling in these two hypodermal cell types. We conditionally activated Wnt signaling in larval animals and performed cell type–specific "mRNA tagging" to enrich for VPC and seam cell–specific mRNAs, and then used microarray analysis to examine gene expression compared to control animals. Two hundred thirty-nine genes activated in response to Wnt signaling were identified, and we characterized 50 genes further. The majority of these genes are expressed in seam and/or vulval lineages during normal development, and reduction of function for nine genes caused defects in the proper division, fate specification, fate execution, or differentiation of seam cells and vulval cells. Therefore, the combination of these techniques was successful at identifying potential cell type–specific Wnt pathway target genes from a small number of cells and at increasing our knowledge of the specification and behavior of these C. elegans larval hypodermal cells. PMID:26048561

MicroRNA-203 Induces Apoptosis by Targeting Bmi-1 in YD-38 Oral Cancer Cells.

PubMed

Kim, Jae-Sung; Choi, Dae Woo; Kim, Chun Sung; Yu, Sun-Kyoung; Kim, Heung-Joong; Go, Dae-San; Lee, Seul Ah; Moon, Sung Min; Kim, Su Gwan; Chun, Hong Sung; Kim, Jeongsun; Kim, Jong-Keun; Kim, DO Kyung

2018-06-01

MicroRNAs (miRNAs) are closely associated with a number of cellular processes, including cell development, differentiation, proliferation, carcinogenesis, and apoptosis. The aim of the present study was to elucidate the molecular mechanisms underlying the tumor suppressor activity of miRNA-203 (miR-203) in YD-38 human oral cancer cells. Polymerase chain reaction analysis, MTT assay, DNA fragmentation assay, fluorescence-activated cell-sorting analysis, gene array, immunoblotting, and luciferase assay were carried out in YD-38 cells. miR-203 expression was significantly down-regulated in YD-38 cells compared to expression levels in normal human oral keratinocytes. miR-203 decreased the viability of YD-38 cells in a time- and dose-dependent manner. In addition, over-expression of miR-203 significantly increased not only DNA segmentation, but also the apoptotic population of YD-38 cells. These results indicate that miR-203 overexpression induces apoptosis in YD-38 cells. Target gene array analysis revealed that the expression of the polycomb complex protein gene Bmi-1, a representative oncogene, was significantly down-regulated by miR-203 in YD-38 cells. Moreover, both mRNA and protein levels of Bmi-1 were significantly reduced in YD-38 cells transfected with miR-203. These results indicate that Bmi-1 is a target gene of miR-203. A luciferase reporter assay confirmed that miR-203 suppressed Bmi-1 expression by directly targeting the 3'-untranslated region. miR-203 induces apoptosis in YD-38 cells by directly targeting Bmi-1, which suggests its possible application as an anti-cancer therapeutic. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Gene targeting in mosquito cells: a demonstration of 'knockout' technology in extrachromosomal gene arrays

PubMed Central

Eggleston, Paul; Zhao, Yuguang

2001-01-01

Background Gene targeting would offer a number of advantages over current transposon-based strategies for insect transformation. These include freedom from both position effects associated with quasi-random integration and concerns over transgene instability mediated by endogenous transposases, independence from phylogenetic restrictions on transposon mobility and the ability to generate gene knockouts. Results We describe here our initial investigations of gene targeting in the mosquito. The target site was a hygromycin resistance gene, stably maintained as part of an extrachromosomal array. Using a promoter-trap strategy to enrich for targeted events, a neomycin resistance gene was integrated into the target site. This resulted in knockout of hygromycin resistance concurrent with the expression of high levels of neomycin resistance from the resident promoter. PCR amplification of the targeted site generated a product that was specific to the targeted cell line and consistent with precise integration of the neomycin resistance gene into the 5' end of the hygromycin resistance gene. Sequencing of the PCR product and Southern analysis of cellular DNA subsequently confirmed this molecular structure. Conclusions These experiments provide the first demonstration of gene targeting in mosquito tissue and show that mosquito cells possess the necessary machinery to bring about precise integration of exogenous sequences through homologous recombination. Further development of these procedures and their extension to chromosomally located targets hold much promise for the exploitation of gene targeting in a wide range of medically and economically important insect species. PMID:11513755

Curcumin: a promising agent targeting cancer stem cells.

PubMed

Zang, Shufei; Liu, Tao; Shi, Junping; Qiao, Liang

2014-01-01

Cancer stem cells are a subset of cells that are responsible for cancer initiation and relapse. They are generally resistant to the current anticancer agents. Successful anticancer therapy must consist of approaches that can target not only the differentiated cancer cells, but also cancer stem cells. Emerging evidence suggested that the dietary agent curcumin exerted its anti-cancer activities via targeting cancer stem cells of various origins such as those of colorectal cancer, pancreatic cancer, breast cancer, brain cancer, and head and neck cancer. In order to enhance the therapeutic potential of curcumin, this agent has been modified or used in combination with other agents in the experimental therapy for many cancers. In this mini-review, we discussed the effect of curcumin and its derivatives in eliminating cancer stem cells and the possible underlying mechanisms.

The mechanism of T-cell mediated cytotoxicity. VI. T-cell projections and their role in target cell killing.

PubMed Central

Sanderson, C J; Glauert, A M

1979-01-01

Electron micrographs of material fixed during the first 10 min of a T-cell cytotoxic system showed T-cell projections and T-cell burrowing into target cells. These observations were made possible by using a system with a very high rate of killing. The projections vary in shape and size, and can push deeply into the target cell, distorting organelles in their path, including the nucleus. The projections contain fine fibrillar material, to the exclusion of organelles. They push the target cell membrane in front of them to form pockets approximating to the shape of the projection. Areas of close contact occur between the projections and the target cell membrane, particularly at the leading edges. The likelihood that these projections develop as a result of contact with specific antigen, and are involved in the cytotoxic mechanism is discussed. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 PMID:311336

Targeting survival pathways in chronic myeloid leukaemia stem cells

PubMed Central

Sinclair, A; Latif, A L; Holyoake, T L

2013-01-01

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder characterized by the presence of a fusion oncogene BCR-ABL, which encodes a protein with constitutive TK activity. The implementation of tyrosine kinase inhibitors (TKIs) marked a major advance in CML therapy; however, there are problems with current treatment. For example, relapse occurs when these drugs are discontinued in the majority of patients who have achieved a complete molecular response on TKI and these agents are less effective in patients with mutations in the BCR-ABL kinase domain. Importantly, TKI can effectively target proliferating mature cells, but do not eradicate quiescent leukaemic stem cells (LSCs), therefore allowing disease persistence despite treatment. It is essential that alternative strategies are used to target the LSC population. BCR-ABL activation is responsible for the modulation of different signalling pathways, which allows the LSC fraction to evade cell death. Several pathways have been shown to be modulated by BCR-ABL, including PI3K/AKT/mTOR, JAK-STAT and autophagy signalling pathways. Targeting components of these survival pathways, alone or in combination with TKI, therefore represents an attractive potential therapeutic approach for targeting the LSC. However, many pathways are also active in normal stem cells. Therefore, potential targets must be validated to effectively eradicate CML stem cells while sparing normal counterparts. This review summarizes the main pathways modulated in CML stem cells, the recent developments and the use of novel drugs to target components in these pathways which may be used to target the LSC population. Linked Articles This article is part of a themed section on Emerging Therapeutic Aspects in Oncology. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-8 PMID:23517124

Tumor-targeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning

PubMed Central

Pegram, Hollie J.; Lee, James C.; Hayman, Erik G.; Imperato, Gavin H.; Tedder, Thomas F.; Sadelain, Michel

2012-01-01

Adoptive cell therapy with tumor-targeted T cells is a promising approach to cancer therapy. Enhanced clinical outcome using this approach requires conditioning regimens with total body irradiation, lymphodepleting chemotherapy, and/or additional cytokine support. However, the need for prior conditioning precludes optimal application of this approach to a significant number of cancer patients intolerant to these regimens. Herein, we present preclinical studies demonstrating that treatment with CD19-specific, chimeric antigen receptor (CAR)–modified T cells that are further modified to constitutively secrete IL-12 are able to safely eradicate established disease in the absence of prior conditioning. We demonstrate in a novel syngeneic tumor model that tumor elimination requires both CD4+ and CD8+ T-cell subsets, autocrine IL-12 stimulation, and subsequent IFNγ secretion by the CAR+ T cells. Importantly, IL-12–secreting, tumor-targeted T cells acquire intrinsic resistance to T regulatory cell–mediated inhibition. Based on these preclinical data, we anticipate that adoptive therapy using CAR-targeted T cells modified to secrete IL-12 will obviate or reduce the need for potentially hazardous conditioning regimens to achieve optimal antitumor responses in cancer patients. PMID:22354001

Breast cancer stem cells, EMT and therapeutic targets

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

Kotiyal, Srishti; Bhattacharya, Susinjan, E-mail: s.bhattacharya@jiit.ac.in

Highlights: • Therapeutic targeting or inhibition of the key molecules of signaling pathways can control growth of breast cancer stem cells (BCSCs). • Development of BCSCs also involves miRNA interactions. • Therapeutic achievement can be done by targeting identified targets in the BCSC pathways. - Abstract: A small heterogeneous population of breast cancer cells acts as seeds to induce new tumor growth. These seeds or breast cancer stem cells (BCSCs) exhibit great phenotypical plasticity which allows them to undergo “epithelial to mesenchymal transition” (EMT) at the site of primary tumor and a future reverse transition. Apart from metastasis they aremore » also responsible for maintaining the tumor and conferring it with drug and radiation resistance and a tendency for post-treatment relapse. Many of the signaling pathways involved in induction of EMT are involved in CSC generation and regulation. Here we are briefly reviewing the mechanism of TGF-β, Wnt, Notch, TNF-α, NF-κB, RTK signalling pathways which are involved in EMT as well as BCSCs maintenance. Therapeutic targeting or inhibition of the key/accessory players of these pathways could control growth of BCSCs and hence malignant cancer. Additionally several miRNAs are dysregulated in cancer stem cells indicating their roles as oncogenes or tumor suppressors. This review also lists the miRNA interactions identified in BCSCs and discusses on some newly identified targets in the BCSC regulatory pathways like SHIP2, nicastrin, Pin 1, IGF-1R, pro-inflammatory cytokines and syndecan which can be targeted for therapeutic achievements.« less

Killing of targets by effector CD8 T cells in the mouse spleen follows the law of mass action

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

Ganusov, Vitaly V

2009-01-01

In contrast with antibody-based vaccines, it has been difficult to measure the efficacy of T cell-based vaccines and to correlate the efficacy of CD8 T cell responses with protection again viral infections. In part, this difficulty is due to poor understanding of the in vivo efficacy of CD8 T cells produced by vaccination. Using a: recently developed experimental method of in vivo cytotoxicity we have investigated quantitative aspects of killing of peptide-pulsed targets by effector and memory CD8 T cells, specific to three epitopes of lymphocytic choriomeningitis virus (LCMV), in the mouse spleen. By analyzing data on killing of targetsmore » with varying number of epitope-specific effector and memory CD8 T cells, we find that killing of targets by effectors follows the law of mass-action, that is the death rate of peptide-pulsed targets is proportional to the frequency of CTLs in the spleen. In contrast, killing of targets by memory CD8 T cells does not follow the mass action law because the death rate of targets saturates at high frequencies of memory CD8 T cells. For both effector and memory cells, we also find little support for the killing term that includes the decrease of the death rate of targets with target cell density. Interestingly, our analysis suggests that at low CD8 T cell frequencies, memory CD8 T cells on the per capita basis are more efficient at killing peptide-pulsed targets than effectors, but at high frequencies, effectors are more efficient killers than memory T cells. Comparison of the estimated killing efficacy of effector T cells with the value that is predicted from theoretical physics and based on motility of T cells in lymphoid tissues, suggests that limiting step in the killing of peptide-pulsed targets is delivering the lethal hit and not finding the target. Our results thus form a basis for quantitative understanding of the process of killing of virus-infected cells by T cell responses in tissues and can be used to correlate the

Enhancement of dendritic cell-based vaccine potency by anti-apoptotic siRNAs targeting key pro-apoptotic proteins in cytotoxic CD8(+) T cell-mediated cell death.

PubMed

Kim, Jin Hee; Kang, Tae Heung; Noh, Kyung Hee; Bae, Hyun Cheol; Kim, Seok-Ho; Yoo, Young Do; Seong, Seung-Yong; Kim, Tae Woo

2009-01-29

Dendritic cells (DCs) have become an important measure for the treatment of malignancies. Current DC preparations, however, generate short-lived DCs because they are subject to cell death from various apoptotic pressures. Antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) is one of the main obstacles to limit the DC-mediated immune priming since CTLs can recognize the target antigen expressing DCs as target cells and kill the DCs. CTLs secret perforin and serine protease granzymes during CTL killing. Perforin and serine protease granzymes induce the release of a number of mitochondrial pro-apoptotic factors, which are controlled by members of the BCL-2 family, such as BAK, BAX and BIM. FasL linking to Fas on DCs triggers the activation of caspase-8, which eventually leads to mitochondria-mediated apoptosis via truncation of BID. In this study, we tried to enhance the DC priming capacity by prolonging DC survival using anti-apoptotic siRNA targeting these key pro-apoptotic molecules in CTL killing. Human papillomavirus (HPV)-16 E7 antigen presenting DCs that were transfected with these anti-apoptotic siRNAs showed increased resistance to T cell-mediated death, leading to enhanced E7-specific CD8(+) T cell activation in vitro and in vivo. Among them, siRNA targeting BIM (siBIM) generated strongest E7-specific E7-specific CD8(+) T cell immunity. More importantly, vaccination with E7 presenting DCs transfected with siBIM was capable of generating a marked therapeutic effect in vaccinated mice. Our data indicate that ex vivo manipulation of DCs with siBIM may represent a plausible strategy for enhancing dendritic cell-based vaccine potency.

Targeting NCK-Mediated Endothelial Cell Front-Rear Polarity Inhibits Neo-Vascularization

PubMed Central

Dubrac, Alexandre; Genet, Gael; Ola, Roxana; Zhang, Feng; Pibouin-Fragner, Laurence; Han, Jinah; Zhang, Jiasheng; Thomas, Jean-Léon; Chedotal, Alain; Schwartz, Martin A.; Eichmann, Anne

2015-01-01

Background Sprouting angiogenesis is a key process driving blood vessel growth in ischemic tissues and an important drug target in a number of diseases, including wet macular degeneration and wound healing. Endothelial cells forming the sprout must develop front-rear polarity to allow sprout extension. The adaptor proteins Nck1 and 2 are known regulators of cytoskeletal dynamics and polarity, but their function in angiogenesis is poorly understood. Here we show that the Nck adaptors are required for endothelial cell front-rear polarity and migration downstream of the angiogenic growth factors VEGF-A and Slit2. Methods and Results Mice carrying inducible, endothelial-specific Nck1/2 deletions fail to develop front-rear polarized vessel sprouts and exhibit severe angiogenesis defects in the postnatal retina and during embryonic development. Inactivation of NCK1 and 2 inhibits polarity by preventing Cdc42 and Pak2 activation by VEGF-A and Slit2. Mechanistically, NCK binding to ROBO1 is required for both Slit2 and VEGF induced front-rear polarity. Selective inhibition of polarized endothelial cell migration by targeting Nck1/2 prevents hypersprouting induced by Notch or Bmp signaling inhibition, as well as pathological ocular neovascularization and wound healing. Conclusions These data reveal a novel signal integration mechanism involving NCK1/2, ROBO1/2 and VEGFR2 that controls endothelial cell front-rear polarity during sprouting angiogenesis. PMID:26659946

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

MiR-615 inhibits cell proliferation, migration and invasion by targeting EGFR in human glioblastoma.

PubMed

Ji, Yanwei; Sun, Qingshan; Zhang, Jianbin; Hu, Haoran

2018-05-15

MiR-615 and epidermal growth factor receptor (EGFR) are associated with a number of disease processes and pathogenesis. However, little is known about the mechanisms of miR-615 and EGFR in human glioblastoma multiforme (GBM). Here, we found that down-regulation of miR-615 expression occurred in GBM tissues and cells, and was inversely correlated with overall survival, relapse-free survival, WHO grade as well as EGFR expression. We further determined that miR-615 functions as a tumor suppressor by inhibiting GBM cell proliferation, cell cycle, migration and invasion, and promoting cell apoptosis. In-vivo assay validated the inhibition effect of miR-615 on tumor growth and EGFR expression. Luciferase reporter assays demonstrated that miR-615 targeted the 3'-untranslated region (3'-UTR) of EGFR. Besides, over-expression of EGFR reversed the inhibition effects of miR-615, while silencing of EGFR aggravated these inhibition effects. In conclusions, we identified that miR-615 plays a tumor suppressor role in GBM cell proliferation, migration and invasion by targeting EGFR expression, and miR-615 may act as a novel biomarker for early diagnosis or therapeutic targets of GBM. Copyright © 2018 Elsevier Inc. All rights reserved.

microRNA-150 Regulates Mobilization and Migration of Bone Marrow-Derived Mononuclear Cells by Targeting Cxcr4

PubMed Central

Tano, Nobuko; Kim, Ha Won; Ashraf, Muhammad

2011-01-01

The interaction between chemokine receptor type 4 (CXCR4) and its ligand, stromal cell-derived factor (SDF)-1, plays an important role in stem cell mobilization and migration in ischemic tissues. MicroRNAs (miRs) are key regulators of stem cell function and are involved in regulation of stem cell survival and differentiation to adopt different cell lineages. In this study, we show that ischemia inhibits the expression of miR-150 in BM-derived mononuclear cells (MNC) and activates its target Cxcr4 gene. Our results show that miR-150/CXCR4 cascade enhances MNC mobilization and migration. By using mouse acute myocardial infarction (MI) model, we found that MNCs in peripheral blood (PB) were increased significantly at day 5 after AMI as compared to control group and the number of CXCR4 positive MNCs both in bone marrow (BM) and PB was also markedly increased after MI. Analysis by microarray-based miRNA profiling and real-time PCR revealed that the expression of miR-150 which targets Cxcr4 gene as predicted was significantly downregulated in BM-MNCs after MI. Abrogation of miR-150 markedly increased CXCR4 protein expression suggesting its target gene. To show that miR-150 regulates MNC mobilization, knockdown of miR-150 in BM-MNCs by specific antisense inhibitor resulted in their higher migration ability in vitro as compared to scramble-transfected MNCs. Furthermore, in vivo BM transplantation of MNCs lacking miR-150 expression by lentiviral vector into the irradiated wild type mice resulted in the increased number of MNCs in PB after AMI as compared to control. In conclusion, this study demonstrates that ischemia mobilizes BM stem cells via miR-150/CXCR4 dependent mechanism and miR-150 may be a novel therapeutic target for stem cell migration to the ischemic tissue for neovascularization and repair. PMID:22039399

The Mechanism of Gene Targeting in Human Somatic Cells

PubMed Central

Kan, Yinan; Ruis, Brian; Lin, Sherry; Hendrickson, Eric A.

2014-01-01

Gene targeting in human somatic cells is of importance because it can be used to either delineate the loss-of-function phenotype of a gene or correct a mutated gene back to wild-type. Both of these outcomes require a form of DNA double-strand break (DSB) repair known as homologous recombination (HR). The mechanism of HR leading to gene targeting, however, is not well understood in human cells. Here, we demonstrate that a two-end, ends-out HR intermediate is valid for human gene targeting. Furthermore, the resolution step of this intermediate occurs via the classic DSB repair model of HR while synthesis-dependent strand annealing and Holliday Junction dissolution are, at best, minor pathways. Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector. Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process. Finally, we demonstrate that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations significantly advance our understanding of HR and gene targeting in human cells. PMID:24699519

Targeted cellular ablation based on the morphology of malignant cells

NASA Astrophysics Data System (ADS)

Ivey, Jill W.; Latouche, Eduardo L.; Sano, Michael B.; Rossmeisl, John H.; Davalos, Rafael V.; Verbridge, Scott S.

2015-11-01

Treatment of glioblastoma multiforme (GBM) is especially challenging due to a shortage of methods to preferentially target diffuse infiltrative cells, and therapy-resistant glioma stem cell populations. Here we report a physical treatment method based on electrical disruption of cells, whose action depends strongly on cellular morphology. Interestingly, numerical modeling suggests that while outer lipid bilayer disruption induced by long pulses (~100 μs) is enhanced for larger cells, short pulses (~1 μs) preferentially result in high fields within the cell interior, which scale in magnitude with nucleus size. Because enlarged nuclei represent a reliable indicator of malignancy, this suggested a means of preferentially targeting malignant cells. While we demonstrate killing of both normal and malignant cells using pulsed electric fields (PEFs) to treat spontaneous canine GBM, we proposed that properly tuned PEFs might provide targeted ablation based on nuclear size. Using 3D hydrogel models of normal and malignant brain tissues, which permit high-resolution interrogation during treatment testing, we confirmed that PEFs could be tuned to preferentially kill cancerous cells. Finally, we estimated the nuclear envelope electric potential disruption needed for cell death from PEFs. Our results may be useful in safely targeting the therapy-resistant cell niches that cause recurrence of GBM tumors.

Targeted cellular ablation based on the morphology of malignant cells

PubMed Central

Ivey, Jill W.; Latouche, Eduardo L.; Sano, Michael B.; Rossmeisl, John H.; Davalos, Rafael V.; Verbridge, Scott S.

2015-01-01

Treatment of glioblastoma multiforme (GBM) is especially challenging due to a shortage of methods to preferentially target diffuse infiltrative cells, and therapy-resistant glioma stem cell populations. Here we report a physical treatment method based on electrical disruption of cells, whose action depends strongly on cellular morphology. Interestingly, numerical modeling suggests that while outer lipid bilayer disruption induced by long pulses (~100 μs) is enhanced for larger cells, short pulses (~1 μs) preferentially result in high fields within the cell interior, which scale in magnitude with nucleus size. Because enlarged nuclei represent a reliable indicator of malignancy, this suggested a means of preferentially targeting malignant cells. While we demonstrate killing of both normal and malignant cells using pulsed electric fields (PEFs) to treat spontaneous canine GBM, we proposed that properly tuned PEFs might provide targeted ablation based on nuclear size. Using 3D hydrogel models of normal and malignant brain tissues, which permit high-resolution interrogation during treatment testing, we confirmed that PEFs could be tuned to preferentially kill cancerous cells. Finally, we estimated the nuclear envelope electric potential disruption needed for cell death from PEFs. Our results may be useful in safely targeting the therapy-resistant cell niches that cause recurrence of GBM tumors. PMID:26596248

Recombinant cell lines expressing shRNA targeting herpes simplex virus 2 VP16 inhibit virus replication.

PubMed

Zhang, Rui; Wang, Yan; Song, Bo; Han, Zhi Qiang; Xu, Yu Ming

2012-01-01

To establish HSV2 VP16 targeting shRNA-expressing cell lines and investigate the antiviral effect of shRNA targeting HSV2 VP16. The cell lines Vero-shRNAs and negative-control Vero-shCON were established. Their inhibition effects on VP16 mRNA expression were tested by real-time fluorescent quantitative polymerase chain reaction (PCR) and their antiviral effects were evaluated by yield reduction assay. The influence of passage numbers on the inhibition ability of cell lines was researched. Vero-shRNA24 targeting the upper stream, Vero-shRNA642 targeting the lower stream and Vero-shCON were established. Vero-shRNA24, Vero-shRNA642 and Vero-shRNA24 + 642 could reduce the VP16 mRNA significantly. Vero-shRNA24 was the most efficient. The HSV2 titers in Vero and Vero-shCON were the highest at 72 h after infection, and started decreasing thereafter. The viral titers of the Vero-shRNA groups reached a peak after 84 h and the highest titers were lower than in the Vero group. The inhibiting effect on VP16 mRNA expression and viral replication of Vero-shRNA24 cell lines of passages 10 and 20 were not significantly different from the primary cell line. Although of no statistical significance, the passage 50 cell line showed decreased inhibiting ability. Recombinant cell lines expressing shRNA targeting HSV2 VP16 were established. They can stably inhibit HSV2 VP16 mRNA expression and viral replication within passage 50. Copyright © 2012 S. Karger AG, Basel.

Pro-Tumoral Inflammatory Myeloid Cells as Emerging Therapeutic Targets.

PubMed

Szebeni, Gabor J; Vizler, Csaba; Nagy, Lajos I; Kitajka, Klara; Puskas, Laszlo G

2016-11-23

Since the observation of Virchow, it has long been known that the tumor microenvironment constitutes the soil for the infiltration of inflammatory cells and for the release of inflammatory mediators. Under certain circumstances, inflammation remains unresolved and promotes cancer development. Here, we review some of these indisputable experimental and clinical evidences of cancer related smouldering inflammation. The most common myeloid infiltrate in solid tumors is composed of myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). These cells promote tumor growth by several mechanisms, including their inherent immunosuppressive activity, promotion of neoangiogenesis, mediation of epithelial-mesenchymal transition and alteration of cellular metabolism. The pro-tumoral functions of TAMs and MDSCs are further enhanced by their cross-talk offering a myriad of potential anti-cancer therapeutic targets. We highlight these main pro-tumoral mechanisms of myeloid cells and give a general overview of their phenotypical and functional diversity, offering examples of possible therapeutic targets. Pharmacological targeting of inflammatory cells and molecular mediators may result in therapies improving patient condition and prognosis. Here, we review experimental and clinical findings on cancer-related inflammation with a major focus on creating an inventory of current small molecule-based therapeutic interventions targeting cancer-related inflammatory cells: TAMs and MDSCs.

