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Sample records for multicellular tumor spheroids

  1. Ontogenetic growth of multicellular tumor spheroids

    NASA Astrophysics Data System (ADS)

    Condat, C. A.; Menchón, S. A.

    2006-11-01

    In ontogenetic growth models, the basal metabolic rate is usually assumed to depend on the individual mass following a power law. Here it is shown that, in the case of multicellular tumor spheroids, the emergence of a necrotic core invalidates this assumption. The implications of this result for spheroid growth are discussed, and a procedure to determine the growth parameters using macroscopic measurements is proposed.

  2. Semiautomatic growth analysis of multicellular tumor spheroids.

    PubMed

    Rodday, Bjoern; Hirschhaeuser, Franziska; Walenta, Stefan; Mueller-Klieser, Wolfgang

    2011-10-01

    Multicellular tumor spheroids (MCTS) are routinely employed as three-dimensional in vitro models to study tumor biology. Cultivation of MCTS in spinner flasks provides better growing conditions, especially with regard to the availability of nutrients and oxygen, when compared with microtiter plates. The main endpoint of drug response experiments is spheroid size. It is common practice to analyze spheroid size manually with a microscope and an ocular micrometer. This requires removal of some spheroids from the flask, which entails major limitations such as loss of MCTS and the risk of contamination. With this new approach, the authors present an efficient and highly reproducible method to analyze the size of complete MCTS populations in culture containers with transparent, flat bottoms. MCTS sediments are digitally scanned and spheroid volumes are calculated by computerized image analysis. The equipment includes regular office hardware (personal computer, flatbed scanner) and software (Adobe Photoshop, Microsoft Excel, ImageJ). The accuracy and precision of the method were tested using industrial precision steel beads with known diameter. In summary, in comparison with other methods, this approach provides benefits in terms of semiautomation, noninvasiveness, and low costs.

  3. Stress Clamp Experiments on Multicellular Tumor Spheroids

    NASA Astrophysics Data System (ADS)

    Montel, Fabien; Delarue, Morgan; Elgeti, Jens; Malaquin, Laurent; Basan, Markus; Risler, Thomas; Cabane, Bernard; Vignjevic, Danijela; Prost, Jacques; Cappello, Giovanni; Joanny, Jean-François

    2011-10-01

    The precise role of the microenvironment on tumor growth is poorly understood. Whereas the tumor is in constant competition with the surrounding tissue, little is known about the mechanics of this interaction. Using a novel experimental procedure, we study quantitatively the effect of an applied mechanical stress on the long-term growth of a spheroid cell aggregate. We observe that a stress of 10 kPa is sufficient to drastically reduce growth by inhibition of cell proliferation mainly in the core of the spheroid. We compare the results to a simple numerical model developed to describe the role of mechanics in cancer progression.

  4. Surface chemistry-mediated penetration and gold nanorod thermotherapy in multicellular tumor spheroids.

    PubMed

    Jin, Shubin; Ma, Xiaowei; Ma, Huili; Zheng, Kaiyuan; Liu, Juan; Hou, Shuai; Meng, Jie; Wang, Paul C; Wu, Xiaochun; Liang, Xing-Jie

    2013-01-01

    We investigated the penetration and thermotherapy efficiency of different surface coated gold nanorods (Au NRs) in multicellular tumor spheroids. The current data show that negatively charged Au NRs, other than positively charged Au NRs, can penetrate deep into the tumor spheroids and achieve a significant thermal therapeutic benefit.

  5. Surface chemistry-mediated penetration and gold nanorod thermotherapy in multicellular tumor spheroids

    NASA Astrophysics Data System (ADS)

    Jin, Shubin; Ma, Xiaowei; Ma, Huili; Zheng, Kaiyuan; Liu, Juan; Hou, Shuai; Meng, Jie; Wang, Paul C.; Wu, Xiaochun; Liang, Xing-Jie

    2012-12-01

    We investigated the penetration and thermotherapy efficiency of different surface coated gold nanorods (Au NRs) in multicellular tumor spheroids. The current data show that negatively charged Au NRs, other than positively charged Au NRs, can penetrate deep into the tumor spheroids and achieve a significant thermal therapeutic benefit.We investigated the penetration and thermotherapy efficiency of different surface coated gold nanorods (Au NRs) in multicellular tumor spheroids. The current data show that negatively charged Au NRs, other than positively charged Au NRs, can penetrate deep into the tumor spheroids and achieve a significant thermal therapeutic benefit. Electronic supplementary information (ESI) available: Materials and methods section. See DOI: 10.1039/c2nr31877f

  6. Surface acoustic streaming in microfluidic system for rapid multicellular tumor spheroids generation

    NASA Astrophysics Data System (ADS)

    AlHasan, Layla; Qi, Aisha; Al-Aboodi, Aswan; Rezk, Amged; Shilton, Richie R.; Chan, Peggy P. Y.; Friend, James; Yeo, Leslie

    2013-12-01

    In this study, we developed a novel and rapid method to generate in vitro three-dimensional (3D) multicellular tumor spheroids using a surface acoustic wave (SAW) device. A SAW device with single-phase unidirectional transducer electrodes (SPUTD) on lithium niobate substrate was fabricated using standing UV photolithography and wet-etching techniques. To generate spheroids, the SAW device was loaded with medium containing human breast carcinoma (BT474) cells, an oscillating electrical signal at resonant frequency was supplied to the SPUDT to generate acoustic radiation in the medium. Spheroids with uniform size and shape can be obtained using this method in less than 1 minute, and the size of the spheroids can be controlled through adjusting the seeding density. The resulting spheroids were used for further cultivation and were monitored using an optical microscope in real time. The viability and actin organization of the spheroids were assessed using live/dead viability staining and actin cytoskeleton staining, respectively. Compared to spheroids generated using the liquid overlay method, the SAW generated spheroids exhibited higher circularity and higher viability. The F-actin filaments of spheroids appear to aggregate compared to that of untreated cells, indicating that mature spheroids can be obtained using this method. This spheroid generating method can be useful for a variety of biological studies and clinical applications.