ISDD: A computational model of particle sedimentation, diffusion and target cell dosimetry for in vitro toxicity studies

PubMed Central

2010-01-01

Background The difficulty of directly measuring cellular dose is a significant obstacle to application of target tissue dosimetry for nanoparticle and microparticle toxicity assessment, particularly for in vitro systems. As a consequence, the target tissue paradigm for dosimetry and hazard assessment of nanoparticles has largely been ignored in favor of using metrics of exposure (e.g. μg particle/mL culture medium, particle surface area/mL, particle number/mL). We have developed a computational model of solution particokinetics (sedimentation, diffusion) and dosimetry for non-interacting spherical particles and their agglomerates in monolayer cell culture systems. Particle transport to cells is calculated by simultaneous solution of Stokes Law (sedimentation) and the Stokes-Einstein equation (diffusion). Results The In vitro Sedimentation, Diffusion and Dosimetry model (ISDD) was tested against measured transport rates or cellular doses for multiple sizes of polystyrene spheres (20-1100 nm), 35 nm amorphous silica, and large agglomerates of 30 nm iron oxide particles. Overall, without adjusting any parameters, model predicted cellular doses were in close agreement with the experimental data, differing from as little as 5% to as much as three-fold, but in most cases approximately two-fold, within the limits of the accuracy of the measurement systems. Applying the model, we generalize the effects of particle size, particle density, agglomeration state and agglomerate characteristics on target cell dosimetry in vitro. Conclusions Our results confirm our hypothesis that for liquid-based in vitro systems, the dose-rates and target cell doses for all particles are not equal; they can vary significantly, in direct contrast to the assumption of dose-equivalency implicit in the use of mass-based media concentrations as metrics of exposure for dose-response assessment. The difference between equivalent nominal media concentration exposures on a μg/mL basis and target cell

Prodrug strategy for cancer cell-specific targeting: A recent overview.

PubMed

Zhang, Xian; Li, Xiang; You, Qidong; Zhang, Xiaojin

2017-10-20

The increasing development of targeted cancer therapy provides extensive possibilities in clinical trials, and numerous strategies have been explored. The prodrug is one of the most promising strategies in targeted cancer therapy to improve the selectivity and efficacy of cytotoxic compounds. Compared with normal tissues, cancer cells are characterized by unique aberrant markers, thus inactive prodrugs targeting these markers are excellent therapeutics to release active drugs, killing cancer cells without damaging normal tissues. In this review, we explore an integrated view of potential prodrugs applied in targeted cancer therapy based on aberrant cancer specific markers and some examples are provided for inspiring new ideas of prodrug strategy for cancer cell-specific targeting. Copyright © 2017. Published by Elsevier Masson SAS.

Longitudinal tracking of subpopulation dynamics and molecular changes during LNCaP cell castration and identification of inhibitors that could target the PSA-/lo castration-resistant cells.

PubMed

Rycaj, Kiera; Cho, Eun Jeong; Liu, Xin; Chao, Hsueh-Ping; Liu, Bigang; Li, Qiuhui; Devkota, Ashwini K; Zhang, Dingxiao; Chen, Xin; Moore, John; Dalby, Kevin N; Tang, Dean G

2016-03-22

We have recently demonstrated that the undifferentiated PSA-/lo prostate cancer (PCa) cell population harbors self-renewing long-term tumor-propagating cells that are refractory to castration, thus representing a therapeutic target. Our goals here are, by using the same lineage-tracing reporter system, to track the dynamic changes of PSA-/lo and PSA+ cells upon castration in vitro, investigate the molecular changes accompanying persistent castration, and develop large numbers of PSA-/lo PCa cells for drug screening. To these ends, we treated LNCaP cells infected with the PSAP-GFP reporter with three regimens of castration, i.e., CDSS, CDSS plus bicalutamide, and MDV3100 continuously for up to ~21 months. We observed that in the first ~7 months, castration led to time-dependent increases in PSA-/lo cells, loss of AR and PSA expression, increased expression of cancer stem cell markers, and many other molecular changes. Meanwhile, castrated LNCaP cells became resistant to high concentrations of MDV3100, chemotherapeutic drugs, and other agents. However, targeted and medium-throughput library screening identified several kinase (e.g., IGF-1R, AKT, PI3K/mTOR, Syk, GSK3) inhibitors as well as the BCL2 inhibitor that could effectively sensitize the LNCaP-CRPC cells to killing. Of interest, LNCaP cells castrated for >7 months showed evidence of cyclic changes in AR and the mTOR/AKT signaling pathways potentially involving epigenetic mechanisms. These observations indicate that castration elicits numerous molecular changes and leads to enrichment of PSA-/lo PCa cells. The ability to generate large numbers of PSA-/lo PCa cells should allow future high-throughput screening to identify novel therapeutics that specifically target this population.

Longitudinal tracking of subpopulation dynamics and molecular changes during LNCaP cell castration and identification of inhibitors that could target the PSA−/lo castration-resistant cells

PubMed Central

Rycaj, Kiera; Cho, Eun Jeong; Liu, Xin; Chao, Hsueh-Ping; Liu, Bigang; Li, Qiuhui; Devkota, Ashwini K.; Zhang, Dingxiao; Chen, Xin; Moore, John; Dalby, Kevin N.; Tang, Dean G.

2016-01-01

We have recently demonstrated that the undifferentiated PSA−/lo prostate cancer (PCa) cell population harbors self-renewing long-term tumor-propagating cells that are refractory to castration, thus representing a therapeutic target. Our goals here are, by using the same lineage-tracing reporter system, to track the dynamic changes of PSA−/lo and PSA+ cells upon castration in vitro, investigate the molecular changes accompanying persistent castration, and develop large numbers of PSA−/lo PCa cells for drug screening. To these ends, we treated LNCaP cells infected with the PSAP-GFP reporter with three regimens of castration, i.e., CDSS, CDSS plus bicalutamide, and MDV3100 continuously for up to ~21 months. We observed that in the first ~7 months, castration led to time-dependent increases in PSA−/lo cells, loss of AR and PSA expression, increased expression of cancer stem cell markers, and many other molecular changes. Meanwhile, castrated LNCaP cells became resistant to high concentrations of MDV3100, chemotherapeutic drugs, and other agents. However, targeted and medium-throughput library screening identified several kinase (e.g., IGF-1R, AKT, PI3K/mTOR, Syk, GSK3) inhibitors as well as the BCL2 inhibitor that could effectively sensitize the LNCaP-CRPC cells to killing. Of interest, LNCaP cells castrated for >7 months showed evidence of cyclic changes in AR and the mTOR/AKT signaling pathways potentially involving epigenetic mechanisms. These observations indicate that castration elicits numerous molecular changes and leads to enrichment of PSA−/lo PCa cells. The ability to generate large numbers of PSA−/lo PCa cells should allow future high-throughput screening to identify novel therapeutics that specifically target this population. PMID:26871947

Fragments of Target Cells are Internalized into Retroviral Envelope Protein-Expressing Cells during Cell-Cell Fusion by Endocytosis

PubMed Central

Izumida, Mai; Kamiyama, Haruka; Suematsu, Takashi; Honda, Eri; Koizumi, Yosuke; Yasui, Kiyoshi; Hayashi, Hideki; Ariyoshi, Koya; Kubo, Yoshinao

2016-01-01

Retroviruses enter into host cells by fusion between viral and host cell membranes. Retroviral envelope glycoprotein (Env) induces the membrane fusion, and also mediates cell-cell fusion. There are two types of cell-cell fusions induced by the Env protein. Fusion-from-within is induced by fusion between viral fusogenic Env protein-expressing cells and susceptible cells, and virions induce fusion-from-without by fusion between adjacent cells. Although entry of ecotropic murine leukemia virus (E-MLV) requires host cell endocytosis, the involvement of endocytosis in cell fusion is unclear. By fluorescent microscopic analysis of the fusion-from-within, we found that fragments of target cells are internalized into Env-expressing cells. Treatment of the Env-expressing cells with an endocytosis inhibitor more significantly inhibited the cell fusion than that of the target cells, indicating that endocytosis in Env-expressing cells is required for the cell fusion. The endocytosis inhibitor also attenuated the fusion-from-without. Electron microscopic analysis suggested that the membrane fusion resulting in fusion-from-within initiates in endocytic membrane dents. This study shows that two types of the viral cell fusion both require endocytosis, and provides the cascade of fusion-from-within. PMID:26834711

Targeted therapies in non-small cell lung carcinoma: what have we achieved so far?

PubMed Central

Houhou, Wissam

2013-01-01

The search for innovative therapeutic agents in non-small cell lung cancer (NSCLC) has witnessed a swift evolution. The number of targeted drugs that can improve patient outcomes with an acceptable safety profile is steadily increasing. In this review, we highlight current drugs that have already been approved or are under evaluation for the treatment of patients with NSCLC, either in monotherapy or combined therapy for both the first- and second-line settings. Experience with drugs targeting the vascular endothelial growth factor and its receptor, as well as the epidermal growth factor receptor is summarized. Moreover, we provide an overview of more novel targets in NSCLC and initial experience with the respective therapeutic agents. PMID:23858333

The therapeutic potential of cell cycle targeting in multiple myeloma.

PubMed

Maes, Anke; Menu, Eline; Veirman, Kim De; Maes, Ken; Vand Erkerken, Karin; De Bruyne, Elke

2017-10-27

Proper cell cycle progression through the interphase and mitosis is regulated by coordinated activation of important cell cycle proteins (including cyclin-dependent kinases and mitotic kinases) and several checkpoint pathways. Aberrant activity of these cell cycle proteins and checkpoint pathways results in deregulation of cell cycle progression, which is one of the key hallmarks of cancer. Consequently, intensive research on targeting these cell cycle regulatory proteins identified several candidate small molecule inhibitors that are able to induce cell cycle arrest and even apoptosis in cancer cells. Importantly, several of these cell cycle regulatory proteins have also been proposed as therapeutic targets in the plasma cell malignancy multiple myeloma (MM). Despite the enormous progress in the treatment of MM the past 5 years, MM still remains most often incurable due to the development of drug resistance. Deregulated expression of the cyclins D is observed in virtually all myeloma patients, emphasizing the potential therapeutic interest of cyclin-dependent kinase inhibitors in MM. Furthermore, other targets have also been identified in MM, such as microtubules, kinesin motor proteins, aurora kinases, polo-like kinases and the anaphase promoting complex/cyclosome. This review will provide an overview of the cell cycle proteins and checkpoint pathways deregulated in MM and discuss the therapeutic potential of targeting proteins or protein complexes involved in cell cycle control in MM.

Cancer stem cell-targeted therapeutics and delivery strategies.

PubMed

Ahmad, Gulzar; Amiji, Mansoor M

2017-08-01

Cancer initiating or stem cells (CSCs) are a small population of cells in the tumor mass, which have been reported to be present in different types of cancers. CSCs usually reside within the tumor and are responsible for reoccurrence of cancer. The imprecise, inaccessible nature and increased efflux of conventional therapeutic drugs make these cells resistant to drugs. We discuss the specific markers for identification of these cells, role of CSCs in chemotherapy resistance and use of different therapeutic means to target them, including elucidation of specific cell markers, exploitation of different signaling pathways and use of nanotechnology. Area covered: This review covers cancer stem cell signaling which are used by these cells to maintain their quiescence, stemness and resistant phenotype, distinct cell surface markers, contribution of these cells in drug resistance, inevitability to cure cancer and use of nanotechnology to overcome this hurdle. Expert opinion: Cancer stem cells are the main culprit of our failure to cure cancer. In order to cure cancer along with other cells types in cancer, cancer stem cells need to be targeted in the tumor bed. Nanotechnology solutions can facilitate clinical translation of the therapeutics along with other emerging technologies to cure cancer.

An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution

NASA Astrophysics Data System (ADS)

Girault, Mathias; Kim, Hyonchol; Arakawa, Hisayuki; Matsuura, Kenji; Odaka, Masao; Hattori, Akihiro; Terazono, Hideyuki; Yasuda, Kenji

2017-01-01

A microfluidic on-chip imaging cell sorter has several advantages over conventional cell sorting methods, especially to identify cells with complex morphologies such as clusters. One of the remaining problems is how to efficiently discriminate targets at the species level without labelling. Hence, we developed a label-free microfluidic droplet-sorting system based on image recognition of cells in droplets. To test the applicability of this method, a mixture of two plankton species with different morphologies (Dunaliella tertiolecta and Phaeodactylum tricornutum) were successfully identified and discriminated at a rate of 10 Hz. We also examined the ability to detect the number of objects encapsulated in a droplet. Single cell droplets sorted into collection channels showed 91 ± 4.5% and 90 ± 3.8% accuracy for D. tertiolecta and P. tricornutum, respectively. Because we used image recognition to confirm single cell droplets, we achieved highly accurate single cell sorting. The results indicate that the integrated method of droplet imaging cell sorting can provide a complementary sorting approach capable of isolating single target cells from a mixture of cells with high accuracy without any staining.

An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution.

PubMed

Girault, Mathias; Kim, Hyonchol; Arakawa, Hisayuki; Matsuura, Kenji; Odaka, Masao; Hattori, Akihiro; Terazono, Hideyuki; Yasuda, Kenji

2017-01-06

A microfluidic on-chip imaging cell sorter has several advantages over conventional cell sorting methods, especially to identify cells with complex morphologies such as clusters. One of the remaining problems is how to efficiently discriminate targets at the species level without labelling. Hence, we developed a label-free microfluidic droplet-sorting system based on image recognition of cells in droplets. To test the applicability of this method, a mixture of two plankton species with different morphologies (Dunaliella tertiolecta and Phaeodactylum tricornutum) were successfully identified and discriminated at a rate of 10 Hz. We also examined the ability to detect the number of objects encapsulated in a droplet. Single cell droplets sorted into collection channels showed 91 ± 4.5% and 90 ± 3.8% accuracy for D. tertiolecta and P. tricornutum, respectively. Because we used image recognition to confirm single cell droplets, we achieved highly accurate single cell sorting. The results indicate that the integrated method of droplet imaging cell sorting can provide a complementary sorting approach capable of isolating single target cells from a mixture of cells with high accuracy without any staining.

An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution

PubMed Central

Girault, Mathias; Kim, Hyonchol; Arakawa, Hisayuki; Matsuura, Kenji; Odaka, Masao; Hattori, Akihiro; Terazono, Hideyuki; Yasuda, Kenji

2017-01-01

A microfluidic on-chip imaging cell sorter has several advantages over conventional cell sorting methods, especially to identify cells with complex morphologies such as clusters. One of the remaining problems is how to efficiently discriminate targets at the species level without labelling. Hence, we developed a label-free microfluidic droplet-sorting system based on image recognition of cells in droplets. To test the applicability of this method, a mixture of two plankton species with different morphologies (Dunaliella tertiolecta and Phaeodactylum tricornutum) were successfully identified and discriminated at a rate of 10 Hz. We also examined the ability to detect the number of objects encapsulated in a droplet. Single cell droplets sorted into collection channels showed 91 ± 4.5% and 90 ± 3.8% accuracy for D. tertiolecta and P. tricornutum, respectively. Because we used image recognition to confirm single cell droplets, we achieved highly accurate single cell sorting. The results indicate that the integrated method of droplet imaging cell sorting can provide a complementary sorting approach capable of isolating single target cells from a mixture of cells with high accuracy without any staining. PMID:28059147

Pros and Cons of Antigen-Presenting Cell Targeted Tumor Vaccines.

PubMed

Goyvaerts, Cleo; Breckpot, Karine

2015-01-01

In therapeutic antitumor vaccination, dendritic cells play the leading role since they decide if, how, when, and where a potent antitumor immune response will take place. Since the disentanglement of the complexity and merit of different antigen-presenting cell subtypes, antitumor immunotherapeutic research started to investigate the potential benefit of targeting these subtypes in situ. This review will discuss which antigen-presenting cell subtypes are at play and how they have been targeted and finally question the true meaning of targeting antitumor-based vaccines.

Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

PubMed

Cha, Jaehyun; Kwon, Inchan

2018-02-27

Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An estimation of the number of cells in the human body.

PubMed

Bianconi, Eva; Piovesan, Allison; Facchin, Federica; Beraudi, Alina; Casadei, Raffaella; Frabetti, Flavia; Vitale, Lorenza; Pelleri, Maria Chiara; Tassani, Simone; Piva, Francesco; Perez-Amodio, Soledad; Strippoli, Pierluigi; Canaider, Silvia

2013-01-01

All living organisms are made of individual and identifiable cells, whose number, together with their size and type, ultimately defines the structure and functions of an organism. While the total cell number of lower organisms is often known, it has not yet been defined in higher organisms. In particular, the reported total cell number of a human being ranges between 10(12) and 10(16) and it is widely mentioned without a proper reference. To study and discuss the theoretical issue of the total number of cells that compose the standard human adult organism. A systematic calculation of the total cell number of the whole human body and of the single organs was carried out using bibliographical and/or mathematical approaches. A current estimation of human total cell number calculated for a variety of organs and cell types is presented. These partial data correspond to a total number of 3.72 × 10(13). Knowing the total cell number of the human body as well as of individual organs is important from a cultural, biological, medical and comparative modelling point of view. The presented cell count could be a starting point for a common effort to complete the total calculation.

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.

T-REX on-demand redox targeting in live cells.

PubMed

Parvez, Saba; Long, Marcus J C; Lin, Hong-Yu; Zhao, Yi; Haegele, Joseph A; Pham, Vanha N; Lee, Dustin K; Aye, Yimon

2016-12-01

This protocol describes targetable reactive electrophiles and oxidants (T-REX)-a live-cell-based tool designed to (i) interrogate the consequences of specific and time-resolved redox events, and (ii) screen for bona fide redox-sensor targets. A small-molecule toolset comprising photocaged precursors to specific reactive redox signals is constructed such that these inert precursors specifically and irreversibly tag any HaloTag-fused protein of interest (POI) in mammalian and Escherichia coli cells. Syntheses of the alkyne-functionalized endogenous reactive signal 4-hydroxynonenal (HNE(alkyne)) and the HaloTag-targetable photocaged precursor to HNE(alkyne) (also known as Ht-PreHNE or HtPHA) are described. Low-energy light prompts photo-uncaging (t 1/2 <1-2 min) and target-specific modification. The targeted modification of the POI enables precisely timed and spatially controlled redox events with no off-target modification. Two independent pathways are described, along with a simple setup to functionally validate known targets or discover novel sensors. T-REX sidesteps mixed responses caused by uncontrolled whole-cell swamping with reactive signals. Modification and downstream response can be analyzed by in-gel fluorescence, proteomics, qRT-PCR, immunofluorescence, fluorescence resonance energy transfer (FRET)-based and dual-luciferase reporters, or flow cytometry assays. T-REX targeting takes 4 h from initial probe treatment. Analysis of targeted redox responses takes an additional 4-24 h, depending on the nature of the pathway and the type of readouts used.

T-REX on-demand redox targeting in live cells

PubMed Central

Parvez, Saba; Long, Marcus J C; Lin, Hong-Yu; Zhao, Yi; Haegele, Joseph A; Pham, Vanha N; Lee, Dustin K; Aye, Yimon

2017-01-01

This protocol describes targetable reactive electrophiles and oxidants (T-REX)—a live-cell-based tool designed to (i) interrogate the consequences of specific and time-resolved redox events, and (ii) screen for bona fide redox-sensor targets. A small-molecule toolset comprising photocaged precursors to specific reactive redox signals is constructed such that these inert precursors specifically and irreversibly tag any HaloTag-fused protein of interest (POI) in mammalian and Escherichia coli cells. Syntheses of the alkyne-functionalized endogenous reactive signal 4-hydroxynonenal (HNE (alkyne)) and the HaloTag-targetable photocaged precursor to HNE (alkyne) (also known as Ht-PreHNE or HtPHA) are described. Low-energy light prompts photo-uncaging (t1/2 <1–2 min) and target-specific modification. The targeted modification of the POI enables precisely timed and spatially controlled redox events with no off-target modification. Two independent pathways are described, along with a simple setup to functionally validate known targets or discover novel sensors. T-REX sidesteps mixed responses caused by uncontrolled whole-cell swamping with reactive signals. Modification and downstream response can be analyzed by in-gel fluorescence, proteomics, qRT-PCR, immunofluorescence, fluorescence resonance energy transfer (FRET)-based and dual-luciferase reporters, or flow cytometry assays. T-REX targeting takes 4 h from initial probe treatment. Analysis of targeted redox responses takes an additional 4–24 h, depending on the nature of the pathway and the type of readouts used. PMID:27809314

Specific targeting and toxicity of sulphonated aluminium phthalocyanine photosensitised liposomes directed to cells by monoclonal antibody in vitro.

PubMed Central

Morgan, J.; Gray, A. G.; Huehns, E. R.

1989-01-01

A partially purified fraction of the water soluble photosensitive dye sulphonated aluminium phthalocyanine (AlSPc) was encapsulated in liposomes which were then linked to a targeting monoclonal antibody 791T/36 using a heterobifunctional linking agent. The photocytotoxic effects of the liposomes were determined on two cell lines bearing an antigen with which the targeting antibody binds: 791T, an osteosarcoma and C170, a colorectal carcinoma; and a control cell line not bearing the antigen; DW-BCL, an Epstein-Barr virus immortalised B-cell line. Antibody dependent cytotoxicity was observed in 791T and C170 cells and was proportional to the number of antigens on the cells, the AlSPc concentration and the time of exposure to activating red light. No significant toxicity was seen using untargeted liposomes, control cells or free AlSPc fraction under similar conditions. Targeted cells and controls kept in the dark also showed no significant toxicity. A possible mechanism of action is postulated and simple adaptations which demonstrate the versatility of the model are discussed. Some suggestions as to the clinical situations to which this system might be applied in the form of photodynamic therapy (PDT) are made. PMID:2930700

A statistical approach to detection of copy number variations in PCR-enriched targeted sequencing data.

PubMed

Demidov, German; Simakova, Tamara; Vnuchkova, Julia; Bragin, Anton

2016-10-22

Multiplex polymerase chain reaction (PCR) is a common enrichment technique for targeted massive parallel sequencing (MPS) protocols. MPS is widely used in biomedical research and clinical diagnostics as the fast and accurate tool for the detection of short genetic variations. However, identification of larger variations such as structure variants and copy number variations (CNV) is still being a challenge for targeted MPS. Some approaches and tools for structural variants detection were proposed, but they have limitations and often require datasets of certain type, size and expected number of amplicons affected by CNVs. In the paper, we describe novel algorithm for high-resolution germinal CNV detection in the PCR-enriched targeted sequencing data and present accompanying tool. We have developed a machine learning algorithm for the detection of large duplications and deletions in the targeted sequencing data generated with PCR-based enrichment step. We have performed verification studies and established the algorithm's sensitivity and specificity. We have compared developed tool with other available methods applicable for the described data and revealed its higher performance. We showed that our method has high specificity and sensitivity for high-resolution copy number detection in targeted sequencing data using large cohort of samples.

Landscape phages and their fusion proteins targeted to breast cancer cells

PubMed Central

Fagbohun, Olusegun A.; Bedi, Deepa; Grabchenko, Natalia I.; Deinnocentes, Patricia A.; Bird, Richard C.; Petrenko, Valery A.

2012-01-01

Breast cancer is a leading cause of death among women in the USA. The efficacy of existing anticancer therapeutics can be improved by targeting them through conjugation with ligands binding to cellular receptors. Recently, we developed a novel drug targeting strategy based on the use of pre-selected cancer-specific ‘fusion pVIII proteins’ (fpVIII), as targeting ligands. To study the efficiency of this approach in animal models, we developed a panel of breast cancer cell-binding phages as a source of targeted fpVIIIs. Two landscape phage peptide libraries (8-mer f8/8 and 9-mer f8/9) were screened to isolate 132 phage variants that recognize breast carcinoma cells MCF-7 and ZR-75-1 and internalize into the cells. When tested for their interaction with the breast cancer cells in comparison with liver cancer cells HepG2, human mammary cells MCF-10A cells and serum, 16 of the phage probes selectively interacted with the breast cancer cells whereas 32 bound both breast and liver cancer cells. The most prominent cancer-specific phage DMPGTVLP, demonstrating sub-nanomolar Kd in interaction with target cells, was used for affinity chromatography of cellular membrane molecules to reveal its potential binding receptor. The isolated protein was identified by direct sequencing as cellular surface nucleolin. This conclusion was confirmed by inhibition of the phage–cell interaction with nucleolin antibodies. Other prominent phage binders VPTDTDYS, VEEGGYIAA, and DWRGDSMDS demonstrate consensus motifs common to previously identified cancer-specific peptides. Isolated phage proteins exhibit inherent binding specificity towards cancer cells, demonstrating the functional activity of the selected fused peptides. The selected phages, their peptide inserts and intact fusion proteins can serve as promising ligands for the development of targeted nanomedicines and their study in model mice with xenograft of human cells MCF-7 and ZR-75-1. PMID:22490956

Modular cell-internalizing aptamer nanostructure enables targeted delivery of large functional RNAs in cancer cell lines.

PubMed

Porciani, David; Cardwell, Leah N; Tawiah, Kwaku D; Alam, Khalid K; Lange, Margaret J; Daniels, Mark A; Burke, Donald H

2018-06-11

Large RNAs and ribonucleoprotein complexes have powerful therapeutic potential, but effective cell-targeted delivery tools are limited. Aptamers that internalize into target cells can deliver siRNAs (<15 kDa, 19-21 nt/strand). We demonstrate a modular nanostructure for cellular delivery of large, functional RNA payloads (50-80 kDa, 175-250 nt) by aptamers that recognize multiple human B cell cancer lines and transferrin receptor-expressing cells. Fluorogenic RNA reporter payloads enable accelerated testing of platform designs and rapid evaluation of assembly and internalization. Modularity is demonstrated by swapping in different targeting and payload aptamers. Both modules internalize into leukemic B cell lines and remained colocalized within endosomes. Fluorescence from internalized RNA persists for ≥2 h, suggesting a sizable window for aptamer payloads to exert influence upon targeted cells. This demonstration of aptamer-mediated, cell-internalizing delivery of large RNAs with retention of functional structure raises the possibility of manipulating endosomes and cells by delivering large aptamers and regulatory RNAs.

Hydroxyl-HIF2-alpha is potential therapeutic target for renal cell carcinomas

PubMed Central

Isono, Takahiro; Chano, Tokuhiro; Yoshida, Tetsuya; Kageyama, Susumu; Kawauchi, Akihiro; Suzaki, Masafumi; Yuasa, Takeshi

2016-01-01

Dormant cancer cells are deprivation-resistant, and cause a number of problems for therapeutic approaches for cancers. Renal cell carcinomas (RCCs) include deprivation-resistant cells that are resistant to various treatments. In this study, the specific characteristics of deprivation-resistant cells were transcriptionally identified by next generation sequencing. The hypoxia-inducible factors (HIF) transcription factor network was significantly enhanced in deprivation-resistant RCCs compared to the sensitive RCCs. Deprivation-resistant RCCs, that had lost Von Hippel-Lindau tumor suppressor expression, expressed hydroxyl-HIF2-alpha in the nucleus, but not sensitive-RCCs. Hydroxyl-HIF-alpha was also expressed in nuclei of RCC tissue samples. Knockdown for HIF2-alpha, but not HIF1-alpha, induced cell death related to a reduction in HIF-related gene expression in deprivation-resistant RCC cells. Chetomin, a nuclear HIF-inhibitor, induced marked level of cytotoxicity in deprivation-resistant cells, similar to the knockdown of HIF2-alpha. Therefore, hydroxyl-HIF2-alpha might be a potential therapeutic target for RCCs. PMID:27822416

The cancer cell adhesion resistome: mechanisms, targeting and translational approaches.