  7. Synchrotron Radiation {mu}-X Ray Fluorescence on Multicellular Tumor Spheroids

    SciTech Connect

    Burattini, E.; Cinque, G.; Bellisola, G.; Fracasso, G.; Colombatti, M.; Monti, F.

    2003-01-24

    Synchrotron Radiation micro X-Ray Fluorescence (SR {mu}-XRF) was applied for the first time to map the trace element content on Multicellular Tumor Spheroids (MTS), i.e. human cell clusters used as an in vitro model for testing micrometastases responses to antitumoral drugs. In particular, immunotoxin molecules composed of a carrier protein (Transferrin) bound to a powerful cytotoxin (Ricin A), were here considered as representatives of a class of therapheutic macromolecules used in cancer theraphy. Spheroids included in polyacrylamide gel and placed inside quartz capillaries were studied at the ESRF ID22 beamline using a 15 keV monochromatic photon microbeam. Elemental maps (of Fe, Cu, Zn and Pb) on four groups of spheroids grown under different conditions were studied: untreated, treated only with the carrier molecule or with the toxin alone, and with the complete immunotoxin molecule (carrier+toxin). The results indicate that the distribution of Zn and, to some extent, Cu in the spheroid cells is homogeneous and independent of the treatment type. Total Reflection X-Ray Fluorescence (TR-XRF) was also applied to quantify the average trace element content in the spheroids. Future developments of the technique are finally outlined on the basis of these preliminary results.

  8. Synchrotron Radiation μ-X Ray Fluorescence on Multicellular Tumor Spheroids

    NASA Astrophysics Data System (ADS)

    Burattini, E.; Cinque, G.; Bellisola, G.; Fracasso, G.; Monti, F.; Colombatti, M.

    2003-01-01

    Synchrotron Radiation micro X-Ray Fluorescence (SR μ-XRF) was applied for the first time to map the trace element content on Multicellular Tumor Spheroids (MTS), i.e. human cell clusters used as an in vitro model for testing micrometastases responses to antitumoral drugs. In particular, immunotoxin molecules composed of a carrier protein (Transferrin) bound to a powerful cytotoxin (Ricin A), were here considered as representatives of a class of therapheutic macromolecules used in cancer theraphy. Spheroids included in polyacrylamide gel and placed inside quartz capillaries were studied at the ESRF ID22 beamline using a 15 keV monochromatic photon microbeam. Elemental maps (of Fe, Cu, Zn and Pb) on four groups of spheroids grown under different conditions were studied: untreated, treated only with the carrier molecule or with the toxin alone, and with the complete immunotoxin molecule (carrier+toxin). The results indicate that the distribution of Zn and, to some extent, Cu in the spheroid cells is homogeneous and independent of the treatment type. Total Reflection X-Ray Fluorescence (TR-XRF) was also applied to quantify the average trace element content in the spheroids. Future developments of the technique are finally outlined on the basis of these preliminary results.

  9. Effect of single-walled carbon nanotubes on tumor cells viability and formation of multicellular tumor spheroids

    NASA Astrophysics Data System (ADS)

    Yakymchuk, Olena M.; Perepelytsina, Olena M.; Dobrydnev, Alexey V.; Sydorenko, Mychailo V.

    2015-03-01

    This paper describes the impact of different concentrations of single-walled carbon nanotubes (SWCNTs) on cell viability of breast adenocarcinoma, MCF-7 line, and formation of multicellular tumor spheroids (MTS). Chemical composition and purity of nanotubes is controlled by Fourier transform infrared spectroscopy. The strength and direction of the influence of SWCNTs on the tumor cell population was assessed by cell counting and measurement of the volume of multicellular tumor spheroids. Effect of SWCNTs on the formation of multicellular spheroids was compared with the results obtained by culturing tumor cells with ultra dispersed diamonds (UDDs). Our results demonstrated that SWCNTs at concentrations ranging from 12.5 to 50 μg/ml did not have cytotoxic influence on tumor cells; instead, they had weak cytostatic effect. The increasing of SWCNTs concentration to 100 to 200 μg/ml stimulated proliferation of tumor cells, especially in suspension fractions. The result of this influence was in formation of more MTS in cell culture with SWCNTs compared with UDDs and control samples. In result, the median volume of MTS after cultivation with SWCNTs at 100 to 200 μg/ml concentrations is 3 to 5 times greater than that in samples which were incubated with the UDDs and is 2.5 times greater than that in control cultures. So, if SWCNTs reduced cell adhesion to substrate and stimulated formation of tumor cell aggregates volume near 7 · 10-3 mm3, at the same time, UDDs reduced adhesion and cohesive ability of cells and stimulated generation of cell spheroids volume no more than 4 · 10-3 mm3. Our results could be useful for the control of cell growth in three-dimensional culture.