PubMed

Dickreuter, Ellen; Cordes, Nils

2017-06-27

Cell adhesion-mediated resistance limits the success of cancer therapies and is a great obstacle to overcome in the clinic. Since the 1990s, where it became clear that adhesion of tumor cells to the extracellular matrix is an important mediator of therapy resistance, a lot of work has been conducted to understand the fundamental underlying mechanisms and two paradigms were deduced: cell adhesion-mediated radioresistance (CAM-RR) and cell adhesion-mediated drug resistance (CAM-DR). Preclinical work has evidently demonstrated that targeting of integrins, adapter proteins and associated kinases comprising the cell adhesion resistome is a promising strategy to sensitize cancer cells to both radiotherapy and chemotherapy. Moreover, the cell adhesion resistome fundamentally contributes to adaptation mechanisms induced by radiochemotherapy as well as molecular drugs to secure a balanced homeostasis of cancer cells for survival and growth. Intriguingly, this phenomenon provides a basis for synthetic lethal targeted therapies simultaneously administered to standard radiochemotherapy. In this review, we summarize current knowledge about the cell adhesion resistome and highlight targeting strategies to override CAM-RR and CAM-DR.

Application of stem cells in targeted therapy of breast cancer: a systematic review.

PubMed

Madjd, Zahra; Gheytanchi, Elmira; Erfani, Elham; Asadi-Lari, Mohsen

2013-01-01

The aim of this systematic review was to investigate whether stem cells could be effectively applied in targeted therapy of breast cancer. A systematic literature search was performed for original articles published from January 2007 until May 2012. Nine studies met the inclusion criteria for phase I or II clinical trials, of which three used stem cells as vehicles, two trials used autologous hematopoetic stem cells and in four trials cancer stem cells were targeted. Mesenchymal stem cells (MSCs) were applied as cellular vehicles to transfer therapeutic agents. Cell therapy with MSC can successfully target resistant cancers. Cancer stem cells were selectively targeted via a proteasome-dependent suicide gene leading to tumor regression. Wnt/β-catenin signaling pathway has been also evidenced to be an attractive CSC-target. This systematic review focused on two different concepts of stem cells and breast cancer marking a turning point in the trials that applied stem cells as cellular vehicles for targeted delivery therapy as well as CSC-targeted therapies. Applying stem cells as targeted therapy could be an effective therapeutic approach for treatment of breast cancer in the clinic and in therapeutic marketing; however this needs to be confirmed with further clinical investigations.

Suppression of lymphocyte proliferation by marijuana components is related to cell number and cell source

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

Klein, T.; Pross, S.; Newton, C.

Conflicting reports have appeared concerning the effect of marijuana components on immune responsiveness. The authors have observed that the effect of cannabinoids on lymphocyte proliferation varied with both the concentration of the drug and the mitogen used. They now report that at a constant concentration of drug, the cannabinoid effect varied from no effect to suppression depending upon the number of cells in culture and the organ source of the cells. Dispersed cell suspensions of mouse lymph node, spleen, and thymus were prepared and cultured at varying cell numbers with either delta-9-tetrahydrocannabinol or 11-hydroxy-delta-9-tetrahydrocannabinol and various mitogens. Lymphocyte proliferation wasmore » analyzed by /sup 3/H-thymidine incorporation. T-lymphocyte mitogen responses in cultures containing high cell numbers were unaffected by the cannabinoids but as cell numbers were reduced a suppression of the response was observed. Furthermore, thymus cells were considerably more susceptible to cannabinoid suppression than cells from either lymph node or spleen. These results suggest that certain lymphocyte subpopulations are more sensitive to cannabinoid suppression and that in addition to drug concentration other variables such as cell number and cell source must be considered when analyzing cannabinoid effects.« less

Bioimaging of Nucleolin Aptamer-Containing 5-(N-benzylcarboxyamide)-2′-deoxyuridine More Capable of Specific Binding to Targets in Cancer Cells

PubMed Central

Lee, Kyue Yim; Kang, Hyungu; Ryu, Sung Ho; Lee, Dong Soo; Lee, Jung Hwan; Kim, Soonhag

2010-01-01

Chemically modified nucleotides have been developed and applied into SELEX procedure to find a novel type of aptamers to fit with targets of interest. In this study, we directly performed chemical modification of 5-(N-benzylcarboxyamide)-2′-deoxyuridine (called 5-BzdU) in the AS1411 aptamer, which binds to the nucleolin protein expressed in cancer cells. Forty-seven compounds of AS1411-containing Cy3-labeled 5-BzdU (called Cy3-(5-BzdU)-modified-AS1411) were synthesized by randomly substituting thymidines one to twelve in AS1411 with Cy3-labeled 5-BzdU. Both statistically quantified fluorescence measurements and confocal imaging analysis demonstrated at least three potential compounds of interest: number 12, 29 and 41 that significantly increased the targeting affinity to cancer cells but no significant activity from normal healthy cells. These results suggest that the position and number of substituents in AS1411 are critical parameters to improve the aptamer function. In this study, we demonstrated that chemical modification of the existing aptamers enhanced the binding and targeting affinity to targets of interest without additional SELEX procedures. PMID:20204158

Concise Review: Emerging Drugs Targeting Epithelial Cancer Stem-Like Cells.

PubMed

Ahmed, Mehreen; Chaudhari, Kritika; Babaei-Jadidi, Roya; Dekker, Lodewijk V; Shams Nateri, Abdolrahman

2017-04-01

Increasing evidence suggests that cancer cell populations contain a small proportion of cells that display stem-like cell properties and which may be responsible for overall tumor maintenance. These cancer stem-like cells (CSCs) appear to have unique tumor-initiating ability and innate survival mechanisms that allow them to resist cancer therapies, consequently promoting relapses. Selective targeting of CSCs may provide therapeutic benefit and several recent reports have indicated this may be possible. In this article, we review drugs targeting CSCs, in selected epithelial cell-derived cancers. Stem Cells 2017;35:839-850. © 2017 AlphaMed Press.

MicroRNA-944 Affects Cell Growth by Targeting EPHA7 in Non-Small Cell Lung Cancer.

PubMed

Liu, Minxia; Zhou, Kecheng; Cao, Yi

2016-09-26

MicroRNAs (miRNAs) have critical roles in lung tumorigenesis and development. To determine aberrantly expressed miRNAs involved in non-small cell lung cancer (NSCLC) and investigate pathophysiological functions and mechanisms, we firstly carried out small RNA deep sequencing in NSCLC cell lines (EPLC-32M1, A549 and 801D) and a human immortalized cell line 16HBE, we then studied miRNA function by cell proliferation and apoptosis. cDNA microarray, luciferase reporter assay and miRNA transfection were used to investigate interaction between the miRNA and target gene. miR-944 was significantly down-regulated in NSCLC and had many putative targets. Moreover, the forced expression of miR-944 significantly inhibited the proliferation of NSCLC cells in vitro. By integrating mRNA expression data and miR-944-target prediction, we disclosed that EPHA7 was a potential target of miR-944, which was further verified by luciferase reporter assay and microRNA transfection. Our data indicated that miR-944 targets EPHA7 in NSCLC and regulates NSCLC cell proliferation, which may offer a new mechanism underlying the development and progression of NSCLC.

Probing Xist RNA Structure in Cells Using Targeted Structure-Seq

PubMed Central

Rutenberg-Schoenberg, Michael; Simon, Matthew D.

2015-01-01

The long non-coding RNA (lncRNA) Xist is a master regulator of X-chromosome inactivation in mammalian cells. Models for how Xist and other lncRNAs function depend on thermodynamically stable secondary and higher-order structures that RNAs can form in the context of a cell. Probing accessible RNA bases can provide data to build models of RNA conformation that provide insight into RNA function, molecular evolution, and modularity. To study the structure of Xist in cells, we built upon recent advances in RNA secondary structure mapping and modeling to develop Targeted Structure-Seq, which combines chemical probing of RNA structure in cells with target-specific massively parallel sequencing. By enriching for signals from the RNA of interest, Targeted Structure-Seq achieves high coverage of the target RNA with relatively few sequencing reads, thus providing a targeted and scalable approach to analyze RNA conformation in cells. We use this approach to probe the full-length Xist lncRNA to develop new models for functional elements within Xist, including the repeat A element in the 5’-end of Xist. This analysis also identified new structural elements in Xist that are evolutionarily conserved, including a new element proximal to the C repeats that is important for Xist function. PMID:26646615

Targeted Nanoparticles for Kidney Cancer Therapy

DTIC Science & Technology

2013-10-01

AD_________________ Award Number: W81XWH-10-1-0434 TITLE: Targeted Nanoparticles for Kidney Cancer Therapy PRINCIPAL...Targeted Nanoparticles for Kidney Cancer Therapy 5b. GRANT NUMBER W81XWH-10-1-0434 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT...lines following treatment with D5 nanotubes. Tthermoablation will be studied initially. Human kidney cancer cells will be injected into the kidney

Cell targeting peptides as smart ligands for targeting of therapeutic or diagnostic agents: a systematic review.

PubMed

Mousavizadeh, Ali; Jabbari, Ali; Akrami, Mohammad; Bardania, Hassan

2017-10-01

Cell targeting peptides (CTP) are small peptides which have high affinity and specificity to a cell or tissue targets. They are typically identified by using phage display and chemical synthetic peptide library methods. CTPs have attracted considerable attention as a new class of ligands to delivery specifically therapeutic and diagnostic agents, because of the fact they have several advantages including easy synthesis, smaller physical sizes, lower immunogenicity and cytotoxicity and their simple and better conjugation to nano-carriers and therapeutic or diagnostic agents compared to conventional antibodies. In this systematic review, we will focus on the basic concepts concerning the use of cell-targeting peptides (CTPs), following the approaches of selecting them from peptide libraries. We discuss several developed strategies for cell-specific delivery of different cargos by CTPs, which are designed for drug delivery and diagnostic applications. Copyright © 2017 Elsevier B.V. All rights reserved.

NK cells are biologic and biochemical targets of 6-mercaptopurine in Crohn's disease patients.

PubMed

Yusung, Susy; McGovern, Dermot; Lin, Lin; Hommes, Daniel; Lagishetty, Venu; Braun, Jonathan

2017-02-01

NK cells, which contribute to immune defense against certain viral infections and neoplasia, are emerging as modifiers of chronic immunologic diseases including transplant rejection and autoimmune diseases. Immunobiology and genetic studies have implicated NK cells as a modifier of Crohn's disease, a condition often treated with thiopurine agents such as 6-mercaptopurine (6-MP). Here, we demonstrate that thiopurines mediate NK cell apoptosis via a caspase 3 and 9 inclusive pathway, and that this process is triggered by thiopurine-mediated inhibition of Rac1. We also show that CD patients in clinical remission maintained on 6-MP have decreased NK cell Rac1 activity, and decreased NK cell numbers in their intestinal biopsies. These observations suggest that thiopurine targeting of NK cells may be a previously unappreciated therapeutic action of these agents in IBD. Copyright © 2016 Elsevier Inc. All rights reserved.

New Small Molecules Targeting Apoptosis and Cell Viability in Osteosarcoma

PubMed Central

Maugg, Doris; Rothenaigner, Ina; Schorpp, Kenji; Potukuchi, Harish Kumar; Korsching, Eberhard; Baumhoer, Daniel; Hadian, Kamyar

2015-01-01

Despite the option of multimodal therapy in the treatment strategies of osteosarcoma (OS), the most common primary malignant bone tumor, the standard therapy has not changed over the last decades and still involves multidrug chemotherapy and radical surgery. Although successfully applied in many patients a large number of patients eventually develop recurrent or metastatic disease in which current therapeutic regimens often lack efficacy. Thus, new therapeutic strategies are urgently needed. In this study, we performed a phenotypic high-throughput screening campaign using a 25,000 small-molecule diversity library to identify new small molecules selectively targeting osteosarcoma cells. We could identify two new small molecules that specifically reduced cell viability in OS cell lines U2OS and HOS, but affected neither hepatocellular carcinoma cell line (HepG2) nor primary human osteoblasts (hOB). In addition, the two compounds induced caspase 3 and 7 activity in the U2OS cell line. Compared to conventional drugs generally used in OS treatment such as doxorubicin, we indeed observed a greater sensitivity of OS cell viability to the newly identified compounds compared to doxorubicin and staurosporine. The p53-negative OS cell line Saos-2 almost completely lacked sensitivity to compound treatment that could indicate a role of p53 in the drug response. Taken together, our data show potential implications for designing more efficient therapies in OS. PMID:26039064

BMI-1 targeting interferes with patient-derived tumor-initiating cell survival and tumor growth in prostate cancer

PubMed Central

Yusuff, Shamila; Davis, Stephani; Flaherty, Kathleen; Huselid, Eric; Patrizii, Michele; Jones, Daniel; Cao, Liangxian; Sydorenko, Nadiya; Moon, Young-Choon; Zhong, Hua; Medina, Daniel J.; Kerrigan, John; Stein, Mark N.; Kim, Isaac Y.; Davis, Thomas W.; DiPaola, Robert S.; Bertino, Joseph R.; Sabaawy, Hatem E.

2016-01-01

Purpose Current prostate cancer (PCa) management calls for identifying novel and more effective therapies. Self-renewing tumor-initiating cells (TICs) hold intrinsic therapy-resistance and account for tumor relapse and progression. As BMI-1 regulates stem cell self-renewal, impairing BMI-1 function for TICs-tailored therapies appears to be a promising approach. Experimental design We have previously developed a combined immunophenotypic and time-of-adherence assay to identify CD49bhiCD29hiCD44hi cells as human prostate TICs. We utilized this assay with patient derived prostate cancer cells and xenograft models to characterize the effects of pharmacological inhibitors of BMI-1. Results We demonstrate that in cell lines and patient-derived TICs, BMI-1 expression is upregulated and associated with stem cell-like traits. From a screened library, we identified a number of post-transcriptional small molecules that target BMI-1 in prostate TICs. Pharmacological inhibition of BMI-1 in patient-derived cells significantly decreased colony formation in vitro and attenuated tumor initiation in vivo, thereby functionally diminishing the frequency of TICs, particularly in cells resistant to proliferation- and androgen receptor (AR)-directed therapies, without toxic effects on normal tissues. Conclusions Our data offer a paradigm for targeting TICs and support the development of BMI-1-targeting therapy for a more effective PCa treatment. PMID:27307599

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.

Bamgineer: Introduction of simulated allele-specific copy number variants into exome and targeted sequence data sets.

PubMed

Samadian, Soroush; Bruce, Jeff P; Pugh, Trevor J

2018-03-01

Somatic copy number variations (CNVs) play a crucial role in development of many human cancers. The broad availability of next-generation sequencing data has enabled the development of algorithms to computationally infer CNV profiles from a variety of data types including exome and targeted sequence data; currently the most prevalent types of cancer genomics data. However, systemic evaluation and comparison of these tools remains challenging due to a lack of ground truth reference sets. To address this need, we have developed Bamgineer, a tool written in Python to introduce user-defined haplotype-phased allele-specific copy number events into an existing Binary Alignment Mapping (BAM) file, with a focus on targeted and exome sequencing experiments. As input, this tool requires a read alignment file (BAM format), lists of non-overlapping genome coordinates for introduction of gains and losses (bed file), and an optional file defining known haplotypes (vcf format). To improve runtime performance, Bamgineer introduces the desired CNVs in parallel using queuing and parallel processing on a local machine or on a high-performance computing cluster. As proof-of-principle, we applied Bamgineer to a single high-coverage (mean: 220X) exome sequence file from a blood sample to simulate copy number profiles of 3 exemplar tumors from each of 10 tumor types at 5 tumor cellularity levels (20-100%, 150 BAM files in total). To demonstrate feasibility beyond exome data, we introduced read alignments to a targeted 5-gene cell-free DNA sequencing library to simulate EGFR amplifications at frequencies consistent with circulating tumor DNA (10, 1, 0.1 and 0.01%) while retaining the multimodal insert size distribution of the original data. We expect Bamgineer to be of use for development and systematic benchmarking of CNV calling algorithms by users using locally-generated data for a variety of applications. The source code is freely available at http://github.com/pughlab/bamgineer.

Number of decidual natural killer cells & macrophages in pre-eclampsia

PubMed Central

Milosevic-Stevanovic, Jelena; Krstic, Miljan; Radovic-Janosevic, Dragana; Popovic, Jasmina; Tasic, Marija; Stojnev, Slavica

2016-01-01

Background & objectives: The process of human placentation is complex and still not well understood. This study was aimed to examine the relationship between clinical features of pre-eclampsia and degree of trophoblastic invasion after its immunohistochemical visualization in the context of possible alterations in the number of natural killer (NK) cells and macrophages in the decidua. Methods: This prospective study included a study group comprising 30 pregnant women with pre-eclampsia delivered by caesarean section and a control group comprising 20 healthy pregnant women also delivered by caesarean section. Samples of placental bed obtained during caesarean section were analyzed after immunohistochemical labelling CD56+ NK cells, CD68+ macrophages and cytokeratin 7 trophoblastic cells. Results: In pre-eclampsia, there was a significantly lower number of CD56+ NK cells in the decidua (P<0.001) and a higher number of CD68+ macrophages (P<0.001) compared to control group. In the subgroup of pre-eclampsia with intrauterine growth retardation (IUGR), a significantly greater number of NK cells (P<0.05) was recorded, as well as an increased number of macrophages, but not significantly compared to pre-eclampsia without IUGR. There was no significant difference in the distribution of these cells in the decidua in relation to the severity of pre-eclampsia. CD56+ NK cells were significantly less (P<0.05) and macrophages were more (P<0.05) in the group with poor trophoblastic invasion. Interpretation & conclusions: Alterations in the number of immune cells in relation to the degree of trophoblastic invasion indicated their role in aetiopathogenesis of pre-eclampsia, while the direct association between their number and severity of pre-eclampsia was not confirmed. PMID:28474619

Nanomedicine: nanoparticles, molecular biosensors, and targeted gene/drug delivery for combined single-cell diagnostics and therapeutics

NASA Astrophysics Data System (ADS)

Prow, Tarl W.; Salazar, Jose H.; Rose, William A.; Smith, Jacob N.; Reece, Lisa; Fontenot, Andrea A.; Wang, Nan A.; Lloyd, R. Stephen; Leary, James F.

2004-07-01

Next generation nanomedicine technologies are being developed to provide for continuous and linked molecular diagnostics and therapeutics. Research is being performed to develop "sentinel nanoparticles" which will seek out diseased (e.g. cancerous) cells, enter those living cells, and either perform repairs or induce those cells to die through apoptosis. These nanoparticles are envisioned as multifunctional "smart drug delivery systems". The nanosystems are being developed as multilayered nanoparticles (nanocrystals, nanocapsules) containing cell targeting molecules, intracellular re-targeting molecules, molecular biosensor molecules, and drugs/enzymes/gene therapy. These "nanomedicine systems" are being constructed to be autonomous, much like present-day vaccines, but will have sophisticated targeting, sensing, and feedback control systems-much more sophisticated than conventional antibody-based therapies. The fundamental concept of nanomedicine is to not to just kill all aberrant cells by surgery, radiation therapy, or chemotherapy. Rather it is to fix cells, when appropriate, one cell-at-a-time, to preserve and re-build organ systems. When cells should not be fixed, such as in cases where an improperly repaired cell might give rise to cancer cells, the nanomedical therapy would be to induce apoptosis in those cells to eliminate them without the damagin bystander effects of the inflammatory immune response system reacting to necrotic cells or those which have died from trauma or injury. The ultimate aim of nanomedicine is to combine diagnostics and therapeutics into "real-time medicine", using where possible in-vivo cytometry techniques for diagnostics and therapeutics. A number of individual components of these multi-component nanoparticles are already working in in-vitro and ex-vivo cell and tissue systems. Work has begun on construction of integrated nanomedical systems.

Optical cell monitoring system for underwater targets

NASA Astrophysics Data System (ADS)

Moon, SangJun; Manzur, Fahim; Manzur, Tariq; Demirci, Utkan

2008-10-01

We demonstrate a cell based detection system that could be used for monitoring an underwater target volume and environment using a microfluidic chip and charge-coupled-device (CCD). This technique allows us to capture specific cells and enumerate these cells on a large area on a microchip. The microfluidic chip and a lens-less imaging platform were then merged to monitor cell populations and morphologies as a system that may find use in distributed sensor networks. The chip, featuring surface chemistry and automatic cell imaging, was fabricated from a cover glass slide, double sided adhesive film and a transparent Polymethlymetacrylate (PMMA) slab. The optically clear chip allows detecting cells with a CCD sensor. These chips were fabricated with a laser cutter without the use of photolithography. We utilized CD4+ cells that are captured on the floor of a microfluidic chip due to the ability to address specific target cells using antibody-antigen binding. Captured CD4+ cells were imaged with a fluorescence microscope to verify the chip specificity and efficiency. We achieved 70.2 +/- 6.5% capturing efficiency and 88.8 +/- 5.4% specificity for CD4+ T lymphocytes (n = 9 devices). Bright field images of the captured cells in the 24 mm × 4 mm × 50 μm microfluidic chip were obtained with the CCD sensor in one second. We achieved an inexpensive system that rapidly captures cells and images them using a lens-less CCD system. This microfluidic device can be modified for use in single cell detection utilizing a cheap light-emitting diode (LED) chip instead of a wide range CCD system.

Biomarker evaluation of face transplant rejection: association of donor T cells with target cell injury.

PubMed

Lian, Christine Guo; Bueno, Ericka M; Granter, Scott R; Laga, Alvaro C; Saavedra, Arturo P; Lin, William M; Susa, Joseph S; Zhan, Qian; Chandraker, Anil K; Tullius, Stefan G; Pomahac, Bohdan; Murphy, George F

2014-06-01

This series of 113 sequential biopsies of full facial transplants provides findings of potential translational significance as well as biological insights that could prompt reexamination of conventional paradigms of effector pathways in skin allograft rejection. Serial biopsies before, during, and after rejection episodes were evaluated for clinicopathological assessment that in selected cases included specific biomarkers for donor-versus-recipient T cells. Histologic evidence of rejection included lymphocyte-associated injury to epidermal rete ridges, follicular infundibula, and dermal microvessels. Surprisingly, during active rejection, immune cells spatially associated with target cell injury consisted abundantly or predominantly of lymphocytes of donor origin with an immunophenotype typical of the resident memory T-cell subset. Current dogma assumes that skin allograft rejection is mediated by recipient T cells that attack epidermal targets, and the association of donor T cells with sites of target cell injury raises questions regarding the potential complexity of immune cell interactions in the rejection process. A more histopathologically refined and immune-based biomarker approach to assessment of rejection of facial transplants is now indicated.

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.

Curcumin suppresses proliferation of colon cancer cells by targeting CDK2.

PubMed

Lim, Tae-Gyu; Lee, Sung-Young; Huang, Zunnan; Lim, Do Young; Chen, Hanyong; Jung, Sung Keun; Bode, Ann M; Lee, Ki Won; Dong, Zigang

2014-04-01

Curcumin, the yellow pigment of turmeric found in Southeast Indian food, is one of the most popular phytochemicals for cancer prevention. Numerous reports have demonstrated modulation of multiple cellular signaling pathways by curcumin and its molecular targets in various cancer cell lines. To identify a new molecular target of curcumin, we used shape screening and reverse docking to screen the Protein Data Bank against curcumin. Cyclin-dependent kinase 2 (CDK2), a major cell-cycle protein, was identified as a potential molecular target of curcumin. Indeed, in vitro and ex vivo kinase assay data revealed a dramatic suppressive effect of curcumin on CDK2 kinase activity. Furthermore, curcumin induced G1 cell-cycle arrest, which is regulated by CDK2 in HCT116 cells. Although the expression levels of CDK2 and its regulatory subunit, cyclin E, were not changed, the phosphorylation of retinoblastoma (Rb), a well-known CDK2 substrate, was reduced by curcumin. Because curcumin induced cell-cycle arrest, we investigated the antiproliferative effect of curcumin on HCT116 colon cancer cells. In this experiment, curcumin suppressed HCT116 cell proliferation effectively. To determine whether CDK2 is a direct target of curcumin, CDK2 expression was knocked down in HCT116 cells. As expected, HCT116 sh-CDK2 cells exhibited G1 arrest and reduced proliferation. Because of the low levels of CDK2 in HCT116 sh-CDK2 cells, the effects of curcumin on G1 arrest and cell proliferation were not substantially relative to HCT116 sh-control cells. From these results, we identified CDK2 as a direct target of curcumin in colon cancer cells.