  10. Low-temperature plasma-induced antiproliferative effects on multi-cellular tumor spheroids

    NASA Astrophysics Data System (ADS)

    Plewa, Joseph-Marie; Yousfi, Mohammed; Frongia, Céline; Eichwald, Olivier; Ducommun, Bernard; Merbahi, Nofel; Lobjois, Valérie

    2014-04-01

    Biomedical applications of low-temperature plasmas are of growing interest, especially in the field of plasma-induced anti-tumor effects. The present work is aimed at investigating the regionalized antiproliferative effects of low-temperature plasmas on a multicellular tumor spheroid (MCTS), a model that mimics the 3D organization and regionalization of a microtumor region. We report that a low-temperature plasma jet, using helium flow in open air, inhibits HCT116 colon carcinoma MCTS growth in a dose-dependent manner. This growth inhibition is associated with the loss of Ki67, and the regionalized accumulation of DNA damage detected by histone H2AX phosphorylation. This regionalized genotoxic effect leads to massive cell death and loss of the MCTS proliferative region. The use of reactive oxygen species (ROS), scavenger N-acetyl cysteine (NAC) and plasma-conditioned media demonstrate that the ROS generated in the media after exposure to low-temperature plasma play a major role in these observed effects. These findings strengthen the interest in the use of MCTS for the evaluation of antiproliferative strategies, and open new perspectives for studies dedicated to demonstrate the potential of low-temperature plasma in cancer therapy.

  11. Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro

    PubMed Central

    Alessandri, Kévin; Sarangi, Bibhu Ranjan; Gurchenkov, Vasily Valérïévitch; Sinha, Bidisha; Kießling, Tobias Reinhold; Fetler, Luc; Rico, Felix; Scheuring, Simon; Lamaze, Christophe; Simon, Anthony; Geraldo, Sara; Vignjević, Danijela; Doméjean, Hugo; Rolland, Leslie; Funfak, Anette; Bibette, Jérôme; Bremond, Nicolas; Nassoy, Pierre

    2013-01-01

    Deciphering the multifactorial determinants of tumor progression requires standardized high-throughput preparation of 3D in vitro cellular assays. We present a simple microfluidic method based on the encapsulation and growth of cells inside permeable, elastic, hollow microspheres. We show that this approach enables mass production of size-controlled multicellular spheroids. Due to their geometry and elasticity, these microcapsules can uniquely serve as quantitative mechanical sensors to measure the pressure exerted by the expanding spheroid. By monitoring the growth of individual encapsulated spheroids after confluence, we dissect the dynamics of pressure buildup toward a steady-state value, consistent with the concept of homeostatic pressure. In turn, these confining conditions are observed to increase the cellular density and affect the cellular organization of the spheroid. Postconfluent spheroids exhibit a necrotic core cemented by a blend of extracellular material and surrounded by a rim of proliferating hypermotile cells. By performing invasion assays in a collagen matrix, we report that peripheral cells readily escape preconfined spheroids and cell–cell cohesivity is maintained for freely growing spheroids, suggesting that mechanical cues from the surrounding microenvironment may trigger cell invasion from a growing tumor. Overall, our technology offers a unique avenue to produce in vitro cell-based assays useful for developing new anticancer therapies and to investigate the interplay between mechanics and growth in tumor evolution. PMID:23980147

  12. Diffusion and binding of monoclonal antibody TNT-1 in multicellular tumor spheroids

    SciTech Connect

    Cheng, F.M.; Hansen, E.B.; Taylor, C.R.; Epstein, A.L. )

    1991-02-06

    Tumor spheroids of HT-29 human colon adenocarcinoma and A375 melanoma were established to investigate the uptake and clearance kinetics of TNT-1, a monoclonal antibody that targets necrotic cells of tumors. Our data reveal that there was rapid uptake of TNT-1 and its F(ab')2 fragment in both spheroid models, whereas an antibody of irrelevant specificity, Lym-1, and its F(ab')2 fragment bound poorly to the spheroids. Unlike previously reported monoclonal antibodies to tumor cell-surface antigens, TNT-1 showed (1) a linear uptake that increased over time without saturation in tumor spheroids and (2) an unexpected uptake by a subpopulation of cells in the viable outer rim of the spheroids. These preclinical studies provide important information concerning the therapeutic potential of TNT monoclonal antibodies for the treatment of cancer and micrometastases.

  13. A Novel Computer-Assisted Approach to evaluate Multicellular Tumor Spheroid Invasion Assay

    PubMed Central

    Cisneros Castillo, Liliana R.; Oancea, Andrei-Dumitru; Stüllein, Christian; Régnier-Vigouroux, Anne

    2016-01-01

    Multicellular tumor spheroids (MCTSs) embedded in a matrix are re-emerging as a powerful alternative to monolayer-based cultures. The primary information gained from a three-dimensional model is the invasiveness of treatment-exposed MCTSs through the acquisition of light microscopy images. The amount and complexity of the acquired data and the bias arisen by their manual analysis are disadvantages calling for an automated, high-throughput analysis. We present a universal algorithm we developed with the scope of being robust enough to handle images of various qualities and various invasion profiles. The novelty and strength of our algorithm lie in: the introduction of a multi-step segmentation flow, where each step is optimized for each specific MCTS area (core, halo, and periphery); the quantification through the density of the two-dimensional representation of a three-dimensional object. This latter offers a fine-granular differentiation of invasive profiles, facilitating a quantification independent of cell lines and experimental setups. Progression of density from the core towards the edges influences the resulting density map thus providing a measure no longer dependent on the sole area size of MCTS, but also on its invasiveness. In sum, we propose a new method in which the concept of quantification of MCTS invasion is completely re-thought. PMID:27731418