Curcumin suppresses proliferation of colon cancer cells by targeting CDK2

PubMed Central

Lim, Tae-Gyu; Lee, Sung-Young; Huang, Zunnan; Lim, Do Young; Chen, Hanyong; Jung, Sung Keun; Bode, Ann M.; Lee, Ki Won; Dong, Zigang

2014-01-01

Curcumin, the yellow pigment of turmeric found in Southeast Indian food, is one of the most popular phytochemicals for cancer prevention. Numerous reports have demonstrated modulation of multiple cellular signaling pathways by curcumin and its molecular targets in various cancer cell lines. To identify a new molecular target of curcumin, we used shape screening and reverse docking to screen the protein data bank against curcumin. Cyclin dependent kinase 2 (CDK2), a major cell cycle protein, was identified as a potential molecular target of curcumin. Indeed, in vitro and ex vivo kinase assay data revealed a dramatic suppressive effect of curcumin on CDK2 kinase activity. Furthermore, curcumin induced G1 cell cycle arrest, which is regulated by CDK2 in HCT116 cells. Although the expression levels of CDK2 and its regulatory subunit, cyclin E, were not changed, the phosphorylation of Rb, a well-known CDK2 substrate, was reduced by curcumin. Because curcumin induced cell cycle arrest, we investigated the anti-proliferative effect of curcumin on HCT116 colon cancer cells. In this experiment, curcumin suppressed HCT116 cell proliferation effectively. To determine if CDK2 is a direct target of curcumin, CDK2 expression was knocked down in HCT116 cells. As expected, HCT116 sh-CDK2 cells exhibited G1 arrest and reduced proliferation. Because of the low levels of CDK2 in HCT116 sh-CDK2 cells, the effects of curcumin on G1 arrest and cell proliferation were not substantial relative to HCT116 sh-control cells. From these results, we identified CDK2 as a direct target of curcumin in colon cancer cells. PMID:24550143

Enhancing Oral Vaccine Potency by Targeting Intestinal M Cells

PubMed Central

Azizi, Ali; Kumar, Ashok; Diaz-Mitoma, Francisco; Mestecky, Jiri

2010-01-01

The immune system in the gastrointestinal tract plays a crucial role in the control of infection, as it constitutes the first line of defense against mucosal pathogens. The attractive features of oral immunization have led to the exploration of a variety of oral delivery systems. However, none of these oral delivery systems have been applied to existing commercial vaccines. To overcome this, a new generation of oral vaccine delivery systems that target antigens to gut-associated lymphoid tissue is required. One promising approach is to exploit the potential of microfold (M) cells by mimicking the entry of pathogens into these cells. Targeting specific receptors on the apical surface of M cells might enhance the entry of antigens, initiating the immune response and consequently leading to protection against mucosal pathogens. In this article, we briefly review the challenges associated with current oral vaccine delivery systems and discuss strategies that might potentially target mouse and human intestinal M cells. PMID:21085599

Colon-targeted delivery of live bacterial cell biotherapeutics including microencapsulated live bacterial cells

PubMed Central

Prakash, Satya; Malgorzata Urbanska, Aleksandra

2008-01-01

There has been an ample interest in delivery of therapeutic molecules using live cells. Oral delivery has been stipulated as best way to deliver live cells to humans for therapy. Colon, in particular, is a part of gastrointestinal (GI) tract that has been proposed to be an oral targeted site. The main objective of these oral therapy procedures is to deliver live cells not only to treat diseases like colorectal cancer, inflammatory bowel disease, and other GI tract diseases like intestinal obstruction and gastritis, but also to deliver therapeutic molecules for overall therapy in various diseases such as renal failure, coronary heart disease, hypertension, and others. This review provides a comprehensive summary of recent advancement in colon targeted live bacterial cell biotherapeutics. Current status of bacterial cell therapy, principles of artificial cells and its potentials in oral delivery of live bacterial cell biotherapeutics for clinical applications as well as biotherapeutic future perspectives are also discussed in our review. PMID:19707368

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.

Vitamin C, a Multi-Tasking Molecule, Finds a Molecular Target in Killing Cancer Cells.

PubMed

Li, Robert

2016-03-01

Early work in the 1970s by Linus Pauling, a twice-honored Nobel laureate, led to his proposal of using high-dose vitamin C to treat cancer patients. Over the past several decades, a number of studies in animal models as well as several small-scale clinical studies have provided substantial support of Linus Pauling's early proposal. Production of reactive oxygen species (ROS) via oxidation of vitamin C appears to be a major underlying event, leading to the selective killing of cancer cells. However, it remains unclear how vitamin C selectively kills cancer cells while sparing normal cells and what the molecular targets of high-dose vitamin C are. In a recent article published in Science (2015 December 11; 350(6266):1391-6. doi: 10.1126/science.aaa5004), Yun et al. reported that vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) through an ROS-dependent mechanism. This work by Yun et al. along with other findings advances our current understanding of the molecular basis of high-dose vitamin C-mediated cancer cell killing, which will likely give an impetus to the continued research efforts aiming to further decipher the novel biochemistry of vitamin C and its unique role in cancer therapy.

A systems biology approach identified different regulatory networks targeted by KSHV miR-K12-11 in B cells and endothelial cells.

PubMed

Yang, Yajie; Boss, Isaac W; McIntyre, Lauren M; Renne, Rolf

2014-08-08

Kaposi's sarcoma associated herpes virus (KSHV) is associated with tumors of endothelial and lymphoid origin. During latent infection, KSHV expresses miR-K12-11, an ortholog of the human tumor gene hsa-miR-155. Both gene products are microRNAs (miRNAs), which are important post-transcriptional regulators that contribute to tissue specific gene expression. Advances in target identification technologies and molecular interaction databases have allowed a systems biology approach to unravel the gene regulatory networks (GRNs) triggered by miR-K12-11 in endothelial and lymphoid cells. Understanding the tissue specific function of miR-K12-11 will help to elucidate underlying mechanisms of KSHV pathogenesis. Ectopic expression of miR-K12-11 differentially affected gene expression in BJAB cells of lymphoid origin and TIVE cells of endothelial origin. Direct miRNA targeting accounted for a small fraction of the observed transcriptome changes: only 29 genes were identified as putative direct targets of miR-K12-11 in both cell types. However, a number of commonly affected biological pathways, such as carbohydrate metabolism and interferon response related signaling, were revealed by gene ontology analysis. Integration of transcriptome profiling, bioinformatic algorithms, and databases of protein-protein interactome from the ENCODE project identified different nodes of GRNs utilized by miR-K12-11 in a tissue-specific fashion. These effector genes, including cancer associated transcription factors and signaling proteins, amplified the regulatory potential of a single miRNA, from a small set of putative direct targets to a larger set of genes. This is the first comparative analysis of miRNA-K12-11's effects in endothelial and B cells, from tissues infected with KSHV in vivo. MiR-K12-11 was able to broadly modulate gene expression in both cell types. Using a systems biology approach, we inferred that miR-K12-11 establishes its GRN by both repressing master TFs and influencing

A new prospect in cancer therapy: targeting cancer stem cells to eradicate cancer.

PubMed

Chen, Li-Sha; Wang, An-Xin; Dong, Bing; Pu, Ke-Feng; Yuan, Li-Hua; Zhu, Yi-Min

2012-12-01

According to the cancer stem cell theory, cancers can be initiated by cancer stem cells. This makes cancer stem cells prime targets for therapeutic intervention. Eradicating cancer stem cells by efficient targeting agents may have the potential to cure cancer. In this review, we summarize recent breakthroughs that have improved our understanding of cancer stem cells, and we discuss the therapeutic strategy of targeting cancer stem cells, a promising future direction for cancer stem cell research.

Glioblastoma Stem Cells as a New Therapeutic Target for Glioblastoma.

PubMed

Kalkan, Rasime

2015-01-01

Primary and secondary glioblastomas (GBMs) are two distinct diseases. The genetic and epigenetic background of these tumors is highly variable. The treatment procedure for these tumors is often unsuccessful because of the cellular heterogeneity and intrinsic ability of the tumor cells to invade healthy tissues. The fatal outcome of these tumors promotes researchers to find out new markers associated with the prognosis and treatment planning. In this communication, the role of glioblastoma stem cells in tumor progression and the malignant behavior of GBMs are summarized with attention to the signaling pathways and molecular regulators that are involved in maintaining the glioblastoma stem cell phenotype. A better understanding of these stem cell-like cells is necessary for designing new effective treatments and developing novel molecular strategies to target glioblastoma stem cells. We discuss hypoxia as a new therapeutic target for GBM. We focus on the inhibition of signaling pathways, which are associated with the hypoxia-mediated maintenance of glioblastoma stem cells, and the knockdown of hypoxia-inducible factors, which could be identified as attractive molecular target approaches for GBM therapeutics.

Monoclonal antibodies targeting non-small cell lung cancer stem-like cells by multipotent cancer stem cell monoclonal antibody library.

PubMed

Cao, Kaiyue; Pan, Yunzhi; Yu, Long; Shu, Xiong; Yang, Jing; Sun, Linxin; Sun, Lichao; Yang, Zhihua; Ran, Yuliang

2017-02-01

Cancer stem cells (CSCs) are a rare subset of cancer cells that play a significant role in cancer initiation, spreading, and recurrence. In this study, a subpopulation of lung cancer stem-like cells (LCSLCs) was identified from non-small cell lung carcinoma cell lines, SPCA-1 and A549, using serum-free suspension sphere-forming culture method. A monoclonal antibody library was constructed using immunized BLAB/c mice with the multipotent CSC cell line T3A-A3. Flow cytometry analysis showed that 33 mAbs targeted antigens can be enriched in sphere cells compared with the parental cells of SPCA-1 and A549 cell lines. Then, we performed functional antibody screening including sphere-forming inhibiting and invasion inhibiting assay. The results showed that two antibodies, 12C7 and 9B8, notably suppressed the self-renewal and invasion of LCSLCs. Fluorescence-activated cell sorting (FACs) found that the positive cells recognized by mAbs, 12C7 or 9B8, displayed features of LCSLCs. Interestingly, we found that these two antibodies recognized different subsets of cells and their combination effect was superior to the individual effect both in vitro and in vivo. Tissue microarrays were applied to detect the expression of the antigens targeted by these two antibodies. The positive expression of 12C7 and 9B8 targeted antigen was 84.4 and 82.5%, respectively, which was significantly higher than that in the non-tumor lung tissues. In conclusion, we screened two potential therapeutic antibodies that target different subsets of LCSLCs.

HLA-targeted flow cytometric sorting of blood cells allows separation of pure and viable microchimeric cell populations.

PubMed

Drabbels, Jos J M; van de Keur, Carin; Kemps, Berit M; Mulder, Arend; Scherjon, Sicco A; Claas, Frans H J; Eikmans, Michael

2011-11-10

Microchimerism is defined by the presence of low levels of nonhost cells in a person. We developed a reliable method for separating viable microchimeric cells from the host environment. For flow cytometric cell sorting, HLA antigens were targeted with human monoclonal HLA antibodies (mAbs). Optimal separation of microchimeric cells (present at a proportion as low as 0.01% in artificial mixtures) was obtained with 2 different HLA mAbs, one targeting the chimeric cells and the other the background cells. To verify purity of separated cell populations, flow-sorted fractions of 1000 cells were processed for DNA analysis by HLA-allele-specific and Y-chromosome-directed real-time quantitative PCR assays. After sorting, PCR signals of chimeric DNA markers in the positive fractions were significantly enhanced compared with those in the presort samples, and they were similar to those in 100% chimeric control samples. Next, we demonstrate applicability of HLA-targeted FACS sorting after pregnancy by separating chimeric maternal cells from child umbilical cord mononuclear cells. Targeting allelic differences with anti-HLA mAbs with FACS sorting allows maximal enrichment of viable microchimeric cells from a background cell population. The current methodology enables reliable microchimeric cell detection and separation in clinical specimens.

Zn(II)-curc targets p53 in thyroid cancer cells.

PubMed

Garufi, Alessia; D'Orazi, Valerio; Crispini, Alessandra; D'Orazi, Gabriella

2015-10-01

TP53 mutation is a common event in many cancers, including thyroid carcinoma. Defective p53 activity promotes cancer resistance to therapies and a more malignant phenotype, acquiring oncogenic functions. Rescuing the function of mutant p53 (mutp53) protein is an attractive anticancer therapeutic strategy. Zn(II)-curc is a novel small molecule that has been shown to target mutp53 protein in several cancer cells, but its effect in thyroid cancer cells remains unclear. Here, we investigated whether Zn(II)-curc could affect p53 in thyroid cancer cells with both p53 mutation (R273H) and wild-type p53. Zn(II)-curc induced mutp53H273 downregulation and reactivation of wild-type functions, such as binding to canonical target promoters and target gene transactivation. This latter effect was similar to that induced by PRIMA-1. In addition, Zn(II)-curc triggered p53 target gene expression in wild-type p53-carrying cells. In combination treatments, Zn(II)-curc enhanced the antitumor activity of chemotherapeutic drugs, in both mutant and wild-type-carrying cancer cells. Taken together, our data indicate that Zn(II)-curc promotes the reactivation of p53 in thyroid cancer cells, providing in vitro evidence for a potential therapeutic approach in thyroid cancers.

ErbB-targeted CAR T-cell immunotherapy of cancer.

PubMed

Whilding, Lynsey M; Maher, John

2015-01-01

Chimeric antigen receptor (CAR) based immunotherapy has been under development for the last 25 years and is now a promising new treatment modality in the field of cancer immunotherapy. The approach involves genetically engineering T cells to target malignant cells through expression of a bespoke fusion receptor that couples an HLA-independent antigen recognition domain to one or more intracellular T-cell activating modules. Multiple clinical trials are now underway in several centers to investigate CAR T-cell immunotherapy of diverse hematologic and solid tumor types. The most successful results have been achieved in the treatment of patients with B-cell malignancies, in whom several complete and durable responses have been achieved. This review focuses on the preclinical and clinical development of CAR T-cell immunotherapy of solid cancers, targeted against members of the ErbB family.

GEM-loaded magnetic albumin nanospheres modified with cetuximab for simultaneous targeting, magnetic resonance imaging, and double-targeted thermochemotherapy of pancreatic cancer cells.

PubMed

Wang, Ling; An, Yanli; Yuan, Chenyan; Zhang, Hao; Liang, Chen; Ding, Fengan; Gao, Qi; Zhang, Dongsheng

2015-01-01

Targeted delivery is a promising strategy to improve the diagnostic imaging and therapeutic effect of cancers. In this paper, novel cetuximab (C225)-conjugated, gemcitabine (GEM)-containing magnetic albumin nanospheres (C225-GEM/MANs) were fabricated and applied as a theranostic nanocarrier to conduct simultaneous targeting, magnetic resonance imaging (MRI), and double-targeted thermochemotherapy against pancreatic cancer cells. Fe3O4 nanoparticles (NPs) and GEM co-loaded albumin nanospheres (GEM/MANs) were prepared, and then C225 was further conjugated to synthesize C225-GEM/MANs. Their morphology, mean particle size, GEM encapsulation ratio, specific cell-binding ability, and thermal dynamic profiles were characterized. The effects of discriminating different EGFR-expressing pancreatic cancer cells (AsPC-1 and MIA PaCa-2) and monitoring cellular targeting effects were assessed by targeted MRI. Lastly, the antitumor efficiency of double/C225/magnetic-targeted and nontargeted thermochemotherapy was compared with chemotherapy alone using 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and flow cytometry (FCM) assay. When treated with targeted nanospheres, AsPC-1 cells showed a significantly less intense MRI T2 signal than MIA PaCa-2 cells, while both cells had similar signal strength when incubated with nontargeted nanospheres. T2 signal intensity was significantly lower when magnetic and C225 targeting were combined, rather than used alone. The inhibitory and apoptotic rates of each thermochemotherapy group were significantly higher than those of the chemotherapy-alone groups. Additionally, both MTT and FCM analysis verified that double-targeted thermochemotherapy had the highest targeted killing efficiency among all groups. The C225-GEM/MANs can distinguish various EGFR-expressing live pancreatic cancer cells, monitor diverse cellular targeting effects using targeted MRI imaging, and efficiently mediate double-targeted thermochemotherapy

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.

Testicular cell junction: a novel target for male contraception.

PubMed

Lee, Nikki P Y; Wong, Elissa W P; Mruk, Dolores D; Cheng, C Yan

2009-01-01

Even though various contraceptive methods are widely available, the number of unwanted pregnancies is still on the rise in developing countries, pressurizing the already resource limited nations. One of the major underlying reasons is the lack of effective, low cost, and safe contraceptives for couples. During the past decade, some studies were performed using animal models to decipher if the Sertoli-germ cell junction in the testis is a target for male fertility regulation. Some of these study models were based on the use of hormones and/or chemicals to disrupt the hypothalamic-pituitary-testicular axis (e.g., androgen-based implants or pills) and others utilized a panel of chemical entities or synthetic peptides to perturb spermatogenesis either reversibly or non-reversibly. Among them, adjudin, a potential male contraceptive, is one of the compounds exerting its action on the unique adherens junctions, known as ectoplasmic specializations, in the testis. Since the testis is equipped with inter-connected cell junctions, an initial targeting of one junction type may affect the others and these accumulative effects could lead to spermatogenic arrest. This review attempts to cover an innovative theme on how male infertility can be achieved by inducing junction instability and defects in the testis, opening a new window of research for male contraceptive development. While it will still take much time and effort of intensive investigation before a product can reach the consumable market, these findings have provided hope for better family planning involving men.

Diurnal rhythms in peripheral blood immune cell numbers of domestic pigs.

PubMed

Engert, Larissa C; Weiler, Ulrike; Pfaffinger, Birgit; Stefanski, Volker; Schmucker, Sonja S

2018-02-01

Diurnal rhythms within the immune system are considered important for immune competence. Until now, they were mostly studied in humans and rodents. However, as the domestic pig is regarded as suitable animal model and due to its importance in agriculture, this study aimed to characterize diurnal rhythmicity in porcine circulating leukocyte numbers. Eighteen pigs were studied over periods of up to 50 h. Cosinor analyses revealed diurnal rhythms in cell numbers of most investigated immune cell populations in blood. Whereas T cell, dendritic cell, and eosinophil counts peaked during nighttime, NK cell and neutrophil counts peaked during daytime. Relative amplitudes of cell numbers in blood differed in T helper cell subtypes with distinctive differentiation states. Mixed model analyses revealed that plasma cortisol concentration was negatively associated with cell numbers of most leukocyte types, except for NK cells and neutrophils. The observed rhythms mainly resemble those found in humans and rodents. Copyright © 2017 Elsevier Ltd. All rights reserved.

Phenotypic high-throughput screening elucidates target pathway in breast cancer stem cell-like cells.

PubMed

Carmody, Leigh C; Germain, Andrew R; VerPlank, Lynn; Nag, Partha P; Muñoz, Benito; Perez, Jose R; Palmer, Michelle A J

2012-10-01

Cancer stem cells (CSCs) are resistant to standard cancer treatments and are likely responsible for cancer recurrence, but few therapies target this subpopulation. Due to the difficulty in propagating CSCs outside of the tumor environment, previous work identified CSC-like cells by inducing human breast epithelial cells into an epithelial-to-mesenchymal transdifferentiated state (HMLE_sh_ECad). A phenotypic screen was conducted against HMLE_sh_ECad with 300 718 compounds from the Molecular Libraries Small Molecule Repository to identify selective inhibitors of CSC growth. The screen yielded 2244 hits that were evaluated for toxicity and selectivity toward an isogenic control cell line. An acyl hydrazone scaffold emerged as a potent and selective scaffold targeting HMLE_sh_ECad. Fifty-three analogues were acquired and tested; compounds ranged in potency from 790 nM to inactive against HMLE_sh_ECad. Of the analogues, ML239 was best-in-class with an IC(50)= 1.18 µM against HMLE_sh_ECad, demonstrated a >23-fold selectivity over the control line, and was toxic to another CSC-like line, HMLE_shTwist, and a breast carcinoma cell line, MDA-MB-231. Gene expression studies conducted with ML239-treated cells showed altered gene expression in the NF-κB pathway in the HMLE_sh_ECad line but not in the isogenic control line. Future studies will be directed toward the identification of ML239 target(s).

Detecting drug-target binding in cells using fluorescence-activated cell sorting coupled with mass spectrometry analysis.

PubMed

Wilson, Kris; Webster, Scott P; Iredale, John P; Zheng, Xiaozhong; Homer, Natalie Z; Pham, Nhan T; Auer, Manfred; Mole, Damian J

2017-12-15

The assessment of drug-target engagement for determining the efficacy of a compound inside cells remains challenging, particularly for difficult target proteins. Existing techniques are more suited to soluble protein targets. Difficult target proteins include those with challenging in vitro solubility, stability or purification properties that preclude target isolation. Here, we report a novel technique that measures intracellular compound-target complex formation, as well as cellular permeability, specificity and cytotoxicity-the toxicity-affinity-permeability-selectivity (TAPS) technique. The TAPS assay is exemplified here using human kynurenine 3-monooxygenase (KMO), a challenging intracellular membrane protein target of significant current interest. TAPS confirmed target binding of known KMO inhibitors inside cells. We conclude that the TAPS assay can be used to facilitate intracellular hit validation on most, if not all intracellular drug targets.

Detecting drug-target binding in cells using fluorescence-activated cell sorting coupled with mass spectrometry analysis

NASA Astrophysics Data System (ADS)

Wilson, Kris; Webster, Scott P.; Iredale, John P.; Zheng, Xiaozhong; Homer, Natalie Z.; Pham, Nhan T.; Auer, Manfred; Mole, Damian J.

2018-01-01

The assessment of drug-target engagement for determining the efficacy of a compound inside cells remains challenging, particularly for difficult target proteins. Existing techniques are more suited to soluble protein targets. Difficult target proteins include those with challenging in vitro solubility, stability or purification properties that preclude target isolation. Here, we report a novel technique that measures intracellular compound-target complex formation, as well as cellular permeability, specificity and cytotoxicity-the toxicity-affinity-permeability-selectivity (TAPS) technique. The TAPS assay is exemplified here using human kynurenine 3-monooxygenase (KMO), a challenging intracellular membrane protein target of significant current interest. TAPS confirmed target binding of known KMO inhibitors inside cells. We conclude that the TAPS assay can be used to facilitate intracellular hit validation on most, if not all intracellular drug targets.

Many si/shRNAs can kill cancer cells by targeting multiple survival genes through an off-target mechanism

PubMed Central

van Dongen, Stijn; Haluck-Kangas, Ashley; Sarshad, Aishe A; Bartom, Elizabeth T; Kim, Kwang-Youn A; Scholtens, Denise M; Hafner, Markus; Zhao, Jonathan C; Murmann, Andrea E

2017-01-01

Over 80% of multiple-tested siRNAs and shRNAs targeting CD95 or CD95 ligand (CD95L) induce a form of cell death characterized by simultaneous activation of multiple cell death pathways preferentially killing transformed and cancer stem cells. We now show these si/shRNAs kill cancer cells through canonical RNAi by targeting the 3’UTR of critical survival genes in a unique form of off-target effect we call DISE (death induced by survival gene elimination). Drosha and Dicer-deficient cells, devoid of most miRNAs, are hypersensitive to DISE, suggesting cellular miRNAs protect cells from this form of cell death. By testing 4666 shRNAs derived from the CD95 and CD95L mRNA sequences and an unrelated control gene, Venus, we have identified many toxic sequences - most of them located in the open reading frame of CD95L. We propose that specific toxic RNAi-active sequences present in the genome can kill cancer cells. PMID:29063830

Towards high-throughput automated targeted femtosecond laser-based transfection of adherent cells

NASA Astrophysics Data System (ADS)

Antkowiak, Maciej; Torres-Mapa, Maria Leilani; Gunn-Moore, Frank; Dholakia, Kishan

2011-03-01

Femtosecond laser induced cell membrane poration has proven to be an attractive alternative to the classical methods of drug and gene delivery. It is a selective, sterile, non-contact technique that offers a highly localized operation, low toxicity and consistent performance. However, its broader application still requires the development of robust, high-throughput and user-friendly systems. We present a system capable of unassisted enhanced targeted optoinjection and phototransfection of adherent mammalian cells with a femtosecond laser. We demonstrate the advantages of a dynamic diffractive optical element, namely a spatial light modulator (SLM) for precise three dimensional positioning of the beam. It enables the implementation of a "point-and-shoot" system in which using the software interface a user simply points at the cell and a predefined sequence of precisely positioned doses can be applied. We show that irradiation in three axial positions alleviates the problem of exact beam positioning on the cell membrane and doubles the number of viably optoinjected cells when compared with a single dose. The presented system enables untargeted raster scan irradiation which provides transfection of adherent cells at the throughput of 1 cell per second.

Cyclophosphamide induces bone marrow to yield higher numbers of precursor dendritic cells in vitro capable of functional antigen presentation to T cells in vivo

PubMed Central

Salem, Mohamed L.; El-Naggar, Sabry A.; Cole, David J.

2009-01-01

We have shown recently that cyclophosphamide (CTX) treatment induced a marked increase in the numbers of immature dendritic cells (DCs) in blood, coinciding with enhanced antigen-specific responses of the adoptively transferred CD8+ T cells. Because this DC expansion was preceded by DC proliferation in bone marrow (BM), we tested whether BM post CTX treatment can generate higher numbers of functional DCs. BM was harvested three days after treatment of C57BL/6 mice with PBS or CTX and cultured with GM-CSF/IL-4 in vitro. Compared with control, BM from CTX-treated mice showed faster generation and yielded higher numbers of DCs with superior activation in response to toll-like receptor (TLR) agonists. Vaccination with peptide-pulsed DCs generated from BM from CTX-treated mice induced comparable adjuvant effects to those induced by control DCs. Taken together, post CTX BM harbors higher numbers of DC precursors capable of differentiating into functional DCs, which be targeted to create host microenvironment riches in activated DCs upon treatment with TLR agonists. PMID:20036354

EGFR-targeted granzyme B expressed in NK cells enhances natural cytotoxicity and mediates specific killing of tumor cells.

PubMed

Oberoi, Pranav; Jabulowsky, Robert A; Bähr-Mahmud, Hayat; Wels, Winfried S

2013-01-01

Natural killer (NK) cells are highly specialized effectors of the innate immune system that hold promise for adoptive cancer immunotherapy. Their cell killing activity is primarily mediated by the pro-apoptotic serine protease granzyme B (GrB), which enters targets cells with the help of the pore-forming protein perforin. We investigated expression of a chimeric GrB fusion protein in NK cells as a means to augment their antitumoral activity. For selective targeting to tumor cells, we fused the epidermal growth factor receptor (EGFR) peptide ligand transforming growth factor α (TGFα) to human pre-pro-GrB. Established human NKL natural killer cells transduced with a lentiviral vector expressed this GrB-TGFα (GrB-T) molecule in amounts comparable to endogenous wildtype GrB. Activation of the genetically modified NK cells by cognate target cells resulted in the release of GrB-T together with endogenous granzymes and perforin, which augmented the effector cells' natural cytotoxicity against NK-sensitive tumor cells. Likewise, GrB-T was released into the extracellular space upon induction of degranulation with PMA and ionomycin. Secreted GrB-T fusion protein displayed specific binding to EGFR-overexpressing tumor cells, enzymatic activity, and selective target cell killing in the presence of an endosomolytic activity. Our data demonstrate that ectopic expression of a targeted GrB fusion protein in NK cells is feasible and can enhance antitumoral activity of the effector cells.

Energy by the Numbers: Fuel Cell Electric Vehicles

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

None

Energy by the Numbers presents fuel cell electric vehicles. Fuel cell electric vehicles (FCEVs) debuted in the U.S. in 2015. Three models are available in the United States. FCEVs run on hydrogen and only emit H2O.

Targeted next-generation sequencing at copy-number breakpoints for personalized analysis of rearranged ends in solid tumors.

PubMed

Kim, Hyun-Kyoung; Park, Won Cheol; Lee, Kwang Man; Hwang, Hai-Li; Park, Seong-Yeol; Sorn, Sungbin; Chandra, Vishal; Kim, Kwang Gi; Yoon, Woong-Bae; Bae, Joon Seol; Shin, Hyoung Doo; Shin, Jong-Yeon; Seoh, Ju-Young; Kim, Jong-Il; Hong, Kyeong-Man

2014-01-01

The concept of the utilization of rearranged ends for development of personalized biomarkers has attracted much attention owing to its clinical applicability. Although targeted next-generation sequencing (NGS) for recurrent rearrangements has been successful in hematologic malignancies, its application to solid tumors is problematic due to the paucity of recurrent translocations. However, copy-number breakpoints (CNBs), which are abundant in solid tumors, can be utilized for identification of rearranged ends. As a proof of concept, we performed targeted next-generation sequencing at copy-number breakpoints (TNGS-CNB) in nine colon cancer cases including seven primary cancers and two cell lines, COLO205 and SW620. For deduction of CNBs, we developed a novel competitive single-nucleotide polymorphism (cSNP) microarray method entailing CNB-region refinement by competitor DNA. Using TNGS-CNB, 19 specific rearrangements out of 91 CNBs (20.9%) were identified, and two polymerase chain reaction (PCR)-amplifiable rearrangements were obtained in six cases (66.7%). And significantly, TNGS-CNB, with its high positive identification rate (82.6%) of PCR-amplifiable rearrangements at candidate sites (19/23), just from filtering of aligned sequences, requires little effort for validation. Our results indicate that TNGS-CNB, with its utility for identification of rearrangements in solid tumors, can be successfully applied in the clinical laboratory for cancer-relapse and therapy-response monitoring.

Targeting Antigens to Dec-205 on Dendritic Cells Induces Immune Protection in Experimental Colitis in Mice

PubMed Central

Wadwa, Munisch; Klopfleisch, Robert; Buer, Jan; Westendorf, Astrid M.

2016-01-01

The endocytotic c-type lectin receptor DEC-205 is highly expressed on immature dendritic cells. In previous studies, it was shown that antigen-targeting to DEC-205 is a useful tool for the induction of antigen-specific Foxp3+ regulatory T cells and thereby can prevent inflammatory processes. However, whether this approach is sufficient to mediate tolerance in mucosal tissues like the gut is unknown. In this study, we established a new mouse model in which the adoptive transfer of naive hemagglutinin (HA)-specific CD4+Foxp3– T cells into VILLIN-HA transgenic mice leads to severe colitis. To analyze if antigen-targeting to DEC-205 could protect against inflammation of the gut, VILLIN-HA transgenic mice were injected with an antibody–antigen complex consisting of the immunogenic HA110–120 peptide coupled to an α-DEC-205 antibody (DEC-HA) before adoptive T cell transfer. DEC-HA-treated mice showed significantly less signs of intestinal inflammation as was demonstrated by reduced loss of body weight and histopathology in the gut. Strikingly, abrogated intestinal inflammation was mediated via the conversion of naive HA-specific CD4+Foxp3– T cells into HA-specific CD4+Foxp3+ regulatory T cells. In this study, we provide evidence that antigen-targeting to DEC-205 can be utilized for the induction of tolerance in mucosal organs that are confronted with large numbers of exogenous antigens. PMID:27141310

Adoptive therapy with CAR redirected T cells: the challenges in targeting solid tumors.

PubMed

Abken, Hinrich

2015-01-01

Recent spectacular success in the adoptive cell therapy of leukemia and lymphoma with chimeric antigen receptor (CAR)-modified T cells raised the expectations that this therapy may be efficacious in a wide range of cancer entities. The expectations are based on the predefined specificity of CAR T cells by an antibody-derived binding domain that acts independently of the natural T-cell receptor, recognizes targets independently of presentation by the major histocompatibility complex and allows targeting toward virtually any cell surface antigen. We here discuss that targeting CAR T cells toward solid tumors faces certain circumstances critical for the therapeutic success. Targeting tumor stroma and taking advantage of TRUCK cells, in other words, CAR T cells with inducible release of a transgenic payload, are some strategies envisaged to overcome current limitations in the near future.

Recombinant immunotoxins and retargeted killer cells: employing engineered antibody fragments for tumor-specific targeting of cytotoxic effectors.

PubMed

Wels, Winfried; Biburger, Markus; Müller, Tina; Dälken, Benjamin; Giesübel, Ulrike; Tonn, Torsten; Uherek, Christoph

2004-03-01

Over the past years, monoclonal antibodies have attracted enormous interest as targeted therapeutics, and a number of such reagents are in clinical use. However, responses could not be achieved in all patients with tumors expressing high levels of the respective target antigens, suggesting that other factors such as limited recruitment of endogenous immune effector mechanisms can also influence treatment outcome. This justifies the search for alternative, potentially more effective reagents. Antibody-toxins and cytolytic effector cells genetically modified to carry antibody-based receptors on the surface, represent such tailor-made targeting vehicles with the potential of improved tumor localization and enhanced efficacy. In this way, advances in recombinant antibody technology have made it possible to circumvent problems inherent in chemical coupling of antibodies and toxins, and have allowed construction via gene fusion of recombinant molecules which combine antibody-mediated recognition of tumor cells with specific delivery of potent protein toxins of bacterial or plant origin. Likewise, recombinant antibody fragments provide the basis for the construction of chimeric antigen receptors that, upon expression in cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells, link antibody-mediated recognition of tumor antigens with these effector cells' potent cytolytic activities, thereby making them promising cellular therapeutics for adoptive cancer therapy. Here, general principles for the derivation of cytotoxic proteins and effector cells with antibody-dependent tumor specificity are summarized, and current strategies to employ these molecules and cells for directed cancer therapy are discussed, focusing mainly on the tumor-associated antigens epidermal growth factor receptor (EGFR) and the closely related ErbB2 (HER2) as targets.

A Synthetic MUC1 Anticancer Vaccine Containing Mannose Ligands for Targeting Macrophages and Dendritic Cells.

PubMed

Glaffig, Markus; Stergiou, Natascha; Hartmann, Sebastian; Schmitt, Edgar; Kunz, Horst

2018-01-08

A MUC1 anticancer vaccine equipped with covalently linked divalent mannose ligands was found to improve the antigen uptake and presentation by targeting mannose-receptor-positive macrophages and dendritic cells. It induced much stronger specific IgG immune responses in mice than the non-mannosylated reference vaccine. Mannose coupling also led to increased numbers of macrophages, dendritic cells, and CD4 + T cells in the local lymph organs. Comparison of di- and tetravalent mannose ligands revealed an increased binding of the tetravalent version, suggesting that higher valency improves binding to the mannose receptor. The mannose-coupled vaccine and the non-mannosylated reference vaccine induced IgG antibodies that exhibited similar binding to human breast tumor cells. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Personalized targeted therapy for esophageal squamous cell carcinoma

PubMed Central

Kang, Xiaozheng; Chen, Keneng; Li, Yicheng; Li, Jianying; D'Amico, Thomas A; Chen, Xiaoxin

2015-01-01

Esophageal squamous cell carcinoma continues to heavily burden clinicians worldwide. Researchers have discovered the genomic landscape of esophageal squamous cell carcinoma, which holds promise for an era of personalized oncology care. One of the most pressing problems facing this issue is to improve the understanding of the newly available genomic data, and identify the driver-gene mutations, pathways, and networks. The emergence of a legion of novel targeted agents has generated much hope and hype regarding more potent treatment regimens, but the accuracy of drug selection is still arguable. Other problems, such as cancer heterogeneity, drug resistance, exceptional responders, and side effects, have to be surmounted. Evolving topics in personalized oncology, such as interpretation of genomics data, issues in targeted therapy, research approaches for targeted therapy, and future perspectives, will be discussed in this editorial. PMID:26167067

Systematic Identification of Combinatorial Drivers and Targets in Cancer Cell Lines

PubMed Central

Tabchy, Adel; Eltonsy, Nevine; Housman, David E.; Mills, Gordon B.

2013-01-01

There is an urgent need to elicit and validate highly efficacious targets for combinatorial intervention from large scale ongoing molecular characterization efforts of tumors. We established an in silico bioinformatic platform in concert with a high throughput screening platform evaluating 37 novel targeted agents in 669 extensively characterized cancer cell lines reflecting the genomic and tissue-type diversity of human cancers, to systematically identify combinatorial biomarkers of response and co-actionable targets in cancer. Genomic biomarkers discovered in a 141 cell line training set were validated in an independent 359 cell line test set. We identified co-occurring and mutually exclusive genomic events that represent potential drivers and combinatorial targets in cancer. We demonstrate multiple cooperating genomic events that predict sensitivity to drug intervention independent of tumor lineage. The coupling of scalable in silico and biologic high throughput cancer cell line platforms for the identification of co-events in cancer delivers rational combinatorial targets for synthetic lethal approaches with a high potential to pre-empt the emergence of resistance. PMID:23577104

Systematic identification of combinatorial drivers and targets in cancer cell lines.

PubMed

Tabchy, Adel; Eltonsy, Nevine; Housman, David E; Mills, Gordon B

2013-01-01

There is an urgent need to elicit and validate highly efficacious targets for combinatorial intervention from large scale ongoing molecular characterization efforts of tumors. We established an in silico bioinformatic platform in concert with a high throughput screening platform evaluating 37 novel targeted agents in 669 extensively characterized cancer cell lines reflecting the genomic and tissue-type diversity of human cancers, to systematically identify combinatorial biomarkers of response and co-actionable targets in cancer. Genomic biomarkers discovered in a 141 cell line training set were validated in an independent 359 cell line test set. We identified co-occurring and mutually exclusive genomic events that represent potential drivers and combinatorial targets in cancer. We demonstrate multiple cooperating genomic events that predict sensitivity to drug intervention independent of tumor lineage. The coupling of scalable in silico and biologic high throughput cancer cell line platforms for the identification of co-events in cancer delivers rational combinatorial targets for synthetic lethal approaches with a high potential to pre-empt the emergence of resistance.

Toward an animal model of cystic fibrosis: Targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells

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

Koller, B.H.; Hyungsuk Kim; Latour, A.M.

1991-12-01

A gene-targeting construct was made containing 7.8 kilobases of DNA spanning exon 10 of the mouse cystic fibrosis transmembrane regulator (CFTR) gene in which part of the exon has been replaced by two neomycin-resistance (Neo) genes driven by different promoters. (This replacement introduces a chain-termination codon at amino acid position 489 in the CFTR sequence). A herpes simplex thymidine kinase gene was on each end of the construct, which was electroporated into embryonic stem (ES) cells. Colonies resistant to G418, or to G418 plus ganciclovir, were selected and screened by Southern blotting or by PCR amplification. Five pools of G418-resistantmore » cells gave PCR products diagnostic of targeting. Four independent clones of ES cells with a disrupted CFTR gene have been isolated from these pools. The frequency of targeting was 1/2500 G418-resistant colonies. This low frequency is not the consequence of marginal expression of the Neo genes in the targeted cells. The CFTR targeting events were clustered among our experiments in a manner suggesting that some unidentified factor(s), possible passage number, influences the recovery of CFTR-targeted cells.« less

Microchimeric cells in systemic lupus erythematosus: targets or innocent bystanders?

PubMed

Stevens, A M

2006-01-01

During pregnancy maternal and fetal cells commute back and forth leading to fetal microchimerism in the mother and maternal microchimerism in the child that can persist for years after the birth. Chimeric fetal and maternal cells can be hematopoietic or can differentiate into somatic cells in multiple organs, potentially acting as targets for 'autoimmunity' and so have been implicated in the pathogenesis of autoimmune diseases that resemble graft-versus-host disease after stem cell transplantation. Fetal cells have been found in women with systemic lupus erythematosus, both in the blood and a target organ, the kidney, suggesting that they may be involved in pathogenesis. Future studies will address how the host immune system normally tolerates maternal and fetal cells or how the balance may change during autoimmunity.

Free Extracellular miRNA Functionally Targets Cells by Transfecting Exosomes from Their Companion Cells.

PubMed

Bryniarski, Krzysztof; Ptak, Wlodzimierz; Martin, Emilia; Nazimek, Katarzyna; Szczepanik, Marian; Sanak, Marek; Askenase, Philip W

2015-01-01

Lymph node and spleen cells of mice doubly immunized by epicutaneous and intravenous hapten application produce a suppressive component that inhibits the action of the effector T cells that mediate contact sensitivity reactions. We recently re-investigated this phenomenon in an immunological system. CD8+ T lymphocyte-derived exosomes transferred suppressive miR-150 to the effector T cells antigen-specifically due to exosome surface coat of antibody light chains made by B1a lymphocytes. Extracellular RNA (exRNA) is protected from plasma RNases by carriage in exosomes or by chaperones. Exosome transfer of functional RNA to target cells is well described, whereas the mechanism of transfer of exRNA free of exosomes remains unclear. In the current study we describe extracellular miR-150, extracted from exosomes, yet still able to mediate antigen-specific suppression. We have determined that this was due to miR-150 association with antibody-coated exosomes produced by B1a cell companions of the effector T cells, which resulted in antigen-specific suppression of their function. Thus functional cell targeting by free exRNA can proceed by transfecting companion cell exosomes that then transfer RNA cargo to the acceptor cells. This contrasts with the classical view on release of RNA-containing exosomes from the multivesicular bodies for subsequent intercellular targeting. This new alternate pathway for transfer of exRNA between cells has distinct biological and immunological significance, and since most human blood exRNA is not in exosomes may be relevant to evaluation and treatment of diseases.

Free Extracellular miRNA Functionally Targets Cells by Transfecting Exosomes from Their Companion Cells

PubMed Central

Bryniarski, Krzysztof; Ptak, Wlodzimierz; Martin, Emilia; Nazimek, Katarzyna; Szczepanik, Marian; Sanak, Marek; Askenase, Philip W.

2015-01-01

Lymph node and spleen cells of mice doubly immunized by epicutaneous and intravenous hapten application produce a suppressive component that inhibits the action of the effector T cells that mediate contact sensitivity reactions. We recently re-investigated this phenomenon in an immunological system. CD8+ T lymphocyte-derived exosomes transferred suppressive miR-150 to the effector T cells antigen-specifically due to exosome surface coat of antibody light chains made by B1a lymphocytes. Extracellular RNA (exRNA) is protected from plasma RNases by carriage in exosomes or by chaperones. Exosome transfer of functional RNA to target cells is well described, whereas the mechanism of transfer of exRNA free of exosomes remains unclear. In the current study we describe extracellular miR-150, extracted from exosomes, yet still able to mediate antigen-specific suppression. We have determined that this was due to miR-150 association with antibody-coated exosomes produced by B1a cell companions of the effector T cells, which resulted in antigen-specific suppression of their function. Thus functional cell targeting by free exRNA can proceed by transfecting companion cell exosomes that then transfer RNA cargo to the acceptor cells. This contrasts with the classical view on release of RNA-containing exosomes from the multivesicular bodies for subsequent intercellular targeting. This new alternate pathway for transfer of exRNA between cells has distinct biological and immunological significance, and since most human blood exRNA is not in exosomes may be relevant to evaluation and treatment of diseases. PMID:25923429

Adoptive immunotherapy for B-cell malignancies with autologous chimeric antigen receptor modified tumor targeted T cells.

PubMed

Park, Jae H; Brentjens, Renier J

2010-04-01

Chemotherapy-resistant B-cell hematologic malignancies may be cured with allogeneic hematopoietic stem cell transplantation (HSCT), demonstrating the potential susceptibility of these tumors to donor T-cell mediated immune responses. However, high rates of transplant-related morbidity and mortality limit this approach. For this reason, there is an urgent need for less-toxic forms of immune-based cellular therapy to treat these malignancies. Adoptive transfer of autologous T cells genetically modified to express chimeric antigen receptors (CARs) targeted to specific tumor-associated antigens represents an attractive means of overcoming the limitations of conventional HSCT. To this end, investigators have generated CARs targeted to various antigens expressed by B-cell malignancies, optimized the design of these CARs to enhance receptor mediated T cell signaling, and demonstrated significant anti-tumor efficacy of the resulting CAR modified T cells both in vitro and in vivo mouse tumor models. These encouraging preclinical data have justified the translation of this approach to the clinical setting with currently 12 open clinical trials and one completed clinical trial treating various B-cell malignancies utilizing CAR modified T cells targeted to either the CD19 or CD20 B-cell specific antigens.

Vesicle-associated membrane protein 7 (VAMP-7) is essential for target cell killing in a natural killer cell line

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

Marcet-Palacios, Marcelo; Odemuyiwa, Solomon O.; Coughlin, Jason J.

2008-02-15

Natural killer cells recognize and induce apoptosis in foreign, transformed or virus-infected cells through the release of perforin and granzymes from secretory lysosomes. Clinically, NK-cell mediated killing is a major limitation to successful allo- and xenotransplantation. The molecular mechanisms that regulate the fusion of granzyme B-containing secretory lysosomes to the plasma membrane in activated NK cells, prior to target cell killing, are not fully understood. Using the NK cell line YT-Indy as a model, we have investigated the expression of SNAP REceptors (SNAREs), both target (t-) and vesicular (v-) SNAREs, and their function in granzyme B-mediated target cell killing. Ourmore » data showed that YT-Indy cells express VAMP-7 and SNAP-23, but not VAMP-2. VAMP-7 was associated with granzyme B-containing lysosomal granules. Using VAMP-7 small interfering RNA (siRNA), we successfully knocked down the expression of VAMP-7 protein in YT-Indy to less than 10% of untreated cells in 24 h. VAMP7-deficient YT-Indy cells activated via co-culture with Jurkat cells released <1 ng/mL of granzyme B, compared to 1.5-2.5 {mu}g/mL from controls. Using Jurkat cells as targets, we showed a 7-fold reduction in NK cell-mediated killing by VAMP-7 deficient YT-Indy cells. Our results show that VAMP-7 is a crucial component of granzyme B release and target cell killing in the NK cell line YT-Indy. Thus, targeting VAMP-7 expression specifically with siRNA, following transplantation, may be a viable strategy for preventing NK cell-mediated transplant rejection, in vivo.« less

Deep magnetic capture of magnetically loaded cells for spatially targeted therapeutics.

PubMed

Huang, Zheyong; Pei, Ning; Wang, Yanyan; Xie, Xinxing; Sun, Aijun; Shen, Li; Zhang, Shuning; Liu, Xuebo; Zou, Yunzeng; Qian, Juying; Ge, Junbo

2010-03-01

Magnetic targeting has recently demonstrated potential in promoting magnetically loaded cell delivery to target lesion, but its application is limited by magnetic attenuation. For deep magnetic capture of cells for spatial targeting therapeutics, we designed a magnetic pole, in which the magnetic field density can be focused at a distance from the pole. As flowing through a tube served as a model of blood vessels, the magnetically loaded mesenchymal stem cells (MagMSCs) were highly enriched at the site distance from the magnetic pole. The cell capture efficiency was positively influenced by the magnetic flux density, and inversely influenced by the flow velocity, and well-fitted with the deductive value by theoretical considerations. It appeared to us that the spatially-focused property of the magnetic apparatus promises a new deep targeting strategy to promote homing and engraftment for cellular therapy. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Controversies in cancer stem cells: targeting embryonic signaling pathways.

PubMed

Takebe, Naoko; Ivy, S Percy

2010-06-15

Selectively targeting cancer stem cells (CSC) or tumor-initiating cells (TIC; from this point onward referred to as CSCs) with novel agents is a rapidly emerging field of oncology. Our knowledge of CSCs and their niche microenvironments remains a nascent field. CSC's critical dependence upon self-renewal makes these regulatory signaling pathways ripe for the development of experimental therapeutic agents. Investigational agents targeting the Notch, Hedgehog, and Wnt pathways are currently in late preclinical development stages, with some early phase 1-2 testing in human subjects. This series of articles will provide an overview and summary of the current state of knowledge of CSCs, their interactive microenvironment, and how they may serve as important targets for antitumor therapies. We also examine the scope and stage of development of early experimental agents that specifically target these highly conserved embryonic signaling pathways. (c) 2010 AACR.

Liver cell-targeted delivery of therapeutic molecules.

PubMed

Kang, Jeong-Hun; Toita, Riki; Murata, Masaharu

2016-01-01

The liver is the largest internal organ in mammals and is involved in metabolism, detoxification, synthesis of proteins and lipids, secretion of cytokines and growth factors and immune/inflammatory responses. Hepatitis, alcoholic or non-alcoholic liver disease, hepatocellular carcinoma, hepatic veno-occlusive disease, and liver fibrosis and cirrhosis are the most common liver diseases. Safe and efficient delivery of therapeutic molecules (drugs, genes or proteins) into the liver is very important to increase the clinical efficacy of these molecules and to reduce their side effects in other organs. Several liver cell-targeted delivery systems have been developed and tested in vivo or ex vivo/in vitro. In this review, we discuss the literature concerning liver cell-targeted delivery systems, with a particular emphasis on the results of in vivo studies.

Metabolic and structural integrity of magnetic nanoparticle-loaded primary endothelial cells for targeted cell therapy.

PubMed

Orynbayeva, Zulfiya; Sensenig, Richard; Polyak, Boris

2015-05-01

To successfully translate magnetically mediated cell targeting from bench to bedside, there is a need to systematically assess the potential adverse effects of magnetic nanoparticles (MNPs) interacting with 'therapeutic' cells. Here, we examined in detail the effects of internalized polymeric MNPs on primary rat endothelial cells' structural intactness, metabolic integrity and proliferation potential. The intactness of cytoskeleton and organelles was studied by fluorescent confocal microscopy, flow cytometry and high-resolution respirometry. MNP-loaded primary endothelial cells preserve intact cytoskeleton and organelles, maintain normal rate of proliferation, calcium signaling and mitochondria energy metabolism. This study provides supportive evidence that MNPs at doses necessary for targeting did not induce significant adverse effects on structural integrity and functionality of primary endothelial cells - potential cell therapy vectors.

Targeting Vaccine-Induced Extrafollicular Pathway of B Cell Differentiation Improves Rabies Postexposure Prophylaxis

PubMed Central

Haley, Shannon L.; Tzvetkov, Evgeni P.; Meuwissen, Samantha; Plummer, Joseph R.

2017-01-01

ABSTRACT Vaccine-induced B cells differentiate along two pathways. The follicular pathway gives rise to germinal centers (GCs) that can take weeks to fully develop. The extrafollicular pathway gives rise to short-lived plasma cells (PCs) that can rapidly secrete protective antibodies within days of vaccination. Rabies virus (RABV) postexposure prophylaxis (PEP) requires rapid vaccine-induced humoral immunity for protection. Therefore, we hypothesized that targeting extrafollicular B cell responses for activation would improve the speed and magnitude of RABV PEP. To test this hypothesis, we constructed, recovered, and characterized a recombinant RABV-based vaccine expressing murine B cell activating factor (BAFF) (rRABV-mBAFF). BAFF is an ideal molecule to improve early pathways of B cell activation, as it links innate and adaptive immunity, promoting potent B cell responses. Indeed, rRABV-mBAFF induced a faster, higher antibody response in mice and enhanced survivorship in PEP settings compared to rRABV. Interestingly, rRABV-mBAFF and rRABV induced equivalent numbers of GC B cells, suggesting that rRABV-mBAFF augmented the extrafollicular B cell pathway. To confirm that rRABV-mBAFF modulated the extrafollicular pathway, we used a signaling lymphocytic activation molecule (SLAM)-associated protein (SAP)-deficient mouse model. In response to antigen, SAP-deficient mice form extrafollicular B cell responses but do not generate GCs. rRABV-mBAFF induced similar anti-RABV antibody responses in SAP-deficient and wild-type mice, demonstrating that BAFF modulated immunity through the extrafollicular and not the GC B cell pathway. Collectively, strategies that manipulate pathways of B cell activation may facilitate the development of a single-dose RABV vaccine that replaces current complicated and costly RABV PEP. IMPORTANCE Effective RABV PEP is currently resource- and cost-prohibitive in regions of the world where RABV is most prevalent. In order to diminish the

Generation of T-cell receptors targeting a genetically stable and immunodominant cytotoxic T-lymphocyte epitope within hepatitis C virus non-structural protein 3.

PubMed

Pasetto, Anna; Frelin, Lars; Brass, Anette; Yasmeen, Anila; Koh, Sarene; Lohmann, Volker; Bartenschlager, Ralf; Magalhaes, Isabelle; Maeurer, Markus; Sällberg, Matti; Chen, Margaret

2012-02-01

Hepatitis C virus (HCV) is a major cause of severe liver disease, and one major contributing factor is thought to involve a dysfunction of virus-specific T-cells. T-cell receptor (TCR) gene therapy with HCV-specific TCRs would increase the number of effector T-cells to promote virus clearance. We therefore took advantage of HLA-A2 transgenic mice to generate multiple TCR candidates against HCV using DNA vaccination followed by generation of stable T-cell-BW (T-BW) tumour hybrid cells. Using this approach, large numbers of non-structural protein 3 (NS3)-specific functional T-BW hybrids can be generated efficiently. These predominantly target the genetically stable HCV genotype 1 NS3(1073-1081) CTL epitope, frequently associated with clearance of HCV in humans. These T-BW hybrid clones recognized the NS3(1073) peptide with a high avidity. The hybridoma effectively recognized virus variants and targeted cells with low HLA-A2 expression, which has not been reported previously. Importantly, high-avidity murine TCRs effectively redirected human non-HCV-specific T-lymphocytes to recognize human hepatoma cells with HCV RNA replication driven by a subgenomic HCV replicon. Taken together, TCR candidates with a range of functional avidities, which can be used to study immune recognition of HCV-positive targets, have been generated. This has implications for TCR-related immunotherapy against HCV.