  14. Azo-Based Iridium(III) Complexes as Multicolor Phosphorescent Probes to Detect Hypoxia in 3D Multicellular Tumor Spheroids

    PubMed Central

    Sun, Lingli; Li, Guanying; Chen, Xiang; Chen, Yu; Jin, Chengzhi; Ji, Liangnian; Chao, Hui

    2015-01-01

    Hypoxia is an important characteristic of malignant solid tumors and is considered as a possible causative factor for serious resistance to chemo- and radiotherapy. The exploration of novel fluorescent probes capable of detecting hypoxia in solid tumors will aid tumor diagnosis and treatment. In this study, we reported the design and synthesis of a series of “off-on” phosphorescence probes for hypoxia detection in adherent and three-dimensional multicellular spheroid models. All of the iridium(III) complexes incorporate an azo group as an azo-reductase reactive moiety to detect hypoxia. Reduction of non-phosphorescent probes Ir1-Ir8 by reductases under hypoxic conditions resulted in the generation of highly phosphorescent corresponding amines for detection of hypoxic regions. Moreover, these probes can penetrate into 3D multicellular spheroids over 100 μm and image the hypoxic regions. Most importantly, these probes display a high selectivity for the detection of hypoxia in 2D cells and 3D multicellular spheroids. PMID:26423609

  15. Impact of multicellular tumor spheroids as an in vivo‑like tumor model on anticancer drug response.

    PubMed

    Galateanu, Bianca; Hudita, Ariana; Negrei, Carolina; Ion, Rodica-Mariana; Costache, Marieta; Stan, Miriana; Nikitovic, Dragana; Hayes, A Wallace; Spandidos, Demetrios A; Tsatsakis, Aristidis M; Ginghina, Octav

    2016-06-01

    The incidence of colorectal cancer is higher in men than in women, amounting to 15% of cancer-related diseases as a whole. As such, undesirable effects, arising from the administration of current chemotherapeutic agents (the FOLFIRI/FOLFOX combinations), which are exerted on the remaining non-cancerous tissues and/or cells, have contributed to the occurrence of resistance to multiple drugs, thus markedly reducing their efficacy. However, the delivery of chemotherapeutic agents may be improved and their action may be more selectively targeted to diseased tissues/cells by means of developing biotechnologies and nano‑techniques. Thus, the current focus is on creating biological tissue and related tumor models, by means of three‑dimensional (3D) spheres, in an attempt to bridge the gap between results obtained in the pre‑clinical phase and promising outcomes obtained in clinical trials. For this purpose, the characterization and use of so‑called 'multicellular tumor spheroids', may prove to be invaluable. In this study, we focus on describing the efficacy of a model 3D system as compared to the traditional 2D tumor spheres in determining drug response, highlighting a potentially greater effect of the drugs following the encapsulation of respective liposomes. The results obtained demonstrate the successful preparation of a suspension of liposomes loaded with folinic acid, oxaliplatin and 5‑fluorouracil (5‑FU), and loaded with meso‑tetra (4‑sulfonatophenyl) porphyrin. Following its use on HT‑29 colorectal cancer cells, an important comparative reduction was noted in the viability of the HT‑29 cells, demonstrating the efficacy of multicellular tumor spheroids carrying liposomes loaded with therapeutic drugs. These findings indicate that the method of drug encapsulation in liposomes may improve the treatment efficacy of chemotherapeutic agents. PMID:27035518

  16. Impact of multicellular tumor spheroids as an in vivo-like tumor model on anticancer drug response

    PubMed Central

    GALATEANU, BIANCA; HUDITA, ARIANA; NEGREI, CAROLINA; ION, RODICA-MARIANA; COSTACHE, MARIETA; STAN, MIRIANA; NIKITOVIC, DRAGANA; HAYES, A. WALLACE; SPANDIDOS, DEMETRIOS A.; TSATSAKIS, ARISTIDIS M.; GINGHINA, OCTAV

    2016-01-01

    The incidence of colorectal cancer is higher in men than in women, amounting to 15% of cancer-related diseases as a whole. As such, undesirable effects, arising from the administration of current chemotherapeutic agents (the FOLFIRI/FOLFOX combinations), which are exerted on the remaining non-cancerous tissues and/or cells, have contributed to the occurrence of resistance to multiple drugs, thus markedly reducing their efficacy. However, the delivery of chemotherapeutic agents may be improved and their action may be more selectively targeted to diseased tissues/cells by means of developing biotechnologies and nano-techniques. Thus, the current focus is on creating biological tissue and related tumor models, by means of three-dimensional (3D) spheres, in an attempt to bridge the gap between results obtained in the pre-clinical phase and promising outcomes obtained in clinical trials. For this purpose, the characterization and use of so-called ‘multicellular tumor spheroids’, may prove to be invaluable. In this study, we focus on describing the efficacy of a model 3D system as compared to the traditional 2D tumor spheres in determining drug response, highlighting a potentially greater effect of the drugs following the encapsulation of respective liposomes. The results obtained demonstrate the successful preparation of a suspension of liposomes loaded with folinic acid, oxaliplatin and 5-fluorouracil (5-FU), and loaded with meso-tetra (4-sulfonatophenyl) porphyrin. Following its use on HT-29 colorectal cancer cells, an important comparative reduction was noted in the viability of the HT-29 cells, demonstrating the efficacy of multicellular tumor spheroids carrying liposomes loaded with therapeutic drugs. These findings indicate that the method of drug encapsulation in liposomes may improve the treatment efficacy of chemotherapeutic agents. PMID:27035518