A 20-Amino Acid Module of Protein Kinase Cϵ Involved in Translocation and Selective Targeting at Cell-Cell Contacts*

PubMed Central

Diouf, Barthélémy; Collazos, Alejandra; Labesse, Gilles; Macari, Françoise; Choquet, Armelle; Clair, Philippe; Gauthier-Rouvière, Cécile; Guérineau, Nathalie C.; Jay, Philippe; Hollande, Frédéric; Joubert, Dominique

2009-01-01

In the pituitary gland, activated protein kinase C (PKC) isoforms accumulate either selectively at the cell-cell contact (α and ϵ) or at the entire plasma membrane (β1 and δ). The molecular mechanisms underlying these various subcellular locations are not known. Here, we demonstrate the existence within PKCϵ of a cell-cell contact targeting sequence (3CTS) that, upon stimulation, is capable of targeting PKCδ, chimerin-α1, and the PKCϵ C1 domain to the cell-cell contact. We show that this selective targeting of PKCϵ is lost upon overexpression of 3CTS fused to a (R-Ahx-R)4 (where Ahx is 6-aminohexanoic acid) vectorization peptide, reflecting a dominant-negative effect of the overexpressed 3CTS on targeting selectivity. 3CTS contains a putative amphipathic α-helix, a 14-3-3-binding site, and the Glu-374 amino acid, involved in targeting selectivity. We show that the integrity of the α-helix is important for translocation but that 14-3-3 is not involved in targeting selectivity. However, PKCϵ translocation is increased when PKCϵ/14-3-3 interaction is abolished, suggesting that phorbol 12-myristate 13-acetate activation may initiate two sets of PKCϵ functions, those depending on 14-3-3 and those depending on translocation to cell-cell contacts. Thus, 3CTS is involved in the modulation of translocation via its 14-3-3-binding site, in cytoplasmic desequestration via the α-helix, and in selective PKCϵ targeting at the cell-cell contact via Glu-374. PMID:19429675

E-selectin liposomal and nanotube-targeted delivery of doxorubicin to circulating tumor cells

PubMed Central

Mitchell, Michael J.; Chen, Christina S.; Ponmudi, Varun; Hughes, Andrew D.; King, Michael R.

2012-01-01

The presence of circulating tumor cells (CTCs) is believed to lead to the formation of secondary tumors via an adhesion cascade involving interaction between adhesion receptors of endothelial cells and ligands on CTCs. Many CTCs express sialylated carbohydrate ligands on their surfaces that adhere to selectin protein found on inflamed endothelial cells. We have investigated the feasibility of using immobilized selectin proteins as a targeting mechanism for CTCs under flow. Herein, targeted liposomal doxorubicin (L-DXR) was functionalized with recombinant human E-selectin (ES) and polyethylene glycol (PEG) to target and kill cancer cells under shear flow, both when immobilized along a microtube device or sheared in a cone-and-plate viscometer in a dilute suspension. Healthy circulating cells such as red blood cells were not targeted by this mechanism and were left to freely circulate, and minimal leukocyte death was observed. Halloysite nanotube (HNT)-coated microtube devices immobilized with nanoscale liposomes significantly enhanced the targeting, capture, and killing of cancer cells. This work demonstrates that E-selectin functionalized L-DXR, sheared in suspension or immobilized onto microtube devices, provides a novel approach to selectively target and deliver chemotherapeutics to CTCs in the bloodstream. PMID:22421423

Integrated DNA/RNA targeted genomic profiling of diffuse large B-cell lymphoma using a clinical assay.

PubMed

Intlekofer, Andrew M; Joffe, Erel; Batlevi, Connie L; Hilden, Patrick; He, Jie; Seshan, Venkatraman E; Zelenetz, Andrew D; Palomba, M Lia; Moskowitz, Craig H; Portlock, Carol; Straus, David J; Noy, Ariela; Horwitz, Steven M; Gerecitano, John F; Moskowitz, Alison; Hamlin, Paul; Matasar, Matthew J; Kumar, Anita; van den Brink, Marcel R; Knapp, Kristina M; Pichardo, Janine D; Nahas, Michelle K; Trabucco, Sally E; Mughal, Tariq; Copeland, Amanda R; Papaemmanuil, Elli; Moarii, Mathai; Levine, Ross L; Dogan, Ahmet; Miller, Vincent A; Younes, Anas

2018-06-12

We sought to define the genomic landscape of diffuse large B-cell lymphoma (DLBCL) by using formalin-fixed paraffin-embedded (FFPE) biopsy specimens. We used targeted sequencing of genes altered in hematologic malignancies, including DNA coding sequence for 405 genes, noncoding sequence for 31 genes, and RNA coding sequence for 265 genes (FoundationOne-Heme). Short variants, rearrangements, and copy number alterations were determined. We studied 198 samples (114 de novo, 58 previously treated, and 26 large-cell transformation from follicular lymphoma). Median number of GAs per case was 6, with 97% of patients harboring at least one alteration. Recurrent GAs were detected in genes with established roles in DLBCL pathogenesis (e.g. MYD88, CREBBP, CD79B, EZH2), as well as notable differences compared to prior studies such as inactivating mutations in TET2 (5%). Less common GAs identified potential targets for approved or investigational therapies, including BRAF, CD274 (PD-L1), IDH2, and JAK1/2. TP53 mutations were more frequently observed in relapsed/refractory DLBCL, and predicted for lack of response to first-line chemotherapy, identifying a subset of patients that could be prioritized for novel therapies. Overall, 90% (n = 169) of the patients harbored a GA which could be explored for therapeutic intervention, with 54% (n = 107) harboring more than one putative target.

Concise Review: Cell Surface N-Linked Glycoproteins as Potential Stem Cell Markers and Drug Targets.

PubMed

Boheler, Kenneth R; Gundry, Rebekah L

2017-01-01

Stem cells and their derivatives hold great promise to advance regenerative medicine. Critical to the progression of this field is the identification and utilization of antibody-accessible cell-surface proteins for immunophenotyping and cell sorting-techniques essential for assessment and isolation of defined cell populations with known functional and therapeutic properties. Beyond their utility for cell identification and selection, cell-surface proteins are also major targets for pharmacological intervention. Although comprehensive cell-surface protein maps are highly valuable, they have been difficult to define until recently. In this review, we discuss the application of a contemporary targeted chemoproteomic-based technique for defining the cell-surface proteomes of stem and progenitor cells. In applying this approach to pluripotent stem cells (PSCs), these studies have improved the biological understanding of these cells, led to the enhanced use and development of antibodies suitable for immunophenotyping and sorting, and contributed to the repurposing of existing drugs without the need for high-throughput screening. The utility of this latter approach was first demonstrated with human PSCs (hPSCs) through the identification of small molecules that are selectively toxic to hPSCs and have the potential for eliminating confounding and tumorigenic cells in hPSC-derived progeny destined for research and transplantation. Overall, the cutting-edge technologies reviewed here will accelerate the development of novel cell-surface protein targets for immunophenotyping, new reagents to improve the isolation of therapeutically qualified cells, and pharmacological studies to advance the treatment of intractable diseases amenable to cell-replacement therapies. Stem Cells Translational Medicine 2017;6:131-138. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

PubMed Central

Unzueta, Ugutz; Céspedes, María Virtudes; Ferrer-Miralles, Neus; Casanova, Isolda; Cedano, Juan; Corchero, José Luis; Domingo-Espín, Joan; Villaverde, Antonio; Mangues, Ramón; Vázquez, Esther

2012-01-01

Background Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4) is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing. Results Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer. Conclusion Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles, and imaging agents. PMID:22923991

miR-1271 promotes non-small-cell lung cancer cell proliferation and invasion via targeting HOXA5

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

Wang, Yongfang; Xu, Lianhong; Jiang, Lixin, E-mail: jianglx66766@163.com

2015-03-13

MicroRNAs (miRNAs) are short, non-coding RNAs (∼22 nt) that play important roles in the pathogenesis of human diseases by negatively regulating numerous target genes at posttranscriptional level. However, the role of microRNAs in lung cancer, particularly non-small-cell lung cancer (NSCLC), has remained elusive. In this study, two microRNAs, miR-1271 and miR-628, and their predicted target genes were identified differentially expressed in NSCLC by analyzing the miRNA and mRNA expression data from NSCLC tissues and their matching normal controls. miR-1271 and its target gene HOXA5 were selected for further investigation. CCK-8 proliferation assay showed that the cell proliferation was promoted by miR-1271more » in NSCLC cells, while miR-1271 inhibitor could significantly inhibited the proliferation of NSCLC cells. Interestingly, migration and invasion assay indicated that overexpression of miR-1271 could significantly promoted the migration and invasion of NSCLC cells, whereas miR-1271 inhibitor could inhibited both cell migration and invasion of NSCLC cells. Western blot showed that miR-1271 suppressed the protein level of HOXA5, and luciferase assays confirmed that miR-1271 directly bound to the 3'untranslated region of HOXA5. This study indicated indicate that miR-1271 regulates NSCLC cell proliferation and invasion, via the down-regulation of HOXA5. Thus, miR-1271 may represent a potential therapeutic target for NSCLC intervention. - Highlights: • Overexpression of miR-1271 promoted proliferation and invasion of NSCLC cells. • miR-1271 inhibitor inhibited the proliferation and invasion of NSCLC cells. • miR-1271 targets 3′ UTR of HOXA5 in NSCLC cells. • miR-1271 negatively regulates HOXA5 in NSCLC cells.« less

MicroRNA-9 regulates non-small cell lung cancer cell invasion and migration by targeting eukaryotic translation initiation factor 5A2.

PubMed

Xu, Guodong; Shao, Guofeng; Pan, Qiaoling; Sun, Lebo; Zheng, Dawei; Li, Minghui; Li, Ni; Shi, Huoshun; Ni, Yiming

2017-01-01

MicroRNAs (miRNAs) play a critical role in cancer development and progression. Bioinformatics analyses has identified eukaryotic translation initiation factor 5A2 (eIF5A2) as a target of miR-9. In this study, we attempted to determine whether miR-9 regulates non-small cell lung cancer (NSCLC) cell invasion and migration by targeting eIF5A2 We examined eIF5A2 expression using reverse transcription-quantitative PCR (RT-qPCR) and subsequently transfected A549 and NCI-H1299 NSCLC cells with a miR-9 mimic or miR-9 inhibitor to determine the migration and invasive capability of the cells via wound healing assay and Transwell invasion assay, respectively. E-cadherin and vimentin expression was detected with western blotting. The miR-9 mimic significantly reduced NSCLC cell invasive and metastatic ability, and the miR-9 inhibitor enhanced NSCLC cell migration activity, increasing the number of migrated cells. There was no significant difference between the negative control siRNA and miR-9 mimic groups after knockdown of eIF5A2; western blotting showed that miR-9 regulated E-cadherin and vimentin expression. These data show that miR-9 regulates NSCLC cell invasion and migration through regulating eIF5A2 expression. Taken together, our findings suggest that the mechanism of miR-9-regulated NSCLC cell invasion and migration may be related to epithelial-mesenchymal transition.

Targeted Cellular Drug Delivery using Tailored Dendritic Nanostructures

NASA Astrophysics Data System (ADS)

Kannan, Rangaramanujam; Kolhe, Parag; Kannan, Sujatha; Lieh-Lai, Mary

2002-03-01

Dendrimers and hyperbranched polymers possess highly branched architectures, with a large number of controllable, tailorble, ‘peripheral’ functionalities. Since the surface chemistry of these materials can be modified with relative ease, these materials have tremendous potential in targeted drug and gene delivery. The large number of end groups can also be tailored to create special affinity to targeted cells, and can also encapsulate drugs and deliver them in a controlled manner. We are developing tailor-modified dendritic systems for drug delivery. Synthesis, in-vitro drug loading, in-vitro drug delivery, and the targeting efficiency to the cell are being studied systematically using a wide variety of experimental tools. Polyamidoamine and Polyol dendrimers, with different generations and end-groups are studied, with drugs such as Ibuprofen and Methotrexate. Our results indicate that a large number of drug molecules can be encapsulated/attached to the dendrimers, depending on the end groups. The drug-encapsulated dendrimer is able to enter the cells rapidly and deliver the drug. Targeting strategies being explored

A perspective on B-cell-targeting therapy for SLE.

PubMed

Looney, R John; Anolik, Jennifer; Sanz, Inaki

2010-02-01

In recent years, large controlled trials have tested several new agents for systemic lupus erythematosus (SLE). Unfortunately, none of these trials has met its primary outcome. This does not mean progress has not been made. In fact, a great deal has been learned about doing clinical trials in lupus and about the biological and clinical effects of the drugs being tested. Many of these drugs were designed to target B cells directly, e.g., rituximab, belimumab, epratuzumab, and transmembrane activator and calcium modulator and cyclophilin ligand interactor-immunoglobulin (TACI-Ig). The enthusiasm for targeting B cells derives from substantial evidence showing the critical role of B cells in murine models of SLE, as well promising results from multiple open trials with rituximab, a chimeric anti-CD20 monoclonal antibody that specifically depletes B cells (Martin and Chan in Immunity 20(5):517-527, 2004; Sobel et al. in J Exp Med 173:1441-1449, 1991; Silverman and Weisman in Arthritis Rheum 48:1484-1492, 2003; Silverman in Arthritis Rheum 52(4):1342, 2005; Shlomchik et al. in Nat Rev Immunol 1:147-153, 2001; Looney et al. in Arthritis Rheum 50:2580-2589, 2004; Lu et al. in Arthritis Rheum 61(4):482-487, 2009; Saito et al. in Lupus 12(10):798-800, 2003; van Vollenhoven et al. in Scand J Rheumatol 33(6):423-427, 2004; Sfikakis et al. Arthritis Rheum 52(2):501-513, 2005). Why have the controlled trials of B-cell-targeting therapies failed to demonstrate efficacy? Were there flaws in design or execution of these trials? Or, were promising animal studies and open trials misleading, as so often happens? This perspective discusses the current state of B-cell-targeting therapies for human lupus and the future development of these therapies.

VEGFR2-targeted fusion antibody improved NK cell-mediated immunosurveillance against K562 cells.

PubMed

Ren, Xueyan; Xie, Wei; Wang, Youfu; Xu, Menghuai; Liu, Fang; Tang, Mingying; Li, Chenchen; Wang, Min; Zhang, Juan

2016-08-01

MHC class I polypeptide-related sequence A (MICA), which is normally expressed on cancer cells, activates NK cells via NK group 2-member D pathway. However, some cancer cells escape NK-mediated immune surveillance by shedding membrane MICA causing immune suppression. To address this issue, we designed an antibody-MICA fusion targeting tumor-specific antigen (vascular endothelial growth factor receptor 2, VEGFR2) based on our patented antibody (mAb04) against VEGFR2. In vitro results demonstrate that the fusion antibody retains both the antineoplastic and the immunomodulatory activity of mAb04. Further, we revealed that it enhanced NK-mediated immunosurveillance against K562 cells through increasing degranulation and cytokine production of NK cells. The overall data suggest our new fusion protein provides a promising approach for cancer-targeted immunotherapy and has prospects for potential application of chronic myeloid leukemia.

Catechol polymers for pH-responsive, targeted drug delivery to cancer cells.

PubMed

Su, Jing; Chen, Feng; Cryns, Vincent L; Messersmith, Phillip B

2011-08-10

A novel cell-targeting, pH-sensitive polymeric carrier was employed in this study for delivery of the anticancer drug bortezomib (BTZ) to cancer cells. Our strategy is based on facile conjugation of BTZ to catechol-containing polymeric carriers that are designed to be taken up selectively by cancer cells through cell surface receptor-mediated mechanisms. The polymer used as a building block in this study was poly(ethylene glycol), which was chosen for its ability to reduce nonspecific interactions with proteins and cells. The catechol moiety was exploited for its ability to bind and release borate-containing therapeutics such as BTZ in a pH-dependent manner. In acidic environments, such as in cancer tissue or the subcellular endosome, BTZ dissociates from the polymer-bound catechol groups to liberate the free drug, which inhibits proteasome function. A cancer-cell-targeting ligand, biotin, was presented on the polymer carriers to facilitate targeted entry of drug-loaded polymer carriers into cancer cells. Our study demonstrated that the cancer-targeting drug-polymer conjugates dramatically enhanced cellular uptake, proteasome inhibition, and cytotoxicity toward breast carcinoma cells in comparison with nontargeting drug-polymer conjugates. The pH-sensitive catechol-boronate binding mechanism provides a chemoselective approach for controlling the release of BTZ in targeted cancer cells, establishing a concept that may be applied in the future toward other boronic acid-containing therapeutics to treat a broad range of diseases. © 2011 American Chemical Society

Targeting stromal glutamine synthetase in tumors disrupts tumor microenvironment-regulated cancer cell growth

USDA-ARS?s Scientific Manuscript database

Reactive stromal cells are an integral part of tumor microenvironment (TME) and interact with cancer cells to regulate their growth. Although targeting stromal cells could be a viable therapy to regulate the communication between TME and cancer cells, identification of stromal targets that make canc...

Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells

PubMed Central

Sloan, Derek D.; Lam, Chia-Ying Kao; Irrinki, Alivelu; Liu, Liqin; Tsai, Angela; Pace, Craig S.; Kaur, Jasmine; Murry, Jeffrey P.; Balakrishnan, Mini; Moore, Paul A.; Johnson, Syd; Nordstrom, Jeffrey L.; Cihlar, Tomas; Koenig, Scott

2015-01-01

HIV reservoirs and production of viral antigens are not eliminated in chronically infected participants treated with combination antiretroviral therapy (cART). Novel therapeutic strategies aiming at viral reservoir elimination are needed to address chronic immune dysfunction and non-AIDS morbidities that exist despite effective cART. The HIV envelope protein (Env) is emerging as a highly specific viral target for therapeutic elimination of the persistent HIV-infected reservoirs via antibody-mediated cell killing. Dual-Affinity Re-Targeting (DART) molecules exhibit a distinct mechanism of action via binding the cell surface target antigen and simultaneously engaging CD3 on cytotoxic T lymphocytes (CTLs). We designed and evaluated Env-specific DARTs (HIVxCD3 DARTs) derived from known antibodies recognizing diverse Env epitopes with or without broadly neutralizing activity. HIVxCD3 DARTs derived from PGT121, PGT145, A32, and 7B2, but not VRC01 or 10E8 antibodies, mediated potent CTL-dependent killing of quiescent primary CD4 T cells infected with diverse HIV isolates. Similar killing activity was also observed with DARTs structurally modified for in vivo half-life extension. In an ex vivo model using cells isolated from HIV-infected participants on cART, combinations of the most potent HIVxCD3 DARTs reduced HIV expression both in quiescent and activated peripheral blood mononuclear cell cultures isolated from HIV-infected participants on suppressive cART. Importantly, HIVxCD3 DARTs did not induce cell-to-cell virus spread in resting or activated CD4 T cell cultures. Collectively, these results provide support for further development of HIVxCD3 DARTs as a promising therapeutic strategy for targeting HIV reservoirs. PMID:26539983

Circulating and disseminated tumor cells: diagnostic tools and therapeutic targets in motion

PubMed Central

Lin, Peter P.; Gires, Olivier

2017-01-01

Enumeration of circulating tumor cells (CTCs) in peripheral blood with the gold standard CellSearchTM has proven prognostic value for tumor recurrence and progression of metastatic disease. Therefore, the further molecular characterization of isolated CTCs might have clinical relevance as liquid biopsy for therapeutic decision-making and to monitor disease progression. The direct analysis of systemic cancer appears particularly important in view of the known disparity in expression of therapeutic targets as well as epithelial-to-mesenchymal transition (EMT)-based heterogeneity between primary and systemic tumor cells, which all substantially complicate monitoring and therapeutic targeting at present. Since CTCs are the potential precursor cells of metastasis, their in-depth molecular profiling should also provide a useful resource for target discovery. The present review will discuss the use of systemically spread cancer cells as liquid biopsy and focus on potential target antigens. PMID:27683128

Targeted delivery of celastrol to mesangial cells is effective against mesangioproliferative glomerulonephritis.

PubMed

Guo, Ling; Luo, Shi; Du, Zhengwu; Zhou, Meiling; Li, Peiwen; Fu, Yao; Sun, Xun; Huang, Yuan; Zhang, Zhirong

2017-10-12

Mesangial cells-mediated glomerulonephritis is a frequent cause of end-stage renal disease. Here, we show that celastrol is effective in treating both reversible and irreversible mesangioproliferative glomerulonephritis in rat models, but find that its off-target distributions cause severe systemic toxicity. We thus target celastrol to mesangial cells using albumin nanoparticles. Celastrol-albumin nanoparticles crosses fenestrated endothelium and accumulates in mesangial cells, alleviating proteinuria, inflammation, glomerular hypercellularity, and excessive extracellular matrix deposition in rat anti-Thy1.1 nephritis models. Celastrol-albumin nanoparticles presents lower drug accumulation than free celastrol in off-target organs and tissues, thereby minimizing celastrol-related systemic toxicity. Celastrol-albumin nanoparticles thus represents a promising treatment option for mesangioproliferative glomerulonephritis and similar glomerular diseases.Mesangial cell-mediated glomerulonephritis is a frequent cause of kidney disease. Here the authors show that celastrol loaded in albumin nanoparticles efficiently targets mesangial cells, and is effective in rat models.

Number of Langerhans cells is decreased in premalignant keratosis and skin cancers.

PubMed

Shevchuk, Z; Filip, A; Shevchuk, V; Kashuba, E

2014-03-01

It was shown earlier that a number of CD207 positive Langerhans cells was lower in basal cell carcinomas than in the normal epidermis. Moreover, benign skin lesions presented a higher number of Langerhans cells when they were compared to malignant tumors. To count Langerhans cells, assessing expression levels of CD1A and CD207 markers in actinic keratosis, basal and squamous cell carcinomas, compared with the normal skin. Comparison of Langerhans cells might give a valuable prognostic marker for skin cancer. Immunohistochemistry and methods of statistics were used. Expression of CD1A and CD207 markers was assessed in tumor samples of actinic keratosis, cutaneous basal and squamous cell carcinomas, in comparison with the normal skin. In each cohort there were 40 patients (and 11 healthy individuals). We have shown that the number of Langerhans cells is considerably lower in cutaneous basal and squamous cell carcinomas, compared with their number in the normal skin (p < 0.0001). CD1A expression correlated with CD207 expression only in the control group. There was no correlation in actinic keratosis, basal and squamous cell carcinoma. This may suggest an alteration of Langerhans cells phenotype in skin neoplastic diseases, making the number of Langerhans cells a valuable prognostic factor for skin tumors.

Identification and validation nucleolin as a target of curcumol in nasopharyngeal carcinoma cells.

PubMed

Wang, Juan; Wu, Jiacai; Li, Xumei; Liu, Haowei; Qin, Jianli; Bai, Zhun; Chi, Bixia; Chen, Xu

2018-06-30

Identification of the specific protein target(s) of a drug is a critical step in unraveling its mechanisms of action (MOA) in many natural products. Curcumol, isolated from well known Chinese medicinal plant Curcuma zedoary, has been shown to possess multiple biological activities. It can inhibit nasopharyngeal carcinoma (NPC) proliferation and induce apoptosis, but its target protein(s) in NPC cells remains unclear. In this study, we employed a mass spectrometry-based chemical proteomics approach reveal the possible protein targets of curcumol in NPC cells. Cellular thermal shift assay (CETSA), molecular docking and cell-based assay was used to validate the binding interactions. Chemical proteomics capturing uncovered that NCL is a target of curcumol in NPC cells, Molecular docking showed that curcumol bound to NCL with an -7.8 kcal/mol binding free energy. Cell function analysis found that curcumol's treatment leads to a degradation of NCL in NPC cells, and it showed slight effects on NP69 cells. In conclusion, our results providing evidences that NCL is a target protein of curcumol. We revealed that the anti-cancer effects of curcumol in NPC cells are mediated, at least in part, by NCL inhibition. Many natural products showed high bioactivity, while their mechanisms of action (MOA) are very poor or completely missed. Understanding the MOA of natural drugs can thoroughly exploit their therapeutic potential and minimize their adverse side effects. Identification of the specific protein target(s) of a drug is a critical step in unraveling its MOA. Compound-centric chemical proteomics is a classic chemical proteomics approach which integrates chemical synthesis with cell biology and mass spectrometry (MS) to identify protein targets of natural products determine the drug mechanism of action, describe its toxicity, and figure out the possible cause of off-target. It is an affinity-based chemical proteomics method to identify small molecule-protein interactions

Viral Capsid DNA Aptamer Conjugates as Multivalent Cell Targeting Vehicles

PubMed Central

Tong, Gary J.; Hsiao, Sonny C.; Carrico, Zachary M.; Francis, Matthew B.

2009-01-01

Nucleic acid aptamers offer significant potential as convenient and evolvable targeting groups for drug delivery. To attach them to the surface of a genome-free viral capsid carrier, an efficient oxidative coupling strategy has been developed. The method involves the periodate-mediated reaction of phenylene diamine substituted oligonucleotides with aniline groups installed on the outer surface of the capsid shells. Up to 60 DNA strands can be attached to each viral capsid with no apparent loss of base-pairing capabilities or protein stability. The ability of the capsids to bind specific cellular targets was demonstrated through the attachment of a 41-nucleotide sequence that targets a tyrosine kinase receptor on Jurkat T cells. After the installation of a fluorescent dye on the capsid interior, capsids bearing the cell-targeting sequence showed significant levels of binding to the cells relative to control samples. Colocalization experiments using confocal microscopy indicated that the capsids were endocytosed and trafficked to lysosomes for degradation. These observations suggest that aptamer-labeled capsids could be used for the targeted drug delivery of acid-labile prodrugs that would be preferentially released upon lysosomal acidification. PMID:19603808

Copy number abnormality of acute lymphoblastic leukemia cell lines based on their genetic subtypes.

PubMed

Tomoyasu, Chihiro; Imamura, Toshihiko; Tomii, Toshihiro; Yano, Mio; Asai, Daisuke; Goto, Hiroaki; Shimada, Akira; Sanada, Masashi; Iwamoto, Shotaro; Takita, Junko; Minegishi, Masayoshi; Inukai, Takeshi; Sugita, Kanji; Hosoi, Hajime

2018-05-21

In this study, we performed genetic analysis of 83 B cell precursor acute lymphoblastic leukemia (B-ALL) cell lines. First, we performed multiplex ligation-dependent probe amplification analysis to identify copy number abnormalities (CNAs) in eight genes associated with B-ALL according to genetic subtype. In Ph + B-ALL cell lines, the frequencies of IKZF1, CDKN2A/2B, BTG1, and PAX5 deletion were significantly higher than those in Ph - B-ALL cell lines. The frequency of CDKN2A/2B deletion in KMT2A rearranged cell lines was significantly lower than that in non-KMT2A rearranged cell lines. These findings suggest that CNAs are correlated with genetic subtype in B-ALL cell lines. In addition, we determined that three B-other ALL cell lines had IKZF1 deletions (YCUB-5, KOPN49, and KOPN75); we therefore performed comprehensive genetic analysis of these cell lines. YCUB-5, KOPN49, and KOPN75 had P2RY8-CRLF2, IgH-CRLF2, and PAX5-ETV6 fusions, respectively. Moreover, targeted capture sequencing revealed that YCUB-5 had JAK2 R683I and KRAS G12D, and KOPN49 had JAK2 R683G and KRAS G13D mutations. These data may contribute to progress in the field of leukemia research.

miRNA-1297 induces cell proliferation by targeting phosphatase and tensin homolog in testicular germ cell tumor cells.

PubMed

Yang, Nian-Qin; Zhang, Jian; Tang, Qun-Ye; Guo, Jian-Ming; Wang, Guo-Min

2014-01-01

To investigate the role of miR-1297 and the tumor suppressor gene PTEN in cell proliferation of testicular germ cell tumors (TGCT). MTT assays were used to test the effect of miR-1297 on proliferation of the NCCIT testicular germ cell tumor cell line. In NCCIT cells, the expression of PTEN was assessed by Western blotting further. In order to confirm target association between miR-1297 and 3'-UTR of PTEN, a luciferase reporter activity assay was employed. Moreover, roles of PTEN in proliferation of NCCIT cells were evaluated by transfection of PTEN siRNA. Proliferation of NCCIT cells was promoted by miR-1297 in a concentration-dependent manner. In addition, miR-1297 could bind to the 3'-UTR of PTEN based on luciferase reporter activity assay, and reduced expression of PTEN at protein level was found. Proliferation of NCCIT cells was significantly enhanced after knockdown of PTEN by siRNA. miR-1297 as a potential oncogene could induce cell proliferation by targeting PTEN in NCCIT cells.

Target cell specific antibody-based photosensitizers for photodynamic therapy

NASA Astrophysics Data System (ADS)

Rosenblum, Lauren T.; Mitsunaga, Makoto; Kakareka, John W.; Morgan, Nicole Y.; Pohida, Thomas J.; Choyke, Peter L.; Kobayashi, Hisataka

2011-03-01

In photodynamic therapy (PDT), localized monochromatic light is used to activate targeted photosensitizers (PS) to induce cellular damage through the generation of cytotoxic species such as singlet oxygen. While first-generation PS passively targeted malignancies, a variety of targeting mechanisms have since been studied, including specifically activatable agents. Antibody internalization has previously been employed as a fluorescence activation system and could potentially enable similar activation of PS. TAMRA, Rhodamine-B and Rhodamine-6G were conjugated to trastuzumab (brand name Herceptin), a humanized monoclonal antibody with specificity for the human epidermal growth factor receptor 2 (HER2), to create quenched PS (Tra-TAM, Tra-RhoB, and Tra-Rho6G). Specific PDT with Tra-TAM and Tra-Rho6G, which formed covalently bound H-dimers, was demonstrated in HER2+ cells: Minimal cell death (<6%) was observed in all treatments of the HER2- cell line (BALB/3T3) and in treatments the HER2+ cell line (3T3/HER2) with light or trastuzumab only. There was significant light-induced cell death in HER2 expressing cells using Tra-TAM (3% dead without light, 20% at 50 J/cm2, 46% at 100 J/cm2) and Tra-Rho6G (5% dead without light, 22% at 50 J/cm2, 46% at 100 J/cm2). No efficacy was observed in treatment with Tra-RhoB, which was also non-specifically taken up by BALB/3T3 cells and which had weaker PS-antibody interactions (as demonstrated by visualization of protein and fluorescence on SDS-PAGE).

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.

TargetLink, a new method for identifying the endogenous target set of a specific microRNA in intact living cells.

PubMed

Xu, Yan; Chen, Yan; Li, Daliang; Liu, Qing; Xuan, Zhenyu; Li, Wen-Hong

2017-02-01

MicroRNAs are small non-coding RNAs acting as posttranscriptional repressors of gene expression. Identifying mRNA targets of a given miRNA remains an outstanding challenge in the field. We have developed a new experimental approach, TargetLink, that applied locked nucleic acid (LNA) as the affinity probe to enrich target genes of a specific microRNA in intact cells. TargetLink also consists a rigorous and systematic data analysis pipeline to identify target genes by comparing LNA-enriched sequences between experimental and control samples. Using miR-21 as a test microRNA, we identified 12 target genes of miR-21 in a human colorectal cancer cell by this approach. The majority of the identified targets interacted with miR-21 via imperfect seed pairing. Target validation confirmed that miR-21 repressed the expression of the identified targets. The cellular abundance of the identified miR-21 target transcripts varied over a wide range, with some targets expressed at a rather low level, confirming that both abundant and rare transcripts are susceptible to regulation by microRNAs, and that TargetLink is an efficient approach for identifying the target set of a specific microRNA in intact cells. C20orf111, one of the novel targets identified by TargetLink, was found to reside in the nuclear speckle and to be reliably repressed by miR-21 through the interaction at its coding sequence.

Cas9-mediated targeting of viral RNA in eukaryotic cells.

PubMed

Price, Aryn A; Sampson, Timothy R; Ratner, Hannah K; Grakoui, Arash; Weiss, David S

2015-05-12

Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems are prokaryotic RNA-directed endonuclease machineries that act as an adaptive immune system against foreign genetic elements. Using small CRISPR RNAs that provide specificity, Cas proteins recognize and degrade nucleic acids. Our previous work demonstrated that the Cas9 endonuclease from Francisella novicida (FnCas9) is capable of targeting endogenous bacterial RNA. Here, we show that FnCas9 can be directed by an engineered RNA-targeting guide RNA to target and inhibit a human +ssRNA virus, hepatitis C virus, within eukaryotic cells. This work reveals a versatile and portable RNA-targeting system that can effectively function in eukaryotic cells and be programmed as an antiviral defense.

Cas9-mediated targeting of viral RNA in eukaryotic cells

PubMed Central

Price, Aryn A.; Sampson, Timothy R.; Ratner, Hannah K.; Grakoui, Arash; Weiss, David S.

2015-01-01

Clustered, regularly interspaced, short palindromic repeats–CRISPR associated (CRISPR-Cas) systems are prokaryotic RNA-directed endonuclease machineries that act as an adaptive immune system against foreign genetic elements. Using small CRISPR RNAs that provide specificity, Cas proteins recognize and degrade nucleic acids. Our previous work demonstrated that the Cas9 endonuclease from Francisella novicida (FnCas9) is capable of targeting endogenous bacterial RNA. Here, we show that FnCas9 can be directed by an engineered RNA-targeting guide RNA to target and inhibit a human +ssRNA virus, hepatitis C virus, within eukaryotic cells. This work reveals a versatile and portable RNA-targeting system that can effectively function in eukaryotic cells and be programmed as an antiviral defense. PMID:25918406

Nandrolone decanoate increases satellite cell numbers in the chicken pectoralis muscle.

PubMed

Allouh, Mohammed Z; Rosser, Benjamin W C

2010-02-01

The anabolic androgenic steroid nandrolone decanoate has minimal androgenic effects and, thus, is widely used to induce muscle hypertrophy in both patients and athletes. Although increases in satellite cell numbers and satellite cells giving rise to new myonuclei are associated with hypertrophy in many experimental models, the relationship between nandrolone and satellite cells is poorly understood. Here we test the hypothesis that nandrolone administration is associated with an increase in satellite cell numbers in muscle. Nandrolone was injected at weekly intervals for four weeks into the right pectoralis muscle of female white leghorn chickens aged 63 days post hatch. Age/size/sex matched control birds received saline injections. The contralateral pectoralis was excised for study from each control and nandrolone treated bird. An antibody against Pax7 and immunocytochemical techniques were used to identify satellite cells. Nandrolone significantly increased mean pectoralis mass by approximately 22%, and mean fiber diameter by about 24%. All satellite cell indices that were quantified increased significantly in chicken pectoralis with administration of nandrolone. Nandrolone injected birds had on average higher satellite cell frequencies (#SC nuclei/all nuclei within basal lamina), number of satellite cells per millimeter of fiber, and satellite cell concentrations (closer together). Myonuclei were further apart (less concentrated) in nandrolone injected muscle. However, an overall increase in myonuclear numbers was revealed by a significantly greater mean number of myonuclei per millimeter of fiber in nandrolone injected muscle. Our results suggest that satellite cells may be key cellular vectors for nandrolone induced muscle fiber hypertrophy.

Targeted adenoviral vectors

NASA Astrophysics Data System (ADS)

Douglas, Joanne T.

The practical implementation of gene therapy in the clinical setting mandates gene delivery vehicles, or vectors, capable of efficient gene delivery selectively to the target disease cells. The utility of adenoviral vectors for gene therapy is restricted by their dependence on the native adenoviral primary cellular receptor for cell entry. Therefore, a number of strategies have been developed to allow CAR-independent infection of specific cell types, including the use of bispecific conjugates and genetic modifications to the adenoviral capsid proteins, in particular the fibre protein. These targeted adenoviral vectors have demonstrated efficient gene transfer in vitro , correlating with a therapeutic benefit in preclinical animal models. Such vectors are predicted to possess enhanced efficacy in human clinical studies, although anatomical barriers to their use must be circumvented.

Pharmacological targets of breast cancer stem cells: a review.

PubMed

Pindiprolu, Sai Kiran S S; Krishnamurthy, Praveen T; Chintamaneni, Pavan Kumar

2018-05-01

Breast cancers contain small population of tumor-initiating cells called breast cancer stem cells (BCSCs), which are spared even after chemotherapy. Recently, BCSCs are implicated to be a cause of metastasis, tumor relapse, and therapy resistance in breast cancer. BCSCs have unique molecular mechanisms, which can be targeted to eliminate them. These include surface biomarkers, proteins involved in self-renewal pathways, drug efflux transporters, apoptotic/antiapoptotic proteins, autophagy, metabolism, and microenvironment regulation. The complex molecular mechanisms behind the survival of BCSCs and pharmacological targets for elimination of BCSCs are described in this review.

[Study on the hepatocytic cell targetability of liposomes].

PubMed

Hou, Xin-pu; Wang, Li; Wang, Xiang-tao; Li, Sha

2003-02-01

To target for hepatocytic cell, liposomes was modified by special ligand. Sterically stabilized liposomes (SSL) was conjugated with asialofeticin (AF), the ligand of asialoglycoprotein receptor (ASGP-R) of hepatocyte. ASGP-R-BLM is the ASGP-R reconstructed on bilayer lipid membrane (BLM). The recognition reaction between AF-SSL and ASGP-R-BLM can be monitored by the varieties of membrane electrical parameters. The targetability of AF-SSL mediated to hepatocyte was detected by radioisotopic labeled in vitro and in vivo. The therapeutic effect of antihepatocarcinoma was observed also. The lifetime of ASGP-R-BLM decreased with the added amount of AF-SSL. It was demonstrated that there was recognition reaction between AF-SSL and ASGP-R-BLM. The combination of AF-SSL with hepatocyte was significantly higher than that of SSL without AF-modified in vitro and in vivo. The survival time of rat for AF-SSL carriered ADM (adriamycin) group was much longer and the toxicities on heart, kidney and lung were lower than those SSL carried ADM group. It is possible to actively target the cell with specific receptor by ligand modified liposomes. The result prvide scientific basis of hepatocyte targeted liposomes.

Selective in vivo metabolic cell-labeling-mediated cancer targeting

PubMed Central

Wang, Hua; Wang, Ruibo; Cai, Kaimin; He, Hua; Liu, Yang; Yen, Jonathan; Wang, Zhiyu; Xu, Ming; Sun, Yiwen; Zhou, Xin; Yin, Qian; Tang, Li; Dobrucki, Iwona T; Dobrucki, Lawrence W; Chaney, Eric J; Boppart, Stephen A; Fan, Timothy M; Lezmi, Stéphane; Chen, Xuesi; Yin, Lichen; Cheng, Jianjun

2017-01-01

Distinguishing cancer cells from normal cells through surface receptors is vital for cancer diagnosis and targeted therapy. Metabolic glycoengineering of unnatural sugars provides a powerful tool to manually introduce chemical receptors onto the cell surface; however, cancer-selective labeling still remains a great challenge. Herein we report the design of sugars that can selectively label cancer cells both in vitro and in vivo. Specifically, we inhibit the cell-labeling activity of tetraacetyl-N-azidoacetylmannosamine (Ac4ManAz) by converting its anomeric acetyl group to a caged ether bond that can be selectively cleaved by cancer-overexpressed enzymes and thus enables the overexpression of azido groups on the surface of cancer cells. Histone deacetylase and cathepsin L-responsive acetylated azidomannosamine, one such enzymatically activatable Ac4ManAz analog developed, mediated cancer-selective labeling in vivo, which enhanced tumor accumulation of a dibenzocyclooctyne–doxorubicin conjugate via click chemistry and enabled targeted therapy against LS174T colon cancer, MDA-MB-231 triple-negative breast cancer and 4T1 metastatic breast cancer in mice. PMID:28192414

Is health literacy related to health behaviors and cell phone usage patterns among the text4baby target population?

PubMed

Poorman, Elisabeth; Gazmararian, Julie; Elon, Lisa; Parker, Ruth

2014-01-01

Text4baby provides educational text messages to pregnant and postpartum women and targets underserved women. The primary purpose of this study is to examine the health behaviors and cell phone usage patterns of a text4baby target population and the associations with health literacy. Pregnant and postpartum women were recruited from two Women, Infant and Children clinics in Atlanta. Women were asked about their demographics, selected pregnancy or postpartum health behaviors, and cell phone usage patterns. Health literacy skills were measured with the English version of the Newest Vital Sign. Multivariable logistic regression was used to examine health behaviors and cell usage patterns by health literacy classification, controlling for commonly accepted confounders. Four hundred sixty-eight women were recruited, and 445 completed the Newest Vital Sign. Of these, 22% had inadequate health literacy, 50% had intermediate health literacy, and 28% had adequate health literacy skills. Compared to adequate health literacy, limited literacy was independently associated with not taking a daily vitamin during pregnancy (OR 3.6, 95% CI: 1.6, 8.5) and never breastfeeding their infant (OR 1.4, 95% CI: 1.1, 1.8). The majority (69.4%) of respondents received nine or more text messages a day prior to enrollment, one in four participants (24.6%) had changed their number within the last six months, and 7.0% of study participants shared a cell phone. Controlling for potentially confounding factors, those with limited health literacy were more likely to share a cell phone than those with adequate health literacy (OR 2.57, 95% CI: 1.79, 3.69). Text4baby messages should be appropriate for low health literacy levels, especially as this population may have higher prevalence of targeted unhealthy behaviors. Text4baby and other mhealth programs targetting low health literacy populations should also be aware of the different ways that these populations use their cell phones, including: sharing

Novel therapeutic Strategies for Targeting Liver Cancer Stem Cells

PubMed Central

Oishi, Naoki; Wang, Xin Wei

2011-01-01

The cancer stem cell (CSC) hypothesis was first proposed over 40 years ago. Advances in CSC isolation were first achieved in hematological malignancies, with the first CSC demonstrated in acute myeloid leukemia. However, using similar strategies and technologies, and taking advantage of available surface markers, CSCs have been more recently demonstrated in a growing range of epithelial and other solid organ malignancies, suggesting that the majority of malignancies are dependent on such a compartment. Primary liver cancer consists predominantly of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). It is believed that hepatic progenitor cells (HPCs) could be the origin of some HCCs and ICCs. Furthermore, stem cell activators such as Wnt/β-catenin, TGF-β, Notch and Hedgehog signaling pathways also expedite tumorigenesis, and these pathways could serve as molecular targets to assist in designing cancer prevention strategies. Recent studies indicate that additional factors such as EpCAM, Lin28 or miR-181 may also contribute to HCC progression by targeting HCC CSCs. Various therapeutic drugs that directly modulate CSCs have been examined in vivo and in vitro. However, CSCs clearly have a complex pathogenesis, with a considerable crosstalk and redundancy in signaling pathways, and hence targeting single molecules or pathways may have a limited benefit for treatment. Many of the key signaling molecules are shared by both CSCs and normal stem cells, which add further challenges for designing molecularly targeted strategies specific to CSCs but sparing normal stem cells to avoid side effects. In addition to the direct control of CSCs, many other factors that are needed for the maintenance of CSCs, such as angiogenesis, vasculogenesis, invasion and migration, hypoxia, immune evasion, multiple drug resistance, and radioresistance, should be taken into consideration when designing therapeutic strategies for HCC. Here we provide a brief review of

Targeting RAS-driven human cancer cells with antibodies to upregulated and essential cell-surface proteins.

PubMed

Martinko, Alexander J; Truillet, Charles; Julien, Olivier; Diaz, Juan E; Horlbeck, Max A; Whiteley, Gordon; Blonder, Josip; Weissman, Jonathan S; Bandyopadhyay, Sourav; Evans, Michael J; Wells, James A

2018-01-23

While there have been tremendous efforts to target oncogenic RAS signaling from inside the cell, little effort has focused on the cell-surface. Here, we used quantitative surface proteomics to reveal a signature of proteins that are upregulated on cells transformed with KRAS G12V , and driven by MAPK pathway signaling. We next generated a toolkit of recombinant antibodies to seven of these RAS-induced proteins. We found that five of these proteins are broadly distributed on cancer cell lines harboring RAS mutations. In parallel, a cell-surface CRISPRi screen identified integrin and Wnt signaling proteins as critical to RAS-transformed cells. We show that antibodies targeting CDCP1, a protein common to our proteomics and CRISPRi datasets, can be leveraged to deliver cytotoxic and immunotherapeutic payloads to RAS-transformed cancer cells and report for RAS signaling status in vivo. Taken together, this work presents a technological platform for attacking RAS from outside the cell. © 2018, Martinko et al.

Bypassing Protein Corona Issue on Active Targeting: Zwitterionic Coatings Dictate Specific Interactions of Targeting Moieties and Cell Receptors.

PubMed

Safavi-Sohi, Reihaneh; Maghari, Shokoofeh; Raoufi, Mohammad; Jalali, Seyed Amir; Hajipour, Mohammad J; Ghassempour, Alireza; Mahmoudi, Morteza

2016-09-07

Surface functionalization strategies for targeting nanoparticles (NP) to specific organs, cells, or organelles, is the foundation for new applications of nanomedicine to drug delivery and biomedical imaging. Interaction of NPs with biological media leads to the formation of a biomolecular layer at the surface of NPs so-called as "protein corona". This corona layer can shield active molecules at the surface of NPs and cause mistargeting or unintended scavenging by the liver, kidney, or spleen. To overcome this corona issue, we have designed biotin-cysteine conjugated silica NPs (biotin was employed as a targeting molecule and cysteine was used as a zwitterionic ligand) to inhibit corona-induced mistargeting and thus significantly enhance the active targeting capability of NPs in complex biological media. To probe the targeting yield of our engineered NPs, we employed both modified silicon wafer substrates with streptavidin (i.e., biotin receptor) to simulate a target and a cell-based model platform using tumor cell lines that overexpress biotin receptors. In both cases, after incubation with human plasma (thus forming a protein corona), cellular uptake/substrate attachment of the targeted NPs with zwitterionic coatings were significantly higher than the same NPs without zwitterionic coating. Our results demonstrated that NPs with a zwitterionic surface can considerably facilitate targeting yield of NPs and provide a promising new type of nanocarriers in biological applications.

A Cell-targeted Photodynamic Nanomedicine Strategy for Head & Neck Cancers

PubMed Central

Master, Alyssa; Malamas, Anthony; Solanki, Rachna; Clausen, Dana M.; Eiseman, Julie L.; Gupta, Anirban Sen

2013-01-01

Photodynamic Therapy (PDT) holds great promise for the treatment of head and neck (H&N) carcinomas where repeated loco-regional therapy often becomes necessary due to the highly aggressive and recurrent nature of the cancers. While interstitial light delivery technologies are being refined for PDT of H&N and other cancers, a parallel clinically relevant research area is the formulation of photosensitizers in nanovehicles that allow systemic administration yet preferential enhanced uptake in the tumor. This approach can render dual-selectivity of PDT, by harnessing both the drug and the light delivery within the tumor. To this end, we report on a cell-targeted nanomedicine approach for the photosensitizer silicon phthalocyanine-4 (Pc 4), by packaging it within polymeric micelles that are surface-decorated with GE11-peptides to promote enhanced cell-selective binding and receptor-mediated internalization in EGFR-overexpressing H&N cancer cells. Using fluorescence spectroscopy and confocal microscopy, we demonstrate in vitro that the EGFR-targeted Pc 4-nanoformulation undergoes faster and higher uptake in EGFR-overexpressing H&N SCC-15 cells. We further demonstrate that this enhanced Pc 4 uptake results in significant cell-killing and drastically reduced post-PDT clonogenicity. Building on this in vitro data, we demonstrate that the EGFR-targeted Pc 4-nanoformulation results in significant intra-tumoral drug uptake and subsequent enhanced PDT response, in vivo, in SCC-15 xenografts in mice. Altogether our results show significant promise towards a cell-targeted photodynamic nanomedicine for effective treatment of H&N carcinomas. PMID:23531079

Innovative T Cell-Targeted Therapy for Ovarian Cancer

DTIC Science & Technology

2012-10-01

from co-culture with EL4 -ROR1neg and EL4 -ROR1+ tumor targets. Ovarian cancer cell lines (A2780, EFO21, EFO27, IGROV1, OC314, and UPN251) were...profiled for ROR1 expression in normoxia (20% O2) and hypoxia (1% O2). Four-hour CRA was used to evaluate cytotoxicity against the OvCa and EL4 tumor...loaded aAPC for negative controls. EL4 is a murine T cell lymphoma cell line used to test specificity of CAR+ T cells with limited allo-reactivity

Antigen sensitivity of CD22-specific chimeric T cell receptors is modulated by target epitope distance from the cell membrane

PubMed Central

James, Scott E.; Greenberg, Philip D.; Jensen, Michael C.; Lin, Yukang; Wang, Jinjuan; Till, Brian G.; Raubitschek, Andrew A.; Forman, Stephen J.; Press, Oliver W.

2008-01-01

We have targeted CD22 as a novel tumor-associated antigen for recognition by human CTL genetically modified to express chimeric T cell receptors (cTCR) recognizing this surface molecule. CD22-specifc cTCR targeting different epitopes of the CD22 molecule promoted efficient lysis of target cells expressing high levels of CD22 with a maximum lytic potential that appeared to decrease as the distance of the target epitope from the target cell membrane increased. Targeting membrane-distal CD22 epitopes with cTCR+ CTL revealed defects in both degranulation and lytic granule targeting. CD22-specific cTCR+ CTL exhibited lower levels of maximum lysis and lower antigen sensitivity than CTL targeting CD20, which has a shorter extracellular domain than CD22. This diminished sensitivity was not a result of reduced avidity of antigen engagement, but instead reflected weaker signaling per triggered cTCR molecule when targeting membrane-distal epitopes of CD22. Both of these parameters were restored by targeting a ligand expressing the same epitope but constructed as a truncated CD22 molecule to approximate the length of a TCR:pMHC complex. The reduced sensitivity of CD22-specific cTCR+ CTL for antigen-induced triggering of effector functions has potential therapeutic applications, as such cells selectively lysed B cell lymphoma lines expressing high levels of CD22 but demonstrated minimal activity against autologous normal B cells, which express lower levels of CD22. Thus, our results demonstrate that cTCR signal strength – and consequently antigen sensitivity – can be modulated by differential choice of target epitopes with respect to distance from the cell membrane, allowing discrimination between targets with disparate antigen density. PMID:18453625

Plasma Membrane Proteomics of Human Breast Cancer Cell Lines Identifies Potential Targets for Breast Cancer Diagnosis and Treatment

PubMed Central

Ziegler, Yvonne S.; Moresco, James J.; Tu, Patricia G.; Yates, John R.; Nardulli, Ann M.

2014-01-01

The use of broad spectrum chemotherapeutic agents to treat breast cancer results in substantial and debilitating side effects, necessitating the development of targeted therapies to limit tumor proliferation and prevent metastasis. In recent years, the list of approved targeted therapies has expanded, and it includes both monoclonal antibodies and small molecule inhibitors that interfere with key proteins involved in the uncontrolled growth and migration of cancer cells. The targeting of plasma membrane proteins has been most successful to date, and this is reflected in the large representation of these proteins as targets of newer therapies. In view of these facts, experiments were designed to investigate the plasma membrane proteome of a variety of human breast cancer cell lines representing hormone-responsive, ErbB2 over-expressing and triple negative cell types, as well as a benign control. Plasma membranes were isolated by using an aqueous two-phase system, and the resulting proteins were subjected to mass spectrometry analysis. Overall, each of the cell lines expressed some unique proteins, and a number of proteins were expressed in multiple cell lines, but in patterns that did not always follow traditional clinical definitions of breast cancer type. From our data, it can be deduced that most cancer cells possess multiple strategies to promote uncontrolled growth, reflected in aberrant expression of tyrosine kinases, cellular adhesion molecules, and structural proteins. Our data set provides a very rich and complex picture of plasma membrane proteins present on breast cancer cells, and the sorting and categorizing of this data provides interesting insights into the biology, classification, and potential treatment of this prevalent and debilitating disease. PMID:25029196

Cell Membrane-Cloaked Nanoparticles for Targeted Therapeutics

NASA Astrophysics Data System (ADS)

Luk, Brian Tsengchi

The advent of nanoparticle-based delivery systems has made a significant impact on clinical patient outcomes. In recent decades, myriad nanoparticle-based therapeutic agents have been developed for the treatment and management of ailments such as cancer, diabetes, pain, bacterial infections, and asthma, among many others. Nanotherapeutics offer many distinct advantages over conventional free drug formulations. For example, nanoparticles are able to accumulate at tumor sites by extravasation through leaky vasculature at tumor sites via the enhanced permeability and retention (EPR) effect; nanoparticles can also be tailored to have desirable characteristics, such as prolonged circulation in the blood stream, improved drug encapsulation, and sustained or triggered drug release. Currently, a growing number of nanoformulations with favorable pharmacological profiles and promising efficacy are being used in clinical trials for the treatment of various cancers. Building on the success of these encouraging clinical results, new engineering strategies have emerged that combine synthetic nanoparticles with natural biomaterials to create nature-inspired biomimetic delivery systems. The work presented in this dissertation focuses on the biointerfacing between synthetic and natural materials, namely in the manifestation of cell membrane-coated nanoparticles. By exploiting the natural functionalities of source cell membranes, cell membrane-cloaked nanoparticles have huge potential in the delivery of therapeutic agents for a variety of applications. The first portion of this thesis will focus on understanding the fundamentals underlying cell membrane coating on synthetic nanoparticles. First introduced in 2011, cell membrane-cloaked nanoparticles showed immediate promise in drug delivery applications, but further understanding was necessary to be able to harness the full potential of the membrane coating platform. The first section provides further insight into the interfacial

WE-G-BRE-03: Dose Painting by Numbers Using Targeted Gold Nanoparticles

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

Altundal, Y; Sajo, E; Korideck, H

Purpose: Homogeneous dose enhancement in tumor cells of lung cancer patients treated with conventional dose of 60–66 Gy in five fractions is limited due to increased risk of toxicity to normal structures. Dose painting by numbers (DPBN) is the prescription of a non-uniform radiation dose distribution in the tumor for each voxel based on the intensity level of that voxel obtained from the tumor image. The purpose of this study is to show that DPBN using targeted gold nanoparticles (GNPs) could enhance conventional doses in the more resistant tumor areas. Methods: Cone beam computed tomography (CBCT) images of GNPs aftermore » intratumoral injection into human tumor were taken at 0, 48, 144 and 160 hours. The dose enhancement in the tumor voxels by secondary electrons from the GNPs was calculated based on analytical microdosimetry methods. The dose enhancement factor (DEF) is the ratio of the doses to the tumor with and without the presence of GNPs. The DEF was calculated for each voxel of the images based on the GNP concentration in the tumor sub-volumes using 6-MV photon spectra obtained using Monte Carlo simulations at 5 cm depth (10×10 cm2 field). Results: The results revealed DEF values of 1.05–2.38 for GNPs concentrations of 1–30 mg/g which corresponds to 12.60 – 28.56 Gy per fraction for delivering 12 Gy per fraction homogenously to lung tumor region. Conclusion: Our preliminary results verify that DPBN could be achieved using GNPs to enhance conventional doses to high risk tumor sub-volumes. In practice, DPBN using GNPs could be achieved due to diffusion of targeted GNPs sustainably released in-situ from radiotherapy biomaterials (e.g. fiducials) coated with polymer film containing the GNPs.« less

Specific elimination of CD133+ tumor cells with targeted oncolytic measles virus.