  17. Receptor tyrosine kinase targeting in multicellular spheroids.

    PubMed

    Breslin, Susan; O'Driscoll, Lorraine

    2015-01-01

    While growing cells as a monolayer is the traditional method for cell culture, the incorporation of multicellular spheroids into experimental design is becoming increasingly popular. This is due to the understanding that cells grown as spheroids tend to replicate the in vivo situation more reliably than monolayer cells. Thus, the use of multicellular spheroids may be more clinically relevant than monolayer cell cultures. Here, we describe methods for multicellular 3D spheroid generation that may be used to provide samples for receptor tyrosine kinase (and other protein) detection. Methods described include the forced-floating poly-HEMA method, the hanging-drop method, and the use of ECM to form multicellular 3D spheroids. PMID:25319898

  18. Changes in Protein Expression in Two Cholangiocarcinoma Cell Lines Undergoing Formation of Multicellular Tumor Spheroids In Vitro

    PubMed Central

    Mischiati, Carlo; Ura, Blendi; Roncoroni, Leda; Elli, Luca; Cervellati, Carlo; Squerzanti, Monica; Conte, Dario; Doneda, Luisa; Polverino de Laureto, Patrizia; de Franceschi, Giorgia; Calza, Roberta; Barrero, Carlos A.; Merali, Salim; Ferrari, Carlo; Bergamini, Carlo M.; Agostinelli, Enzo

    2015-01-01

    Epithelial-to-Mesenchymal Transition (EMT) is relevant in malignant growth and frequently correlates with worsening disease progression due to its implications in metastases and resistance to therapeutic interventions. Although EMT is known to occur in several types of solid tumors, the information concerning tumors arising from the epithelia of the bile tract is still limited. In order to approach the problem of EMT in cholangiocarcinoma, we decided to investigate the changes in protein expression occurring in two cell lines under conditions leading to growth as adherent monolayers or to formation of multicellular tumor spheroids (MCTS), which are considered culture models that better mimic the growth characteristics of in-vivo solid tumors. In our system, changes in phenotypes occur with only a decrease in transmembrane E-cadherin and vimentin expression, minor changes in the transglutaminase protein/activity but with significant differences in the proteome profiles, with declining and increasing expression in 6 and in 16 proteins identified by mass spectrometry. The arising protein patterns were analyzed based on canonical pathways and network analysis. These results suggest that significant metabolic rearrangements occur during the conversion of cholangiocarcinomas cells to the MCTS phenotype, which most likely affect the carbohydrate metabolism, protein folding, cytoskeletal activity, and tissue sensitivity to oxygen. PMID:25756965

  19. Tumor-penetrating peptide fused EGFR single-domain antibody enhances cancer drug penetration into 3D multicellular spheroids and facilitates effective gastric cancer therapy

    PubMed Central

    Sha, Huizi; Zou, Zhengyun; Xin, Kai; Bian, Xinyu; Cai, Xueting; Lu, Wuguang; Chen, Jiao; Chen, Gang; Huang, Leaf; Blair, Andrew M.; Cao, Peng; Liu, Baorui

    2016-01-01

    Human tumors, including gastric cancer, frequently express high levels of epidermal growth factor receptors (EGFRs), which are associated with a poor prognosis. Targeted delivery of anticancer drugs to cancerous tissues shows potential in sparing unaffected tissues. However, it has been a major challenge for drug penetration in solid tumor tissues due to the complicated tumor microenvironment. We have constructed a recombinant protein named anti-EGFR-iRGD consisting of an anti-EGFR VHH (the variable domain from the heavy chain of the antibody) fused to iRGD, a tumor-specific binding peptide with high permeability. Anti-EGFR-iRGD, which targets EGFR and αvβ3, spreads extensively throughout both the multicellular spheroids and the tumor mass. The recombinant protein anti-EGFR-iRGD also exhibited antitumor activity in tumor cell lines, multicellular spheroids, and mice. Moreover, anti-EGFR-iRGD could improve anticancer drugs, such as doxorubicin (DOX), bevacizumab, nanoparticle permeability and efficacy in multicellular spheroids. This study draws attention to the importance of iRGD peptide in the therapeutic approach of anti-EGFR-iRGD. As a consequence, anti-EGFR-iRGD could be a drug candidate for cancer treatment and a useful adjunct of other anticancer drugs. PMID:25553823

  20. Short and long time effects of low temperature Plasma Activated Media on 3D multicellular tumor spheroids

    NASA Astrophysics Data System (ADS)

    Judée, Florian; Fongia, Céline; Ducommun, Bernard; Yousfi, Mohammed; Lobjois, Valérie; Merbahi, Nofel

    2016-02-01

    This work investigates the regionalized antiproliferative effects of plasma-activated medium (PAM) on colon adenocarcinoma multicellular tumor spheroid (MCTS), a model that mimics 3D organization and regionalization of a microtumor region. PAM was generated by dielectric barrier plasma jet setup crossed by helium carrier gas. MCTS were transferred in PAM at various times after plasma exposure up to 48 hours and effect on MCTS growth and DNA damage were evaluated. We report the impact of plasma exposure duration and delay before transfer on MCTS growth and DNA damage. Local accumulation of DNA damage revealed by histone H2AX phosphorylation is observed on outermost layers and is dependent on plasma exposure. DNA damage is completely reverted by catalase addition indicating that H2O2 plays major role in observed genotoxic effect while growth inhibitory effect is maintained suggesting that it is due to others reactive species. SOD and D-mannitol scavengers also reduced DNA damage by 30% indicating that and OH* are involved in H2O2 formation. Finally, PAM is able to retain its cytotoxic and genotoxic activity upon storage at +4 °C or ‑80 °C. These results suggest that plasma activated media may be a promising new antitumor strategy for colorectal cancer tumors.

  1. Short and long time effects of low temperature Plasma Activated Media on 3D multicellular tumor spheroids

    PubMed Central

    Judée, Florian; Fongia, Céline; Ducommun, Bernard; Yousfi, Mohammed; Lobjois, Valérie; Merbahi, Nofel

    2016-01-01

    This work investigates the regionalized antiproliferative effects of plasma-activated medium (PAM) on colon adenocarcinoma multicellular tumor spheroid (MCTS), a model that mimics 3D organization and regionalization of a microtumor region. PAM was generated by dielectric barrier plasma jet setup crossed by helium carrier gas. MCTS were transferred in PAM at various times after plasma exposure up to 48 hours and effect on MCTS growth and DNA damage were evaluated. We report the impact of plasma exposure duration and delay before transfer on MCTS growth and DNA damage. Local accumulation of DNA damage revealed by histone H2AX phosphorylation is observed on outermost layers and is dependent on plasma exposure. DNA damage is completely reverted by catalase addition indicating that H2O2 plays major role in observed genotoxic effect while growth inhibitory effect is maintained suggesting that it is due to others reactive species. SOD and D-mannitol scavengers also reduced DNA damage by 30% indicating that and OH* are involved in H2O2 formation. Finally, PAM is able to retain its cytotoxic and genotoxic activity upon storage at +4 °C or −80 °C. These results suggest that plasma activated media may be a promising new antitumor strategy for colorectal cancer tumors. PMID:26898904

  2. Short and long time effects of low temperature Plasma Activated Media on 3D multicellular tumor spheroids

    NASA Astrophysics Data System (ADS)

    Judée, Florian; Fongia, Céline; Ducommun, Bernard; Yousfi, Mohammed; Lobjois, Valérie; Merbahi, Nofel

    2016-02-01

    This work investigates the regionalized antiproliferative effects of plasma-activated medium (PAM) on colon adenocarcinoma multicellular tumor spheroid (MCTS), a model that mimics 3D organization and regionalization of a microtumor region. PAM was generated by dielectric barrier plasma jet setup crossed by helium carrier gas. MCTS were transferred in PAM at various times after plasma exposure up to 48 hours and effect on MCTS growth and DNA damage were evaluated. We report the impact of plasma exposure duration and delay before transfer on MCTS growth and DNA damage. Local accumulation of DNA damage revealed by histone H2AX phosphorylation is observed on outermost layers and is dependent on plasma exposure. DNA damage is completely reverted by catalase addition indicating that H2O2 plays major role in observed genotoxic effect while growth inhibitory effect is maintained suggesting that it is due to others reactive species. SOD and D-mannitol scavengers also reduced DNA damage by 30% indicating that and OH* are involved in H2O2 formation. Finally, PAM is able to retain its cytotoxic and genotoxic activity upon storage at +4 °C or -80 °C. These results suggest that plasma activated media may be a promising new antitumor strategy for colorectal cancer tumors.

  3. Short and long time effects of low temperature Plasma Activated Media on 3D multicellular tumor spheroids.

    PubMed

    Judée, Florian; Fongia, Céline; Ducommun, Bernard; Yousfi, Mohammed; Lobjois, Valérie; Merbahi, Nofel

    2016-01-01

    This work investigates the regionalized antiproliferative effects of plasma-activated medium (PAM) on colon adenocarcinoma multicellular tumor spheroid (MCTS), a model that mimics 3D organization and regionalization of a microtumor region. PAM was generated by dielectric barrier plasma jet setup crossed by helium carrier gas. MCTS were transferred in PAM at various times after plasma exposure up to 48 hours and effect on MCTS growth and DNA damage were evaluated. We report the impact of plasma exposure duration and delay before transfer on MCTS growth and DNA damage. Local accumulation of DNA damage revealed by histone H2AX phosphorylation is observed on outermost layers and is dependent on plasma exposure. DNA damage is completely reverted by catalase addition indicating that H2O2 plays major role in observed genotoxic effect while growth inhibitory effect is maintained suggesting that it is due to others reactive species. SOD and D-mannitol scavengers also reduced DNA damage by 30% indicating that O(2)(-)* and OH* are involved in H2O2 formation. Finally, PAM is able to retain its cytotoxic and genotoxic activity upon storage at +4 °C or -80 °C. These results suggest that plasma activated media may be a promising new antitumor strategy for colorectal cancer tumors. PMID:26898904

  4. Visualizing the effect of tumor microenvironments on radiation-induced cell kinetics in multicellular spheroids consisting of HeLa cells

    SciTech Connect

    Kaida, Atsushi; Miura, Masahiko

    2013-10-04

    Highlights: •We visualized radiation-induced cell kinetics in spheroids. •HeLa-Fucci cells were used for detection of cell-cycle changes. •Radiation-induced G2 arrest was prolonged in the spheroid. •The inner and outer cell fractions behaved differently. -- Abstract: In this study, we visualized the effect of tumor microenvironments on radiation-induced tumor cell kinetics. For this purpose, we utilized a multicellular spheroid model, with a diameter of ∼500 μm, consisting of HeLa cells expressing the fluorescent ubiquitination-based cell-cycle indicator (Fucci). In live spheroids, a confocal laser scanning microscope allowed us to clearly monitor cell kinetics at depths of up to 60 μm. Surprisingly, a remarkable prolongation of G2 arrest was observed in the outer region of the spheroid relative to monolayer-cultured cells. Scale, an aqueous reagent that renders tissues optically transparent, allowed visualization deeper inside spheroids. About 16 h after irradiation, a red fluorescent cell fraction, presumably a quiescent G0 cell fraction, became distinct from the outer fraction consisting of proliferating cells, most of which exhibited green fluorescence indicative of G2 arrest. Thereafter, the red cell fraction began to emit green fluorescence and remained in prolonged G2 arrest. Thus, for the first time, we visualized the prolongation of radiation-induced G2 arrest in spheroids and the differences in cell kinetics between the outer and inner fractions.