PubMed

Bach, Patricia; Abel, Tobias; Hoffmann, Christopher; Gal, Zoltan; Braun, Gundula; Voelker, Iris; Ball, Claudia R; Johnston, Ian C D; Lauer, Ulrich M; Herold-Mende, Christel; Mühlebach, Michael D; Glimm, Hanno; Buchholz, Christian J

2013-01-15

Tumor-initiating cells (TIC) are critical yet evasive targets for the development of more effective antitumoral strategies. The cell surface marker CD133 is frequently used to identify TICs of various tumor entities, including hepatocellular cancer and glioblastoma. Here, we describe oncolytic measles viruses (MV) retargeted to CD133. The viruses, termed MV-141.7 and MV-AC133, infected and selectively lysed CD133(+) tumor cells. Both viruses exerted strong antitumoral effects on human hepatocellular carcinoma growing subcutaneously or multifocally in the peritoneal cavity of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Notably, the CD133-targeted viruses were more effective in prolonging survival than the parental MV-NSe, which is currently assessed as oncolytic agent in clinical trials. Interestingly, target receptor overexpression or increased spreading kinetics through tumor cells were excluded as being causative for the enhanced oncolytic activity of CD133-targeted viruses. MV-141.7 was also effective in mouse models of orthotopic glioma tumor spheres and primary colon cancer. Our results indicate that CD133-targeted measles viruses selectively eliminate CD133(+) cells from tumor tissue, offering a key tool for research in tumor biology and cancer therapy.

Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells.

PubMed

Sato, Hiroshi; Kato, Hiroki; Yamaza, Haruyoshi; Masuda, Keiji; Nguyen, Huong Thi Nguyen; Pham, Thanh Thi Mai; Han, Xu; Hirofuji, Yuta; Nonaka, Kazuaki

2017-01-01

Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk) gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV). Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

Therapeutic targeting of the p53 pathway in cancer stem cells

PubMed Central

Prabhu, Varun V.; Allen, Joshua E.; Hong, Bo; Zhang, Shengliang; Cheng, Hairong; El-Deiry, Wafik S.

2013-01-01

Introduction Cancer stem cells are a high profile drug target for cancer therapeutics due to their indispensable role in cancer progression, maintenance, and therapeutic resistance. Restoring wild-type p53 function is an attractive new therapeutic approach for the treatment of cancer due to the well-described powerful tumor suppressor function of p53. As emerging evidence intimately links p53 and stem cell biology, this approach also provides an opportunity to target cancer stem cells. Areas covered Therapeutic approaches to restore the function of wild-type p53, cancer and normal stem cell biology in relation to p53, and the downstream effects of p53 on cancer stem cells. Expert opinion The restoration of wild-type p53 function by targeting p53 directly, its interacting proteins, or its family members holds promise as a new class of cancer therapies. This review examines the impact that such therapies may have on normal and cancer stem cells based on the current evidence linking p53 signaling with these populations. PMID:22998602

Eliminating Cancer Stem Cells by Targeting Embryonic Signaling Pathways.

PubMed

Oren, Ohad; Smith, B Douglas

2017-02-01

Dramatic advances have been made in the understanding of cancer over the past decade. Prime among those are better appreciation of the biology of cancer and the development of targeted therapies. Despite these improvements, however, most tumors remain refractory to anti-cancer medications and frequently recur. Cancer Stem Cells (CSCs), which in some cases express markers of pluripotency (e.g., Oct-4), share many of the molecular features of normal stem cells. These cells have been hypothesised to play a role in tumor resistance and relapse. They exhibit dependence on many primitive regulatory pathways and may be best viewed in the context of embryonic signaling pathways. In this article, we review important embryonic signaling cascades and their differential expression in CSCs. We also discuss these pathways as actionable targets for novel therapies in hopes that eliminating cancer stem cells will lead to an improvement in overall survival for patients.

DEVELOPMENTAL NEUROTOXICITY OF ORGANOPHOSPHATES TARGETS CELL CYCLE AND APOPTOSIS, REVEALED BY TRANSCRIPTIONAL PROFILES IN VIVO AND IN VITRO

PubMed Central

Slotkin, Theodore A.; Seidler, Frederic J.

2012-01-01

Developmental organophosphate exposure reduces the numbers of neural cells, contributing to neurobehavioral deficits. We administered chlorpyrifos or diazinon to newborn rats on postnatal days 1–4, in doses straddling the threshold for barely-detectable cholinesterase, and evaluated gene expression in the cell cycle and apoptosis pathways on postnatal day 5. Both organophosphates evoked transcriptional changes in 20–25% of the genes in each category; chlorpyrifos and diazinon targeted the same genes, with similar magnitudes of change, as evidenced by high concordance. Furthermore, the same effects were obtained with doses above or below the threshold for cholinesterase inhibition, indicating a mechanism unrelated to anticholinesterase actions. We then evaluated the effects of chlorpyrifos in undifferentiated and differentiating PC12 cells and found even greater targeting of cell cycle and apoptosis genes, affecting up to 40% of all genes in the pathways. Notably, the genes affected in undifferentiated cells were not concordant with those in differentiating cells, pointing to dissimilar outcomes dependent on developmental stage. The in vitro model successfully identified 60–70% of the genes affected by chlorpyrifos in vivo, indicating that the effects are exerted directly on developing neural cells. Our results show that organophosphates target the genes regulating the cell cycle and apoptosis in the developing brain and in neuronotypic cells in culture, with the pattern of vulnerability dependent on the specific stage of development. Equally important, these effects do not reflect actions on cholinesterase and operate at exposures below the threshold for any detectable inhibition of this enzyme. PMID:22222554

Developmental neurotoxicity of organophosphates targets cell cycle and apoptosis, revealed by transcriptional profiles in vivo and in vitro.

PubMed

Slotkin, Theodore A; Seidler, Frederic J

2012-03-01

Developmental organophosphate exposure reduces the numbers of neural cells, contributing to neurobehavioral deficits. We administered chlorpyrifos or diazinon to newborn rats on postnatal days 1-4, in doses straddling the threshold for barely-detectable cholinesterase inhibition, and evaluated gene expression in the cell cycle and apoptosis pathways on postnatal day 5. Both organophosphates evoked transcriptional changes in 20-25% of the genes in each category; chlorpyrifos and diazinon targeted the same genes, with similar magnitudes of change, as evidenced by high concordance. Furthermore, the same effects were obtained with doses above or below the threshold for cholinesterase inhibition, indicating a mechanism unrelated to anticholinesterase actions. We then evaluated the effects of chlorpyrifos in undifferentiated and differentiating PC12 cells and found even greater targeting of cell cycle and apoptosis genes, affecting up to 40% of all genes in the pathways. Notably, the genes affected in undifferentiated cells were not concordant with those in differentiating cells, pointing to dissimilar outcomes dependent on developmental stage. The in vitro model successfully identified 60-70% of the genes affected by chlorpyrifos in vivo, indicating that the effects are exerted directly on developing neural cells. Our results show that organophosphates target the genes regulating the cell cycle and apoptosis in the developing brain and in neuronotypic cells in culture, with the pattern of vulnerability dependent on the specific stage of development. Equally important, these effects do not reflect actions on cholinesterase and operate at exposures below the threshold for any detectable inhibition of this enzyme. Copyright © 2011 Elsevier Inc. All rights reserved.

Kif3a Controls Murine Nephron Number Via GLI3 Repressor, Cell Survival, and Gene Expression in a Lineage-Specific Manner

PubMed Central

Chi, Lijun; Galtseva, Alevtina; Chen, Lin; Mo, Rong; Hui, Chi-chung; Rosenblum, Norman D.

2013-01-01

The primary cilium is required during early embryo patterning, epithelial tubulogenesis, and growth factor-dependent signal transduction. The requirement for primary cilia during renal epithelial-mesenchymal tissue interactions that give rise to nephrons is undefined. Here, we used Cre-mediated recombination to generate mice with Kif3a deficiency targeted to the ureteric and/or metanephric mesenchyme cell lineages in the embryonic kidney. Gradual loss of primary cilia in either lineage leads to a phenotype of reduced nephron number. Remarkably, in addition to cyst formation, loss of primary cilia in the ureteric epithelial cell leads to decreased expression of Wnt11 and Ret and reduced ureteric branching. Constitutive expression of GLI3 repressor (Gli3Δ699/+) rescues these abnormalities. In embryonic metanephric mesenchyme cells, Kif3a deficiency limits survival of nephrogenic progenitor cells and expression of genes required for nephron formation. Together, our data demonstrate that Kif3a controls nephron number via distinct cell lineage-specific mechanisms. PMID:23762375

PEGylated anticancer-carbon nanotubes complex targeting mitochondria of lung cancer cells

NASA Astrophysics Data System (ADS)

Kim, Sang-Woo; Lee, Yeon Kyung; Lee, Jong Yeon; Hong, Jeong Hee; Khang, Dongwoo

2017-11-01

Although activating apoptosis in cancer cells by targeting the mitochondria is an effective strategy for cancer therapy, insufficient targeting of the mitochondria in cancer cells restricts the availability in clinical treatment. Here, we report on a polyethylene glycol-coated carbon nanotube (CNT)-ABT737 nanodrug that improves the mitochondrial targeting of lung cancer cells. The polyethylene glycol-coated CNT-ABT737 nanodrug internalized into the early endosomes via macropinocytosis and clathrin-mediated endocytosis in advance of early endosomal escape and delivered into the mitochondria. Cytosol release of the nanodrug led to apoptosis of lung cancer cells by abruption of the mitochondrial membrane potential, inducing Bcl-2-mediated apoptosis and generating intracellular reactive oxygen species. As such, this study provides an effective strategy for increasing the anti-lung cancer efficacy by increasing mitochondria accumulation rate of cytosol released anticancer nanodrugs.

Folate receptor‐targeted aminoglycoside‐derived polymers for transgene expression in cancer cells

PubMed Central

Godeshala, Sudhakar; Nitiyanandan, Rajeshwar; Thompson, Brian; Goklany, Sheba; Nielsen, David R.

2016-01-01

Abstract Targeted delivery of anticancer therapeutics can potentially overcome the limitations associated with current chemotherapeutic regimens. Folate receptors are overexpressed in several cancers, including ovarian, triple‐negative breast and bladder cancers, making them attractive for targeted delivery of nucleic acid therapeutics to these tumors. This work describes the synthesis, characterization and evaluation of folic acid‐conjugated, aminoglycoside‐derived polymers for targeted delivery of transgenes to breast and bladder cancer cell lines. Transgene expression was significantly higher with FA‐conjugated aminoglycoside‐derived polymers than with Lipofectamine, and these polymers demonstrated minimal cytotoxicty. Competitive inhibition using free folic acid significantly reduced transgene expression efficacy of folate‐targeted polymers, suggesting a role for folate receptor‐mediated uptake. High efficacy FA‐targeted polymers were employed to deliver a plasmid expressing the TRAIL protein, which induced death in cancer cells. These results indicate that FA‐conjugated aminoglycoside‐derived polymers are promising for targeted delivery of nucleic acids to cancer cells that overexpress folate receptors. PMID:29313013

Update on B-cell targeted therapies for systemic lupus erythematosus.

PubMed

Mok, Chi Chiu

2014-06-01

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by flares and remission, leading to accrual of organ damage over time as a result of persistent tissue inflammation and treatment-related complications. Novel therapies aiming at better treatment response and fewer adverse effects are being tested in the pipeline. This review summarizes the B-cell abnormalities observed in patients with SLE, and updates recent data on the efficacy and safety of B-cell targeted therapies in the treatment of SLE. The pitfalls of clinical trial design and future directions of the development of SLE therapeutics are discussed. The variability of clinical response to treatment in SLE reflects the clinical and immunological heterogeneity of the disease. The treatment plan for patients with SLE should be individualized with the aim of eradicating disease activity, preventing flares and minimizing treatment-related complications. Despite the disappointment of recent clinical trials, B-cell remains the promising target of future SLE therapies. Results from ongoing clinical trials on B-cell targeted biological agents are eagerly awaited.

Targeting Vaccine-Induced Extrafollicular Pathway of B Cell Differentiation Improves Rabies Postexposure Prophylaxis.

PubMed

Haley, Shannon L; Tzvetkov, Evgeni P; Meuwissen, Samantha; Plummer, Joseph R; McGettigan, James P

2017-04-15

Vaccine-induced B cells differentiate along two pathways. The follicular pathway gives rise to germinal centers (GCs) that can take weeks to fully develop. The extrafollicular pathway gives rise to short-lived plasma cells (PCs) that can rapidly secrete protective antibodies within days of vaccination. Rabies virus (RABV) postexposure prophylaxis (PEP) requires rapid vaccine-induced humoral immunity for protection. Therefore, we hypothesized that targeting extrafollicular B cell responses for activation would improve the speed and magnitude of RABV PEP. To test this hypothesis, we constructed, recovered, and characterized a recombinant RABV-based vaccine expressing murine B cell activating factor (BAFF) (rRABV-mBAFF). BAFF is an ideal molecule to improve early pathways of B cell activation, as it links innate and adaptive immunity, promoting potent B cell responses. Indeed, rRABV-mBAFF induced a faster, higher antibody response in mice and enhanced survivorship in PEP settings compared to rRABV. Interestingly, rRABV-mBAFF and rRABV induced equivalent numbers of GC B cells, suggesting that rRABV-mBAFF augmented the extrafollicular B cell pathway. To confirm that rRABV-mBAFF modulated the extrafollicular pathway, we used a signaling lymphocytic activation molecule (SLAM)-associated protein (SAP)-deficient mouse model. In response to antigen, SAP-deficient mice form extrafollicular B cell responses but do not generate GCs. rRABV-mBAFF induced similar anti-RABV antibody responses in SAP-deficient and wild-type mice, demonstrating that BAFF modulated immunity through the extrafollicular and not the GC B cell pathway. Collectively, strategies that manipulate pathways of B cell activation may facilitate the development of a single-dose RABV vaccine that replaces current complicated and costly RABV PEP. IMPORTANCE Effective RABV PEP is currently resource- and cost-prohibitive in regions of the world where RABV is most prevalent. In order to diminish the requirements for

Prostate-specific membrane antigen-directed nanoparticle targeting for extreme nearfield ablation of prostate cancer cells.

PubMed

Lee, Seung S; Roche, Philip Jr; Giannopoulos, Paresa N; Mitmaker, Elliot J; Tamilia, Michael; Paliouras, Miltiadis; Trifiro, Mark A

2017-03-01

Almost all biological therapeutic interventions cannot overcome neoplastic heterogeneity. Physical ablation therapy is immune to tumor heterogeneity, but nearby tissue damage is the limiting factor in delivering lethal doses. Multi-walled carbon nanotubes offer a number of unique properties: chemical stability, photonic properties including efficient light absorption, thermal conductivity, and extensive surface area availability for covalent chemical ligation. When combined together with a targeting moiety such as an antibody or small molecule, one can deliver highly localized temperature increases and cause extensive cellular damage. We have functionalized multi-walled carbon nanotubes by conjugating an antibody against prostate-specific membrane antigen. In our in vitro studies using prostate-specific membrane antigen-positive LNCaP prostate cancer cells, we have effectively demonstrated cell ablation of >80% with a single 30-s exposure to a 2.7-W, 532-nm laser for the first time without bulk heating. We also confirmed the specificity and selectivity of prostate-specific membrane antigen targeting by assessing prostate-specific membrane antigen-null PC3 cell lines under the same conditions (<10% cell ablation). This suggests that we can achieve an extreme nearfield cell ablation effect, thus restricting potential tissue damage when transferred to in vivo clinical applications. Developing this new platform will introduce novel approaches toward current therapeutic modalities and will usher in a new age of effective cancer treatment squarely addressing tumoral heterogeneity.

Analysis of gene expression in small numbers of purified hemopoietic progenitor cells by RT-PCR.

PubMed

Ziegler, B L; Lamping, C P; Thoma, S J; Fliedner, T M

1995-05-01

Primitive hemopoietic stem cells represent the most probable targets for genetic alterations due to exposure to ionizing irradiation or chemical carcinogens. We have applied a two-step protocol for the purification of CD34+HLA-DR-/low hemopoietic progenitor cells from cord blood (CB). CD34+ cells were isolated by monoclonal antibody (mAb) against CD34 (My10) and immunomagnetic beads. Beads were cleaved off the CD34+ cells by enzymatic treatment with chymopapain. Due to chymopapain-resistance of epitopes recognized by the used mAbs purity control of CD34+ cells and separation into CD34+HLA-DR-/low and CD34+HLA-DR+ subsets could be performed by using flow cytometry. Two miniaturized procedures were applied to isolate poly(A)+ mRNA for the reverse transcription polymerase chain reaction (RT-PCR) from small numbers of CD34+HLA-DR-/low cells. In five experiments, the mean purity of immunomagnetically isolated CD34+ cells was 93.8% +/- 3.9. Flow cytometry sorting of CD34+ cells resulted in pure CD34+HLA-DR-/low populations (purity > 98.8%; range 98.8% to 99.9%; viability > 96%) with an average yield of 2600 +/- 800 cells/5 x 10(7) low density CB cells. By RT-PCR using both poly(A)+ mRNA isolation procedures, sequences corresponding to CD34 and beta 2-microglobulin were amplified from as few as 20 cells. Furthermore, a sequence-independent RT-PCR (SIP-RT-PCR) was applied to amplify the cDNA derived from five erythroblasts isolated from a burst-forming unit-erythroid (BFU-E). Upon hybridization, full-length c-fos message was detected in the SIP-RT-PCR amplified material. Our data demonstrate that gene expression can be detected at the transcriptional level in small numbers of hemopoietic progenitor cells. In addition, the SIP-RT-PCR may allow the amplification of unique mRNA species when subtractive hybridization procedures are performed. The presented data should be useful to analyze gene expression in rare subsets of radiation-exposed immature hemopoietic stem

The Endocytic Adaptor Eps15 Controls Marginal Zone B Cell Numbers

PubMed Central

Pozzi, Benedetta; Amodio, Stefania; Lucano, Caterina; Sciullo, Anna; Ronzoni, Simona; Castelletti, Daniela; Adler, Thure; Treise, Irina; Betsholtz, Ingrid Holmberg; Rathkolb, Birgit; Busch, Dirk H.; Wolf, Eckhard; Fuchs, Helmut; Gailus-Durner, Valérie; de Angelis, Martin Hrabě; Betsholtz, Christer; Casola, Stefano; Di Fiore, Pier Paolo; Offenhäuser, Nina

2012-01-01

Eps15 is an endocytic adaptor protein involved in clathrin and non-clathrin mediated endocytosis. In Caenorhabditis elegans and Drosophila melanogaster lack of Eps15 leads to defects in synaptic vesicle recycling and synapse formation. We generated Eps15-KO mice to investigate its function in mammals. Eps15-KO mice are born at the expected Mendelian ratio and are fertile. Using a large-scale phenotype screen covering more than 300 parameters correlated to human disease, we found that Eps15-KO mice did not show any sign of disease or neural deficits. Instead, altered blood parameters pointed to an immunological defect. By competitive bone marrow transplantation we demonstrated that Eps15-KO hematopoietic precursor cells were more efficient than the WT counterparts in repopulating B220+ bone marrow cells, CD19− thymocytes and splenic marginal zone (MZ) B cells. Eps15-KO mice showed a 2-fold increase in MZ B cell numbers when compared with controls. Using reverse bone marrow transplantation, we found that Eps15 regulates MZ B cell numbers in a cell autonomous manner. FACS analysis showed that although MZ B cells were increased in Eps15-KO mice, transitional and pre-MZ B cell numbers were unaffected. The increase in MZ B cell numbers in Eps15 KO mice was not dependent on altered BCR signaling or Notch activity. In conclusion, in mammals, the endocytic adaptor protein Eps15 is a regulator of B-cell lymphopoiesis. PMID:23226392

T-cell triggering thresholds are modulated by the number of antigen within individual T-cell receptor clusters

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

Manz, Boryana N.; Jackson, Bryan L.; Petit, Rebecca S.

2011-05-31

T cells react to extremely small numbers of activating agonist peptides. Spatial organization of T-cell receptors (TCR) and their peptide-major histocompatibility complex (pMHC) ligands into microclusters is correlated with T-cell activation. In this study, we have designed an experimental strategy that enables control over the number of agonist peptides per TCR cluster, without altering the total number engaged by the cell. Supported membranes, partitioned with grids of barriers to lateral mobility, provide an effective way of limiting the total number of pMHC ligands that may be assembled within a single TCR cluster. Observations directly reveal that restriction of pMHC contentmore » within individual TCR clusters can decrease T-cell sensitivity for triggering initial calcium flux at fixed total pMHC density. Further analysis suggests that triggering thresholds are determined by the number of activating ligands available to individual TCR clusters, not by the total number encountered by the cell. Results from a series of experiments in which the overall agonist density and the maximum number of agonist per TCR cluster are independently varied in primary T cells indicate that the most probable minimal triggering unit for calcium signaling is at least four pMHC in a single cluster for this system. In conclusion, this threshold is unchanged by inclusion of coagonist pMHC, but costimulation of CD28 by CD80 can modulate the threshold lower.« less

Identification of Novel Targets for Lung Cancer Therapy Using an Induced Pluripotent Stem Cell Model.

PubMed

Shukla, Vivek; Rao, Mahadev; Zhang, Hongen; Beers, Jeanette; Wangsa, Darawalee; Wangsa, Danny; Buishand, Floryne O; Wang, Yonghong; Yu, Zhiya; Stevenson, Holly; Reardon, Emily; McLoughlin, Kaitlin C; Kaufman, Andrew; Payabyab, Eden; Hong, Julie A; Zhang, Mary; Davis, Sean R; Edelman, Daniel C; Chen, Guokai; Miettinen, Markku; Restifo, Nicholas; Ried, Thomas; Meltzer, Paul S; Schrump, David S

2018-04-01

small cell lung cancer lines and specimens. Overexpression of the additional sex combs like-3 gene correlated with increased genomic copy number in small cell lung cancer lines. Knock-down of the additional sex combs like-3 gene inhibited proliferation, clonogenicity, and teratoma formation by lung induced pluripotent stem cells and significantly diminished in vitro clonogenicity and growth of small cell lung cancer cells in vivo. Collectively, these studies highlight the potential utility of this lung induced pluripotent stem cell model for elucidating epigenetic mechanisms contributing to pulmonary carcinogenesis and suggest that additional sex combs like-3 is a novel target for small cell lung cancer therapy.

Construction of ultrasonic nanobubbles carrying CAIX polypeptides to target carcinoma cells derived from various organs.

PubMed

Zhu, Lianhua; Guo, Yanli; Wang, Luofu; Fan, Xiaozhou; Xiong, Xingyu; Fang, Kejing; Xu, Dan

2017-09-29

Ultrasound molecular imaging is a novel diagnostic approach for tumors, whose key link is the construction of targeted ultrasound contrast agents. However, available targeted ultrasound contrast agents for molecular imaging of tumors are only achieving imaging in blood pool or one type tumor. No targeted ultrasound contrast agents have realized targeted ultrasound molecular imaging of tumor parenchymal cells in a variety of solid tumors so far. Carbonic anhydrase IX (CAIX) is highly expressed on cell membranes of various malignant solid tumors, so it's a good target for ultrasound molecular imaging. Here, targeted nanobubbles carrying CAIX polypeptides for targeted binding to a variety of malignant tumors were constructed, and targeted binding ability and ultrasound imaging effect in different types of tumors were evaluated. The mean diameter of lipid targeted nanobubbles was (503.7 ± 78.47) nm, and the polypeptides evenly distributed on the surfaces of targeted nanobubbles, which possessed the advantages of homogenous particle size, high stability, and good safety. Targeted nanobubbles could gather around CAIX-positive cells (786-O and Hela cells), while they cannot gather around CAIX-negative cells (BxPC-3 cells) in vitro, and the affinity of targeted nanobubbles to CAIX-positive cells were significantly higher than that to CAIX-negative cells (P < 0.05). Peak intensity and duration time of targeted nanobubbles and blank nanobubbles were different in CAIX-positive transplanted tumor tissues in vivo (P < 0.05). Moreover, targeted nanobubbles in CAIX-positive transplanted tumor tissues produced higher peak intensity and longer duration time than those in CAIX-negative transplanted tumor tissues (P < 0.05). Finally, immunofluorescence not only confirmed targeted nanobubbles could pass through blood vessels to enter in tumor tissue spaces, but also clarified imaging differences of targeted nanobubbles in different types of transplanted tumor tissues

Self-focusing therapeutic gene delivery with intelligent gene vector swarms: intra-swarm signalling through receptor transgene expression in targeted cells.

PubMed