  5. Increased nanoparticle penetration in collagenase-treated multicellular spheroids

    PubMed Central

    Goodman, Thomas T; Olive, Peggy L; Pun, Suzie H

    2007-01-01

    The extracellular matrix of solid tumors presents a transport barrier that restricts nanoparticle penetration, thereby limiting the efficacy of nanosized delivery vehicles for cancer imaging and therapy. In this study, the effect of nanoparticle size and collagenase treatment on penetration of carboxylated polystyrene nanoparticles was systematically assessed in a multicellular spheroid model. Penetration of the nanoparticles into the spheroid core was limited to particles smaller than 100 nm. Collagenase treatment of spheroids resulted in significantly increased penetration of nanoparticles up to 100 nm with only a minor increase in particle penetration observed for particles larger than 100 nm. Collagenase was immobilized onto the surface of nanoparticles for site-specific degradation of ECM proteins. Collagenase-coated, 100 nm nanoparticles demonstrated a 4-fold increase in the number of particles delivered to the spheroid core compared with control nanoparticles. Thus, nanoparticle delivery to solid tumors may be substantially improved by the incorporation of ECM-modulating enzymes in the delivery formulation. PMID:17722554

  6. Quantitative Determination of Irinotecan and the Metabolite SN-38 by Nanoflow Liquid Chromatography-Tandem Mass Spectrometry in Different Regions of Multicellular Tumor Spheroids

    PubMed Central

    Liu, Xin; Hummon, Amanda B.

    2015-01-01

    A new and simple method was developed to evaluate the distribution of therapeutics in three-dimensional multicellular tumor spheroids (MCTS) by combining serial trypsinization and nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS). This methodology was validated with quantitative measurements of irinotecan and its bioactive metabolite, SN-38, in distinct spatial regions of HCT 116 MCTS. Irinotecan showed a time-dependent permeability into MCTS with most of the drug accumulating in the core after 24 hours of treatment. The amount of SN-38 detected was 30 times lower than that of the parent drug, and was more abundant in the outer rim and intermediate regions of MCTS where proliferating cells were present. This method can be used to investigate novel and established drugs. It enables investigation of drug penetration properties and identification of metabolites with spatial specificity in MCTS. The new approach has great value in facilitating the drug evaluation process. PMID:25604392

  7. Quantitative Determination of Irinotecan and the Metabolite SN-38 by Nanoflow Liquid Chromatography-Tandem Mass Spectrometry in Different Regions of Multicellular Tumor Spheroids

    NASA Astrophysics Data System (ADS)

    Liu, Xin; Hummon, Amanda B.

    2015-04-01

    A new and simple method was developed to evaluate the distribution of therapeutics in three-dimensional multicellular tumor spheroids (MCTS) by combining serial trypsinization and nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS). This methodology was validated with quantitative measurements of irinotecan and its bioactive metabolite, SN-38, in distinct spatial regions of HCT 116 MCTS. Irinotecan showed a time-dependent permeability into MCTS with most of the drug accumulating in the core after 24 h of treatment. The amount of SN-38 detected was 30 times lower than that of the parent drug, and was more abundant in the outer rim and intermediate regions of MCTS where proliferating cells were present. This method can be used to investigate novel and established drugs. It enables investigation of drug penetration properties and identification of metabolites with spatial specificity in MCTS. The new approach has great value in facilitating the drug evaluation process.

  8. GPI/AMF inhibition blocks the development of the metastatic phenotype of mature multi-cellular tumor spheroids.

    PubMed

    Gallardo-Pérez, Juan Carlos; Rivero-Segura, Nadia Alejandra; Marín-Hernández, Alvaro; Moreno-Sánchez, Rafael; Rodríguez-Enríquez, Sara

    2014-06-01

    Epithelial-mesenchymal transition (EMT) and cellular invasiveness are two pivotal processes for the development of metastatic tumor phenotypes. The metastatic profile of non-metastatic MCF-7 cells growing as multi-cellular tumor microspheroids (MCTSs) was analyzed by determining the contents of the EMT, invasive and migratory proteins, as well as their migration and invasiveness potential and capacity to secrete active cytokines such as the glucose phosphate isomerase/AMF (GPI/AMF). As for the control, the same analysis was also performed in MCF-7 and MDA-MB-231 (highly metastatic, MDA) monolayer cells, and in stage IIIB and IV human metastatic breast biopsies. The proliferative cell layers (PRL) of mature MCF-7 MCTSs, MDA monolayer cells and metastatic biopsies exhibited increased cellular contents (2-15 times) of EMT (β-catenin, SNAIL), migratory (vimentin, cytokeratin, and fibronectin) and invasive (MMP-1, VEGF) proteins versus MCF-7 monolayer cells, quiescent cell layers of mature MCF-7 MCTS and non-metastatic breast biopsies. The increase in metastatic proteins correlated with substantially elevated cellular abilities for migration (18-times) and invasiveness (13-times) and with the higher level (6-times) of the cytokine GPI/AMF in the extracellular medium of PRL, as compared to MCF-7 monolayer cells. Interestingly, the addition of the GPI/AMF inhibitors erythrose-4-phosphate or 6-phosphogluconate at micromolar doses significantly decreased its extracellular activity (>80%), with a concomitant diminution in the metastatic protein content and migratory tumor cell capacity, and with no inhibitory effect on tumor lactate production or toxicity on 3T3 mouse fibroblasts. The present findings provide new insights into the discovery of metabolic inhibitors to be used as complementary therapy against metastatic and aggressive tumors.

  9. Response of 9L rat brain tumor multicellular spheroids to single and fractionated doses of 1,3-bis(2-chloroethyl)-1-nitrosourea.

    PubMed

    Sano, Y; Hoshino, T; Barker, M; Deen, D F

    1984-02-01

    This study was designed to examine the relative effect of each of four fractions of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) against 9L rat brain tumor multicellular spheroids and to compare the results of the cell survival and growth delay assays. Similar levels of cell kill resulted when BCNU was administered either as single fractions of 1.5, 3.0, 4.5, or 6.0 micrograms/ml for 1 hr or as one to four fractions of 1.5 micrograms/ml that were administered sequentially for 1 hr each. Survival was increased if the assay was delayed until 24 hr after drug treatment, which indicates that 9L cells in spheroids recover from BCNU-induced potentially lethal damage. When BCNU was administered in 1.5-micrograms/ml fractions, plating efficiencies depended markedly on the interval between the fractions. The 12-hr protocol produced an overall higher cell kill. Fractionation schedules of 24 and 36 hr produced less cell kill than did the other schedules. Survival plateaued for the last three treatments with BCNU in the 36-hr schedule. Cells in S phase at the time of administration of the initial 1.5-micrograms/ml fraction of BCNU moved into G1- and G2-M phases by 12 hr after treatment. For time periods longer than 12 hr, cells began to appear in the BCNU-resistant S phase. Thus, the movement of cells into the drug-sensitive and -resistant phases after the first fraction correlates well with the corresponding overall cytotoxic effect produced by treatment with the combined BCNU (1.5 micrograms/ml) fractions. For a higher concentration (3.0 micrograms/ml for 1 hr), maximum cell kill was reached within the 12- to 18-hr interval, after which cell kill plateaued. Cells were not found in the S-phase fraction 12 to 36 hr after the first treatment with 3.0 micrograms/ml; maximum cell kill for the fractionated protocols resulted at these times. Therefore, BCNU, which is classified as a cell cycle-nonspecific drug, can induce a partial synchrony in 9L spheroid cells, which determines

  10. Droplet-based microfluidic system to form and separate multicellular spheroids using magnetic nanoparticles.

    PubMed

    Yoon, Sungjun; Kim, Jeong Ah; Lee, Seung Hwan; Kim, Minsoo; Park, Tai Hyun

    2013-04-21

    The importance of creating a three-dimensional (3-D) multicellular spheroid has recently been gaining attention due to the limitations of monolayer cell culture to precisely mimic in vivo structure and cellular interactions. Due to this emerging interest, researchers have utilized new tools, such as microfluidic devices, that allow high-throughput and precise size control to produce multicellular spheroids. We have developed a droplet-based microfluidic system that can encapsulate both cells and magnetic nanoparticles within alginate beads to mimic the function of a multicellular tumor spheroid. Cells were entrapped within the alginate beads along with magnetic nanoparticles, and the beads of a relatively uniform size (diameters of 85% of the beads were 170-190 μm) were formed in the oil phase. These beads were passed through parallel streamlines of oil and culture medium, where the beads were magnetically transferred into the medium phase from the oil phase using an external magnetic force. This microfluidic chip eliminates additional steps for collecting the spheroids from the oil phase and transferring them to culture medium. Ultimately, the overall spheroid formation process can be achieved on a single microchip. PMID:23426090

  11. Study of the Chemotactic Response of Multicellular Spheroids in a Microfluidic Device.

    PubMed

    Ayuso, Jose M; Basheer, Haneen A; Monge, Rosa; Sánchez-Álvarez, Pablo; Doblaré, Manuel; Shnyder, Steven D; Vinader, Victoria; Afarinkia, Kamyar; Fernández, Luis J; Ochoa, Ignacio

    2015-01-01

    We report the first application of a microfluidic device to observe chemotactic migration in multicellular spheroids. A microfluidic device was designed comprising a central microchamber and two lateral channels through which reagents can be introduced. Multicellular spheroids were embedded in collagen and introduced to the microchamber. A gradient of fetal bovine serum (FBS) was established across the central chamber by addition of growth media containing serum into one of the lateral channels. We observe that spheroids of oral squamous carcinoma cells OSC-19 invade collectively in the direction of the gradient of FBS. This invasion is more directional and aggressive than that observed for individual cells in the same experimental setup. In contrast to spheroids of OSC-19, U87-MG multicellular spheroids migrate as individual cells. A study of the exposure of spheroids to the chemoattractant shows that the rate of diffusion into the spheroid is slow and thus, the chemoattractant wave engulfs the spheroid before diffusing through it. PMID:26444904

  12. Study of the Chemotactic Response of Multicellular Spheroids in a Microfluidic Device

    PubMed Central

    Ayuso, Jose M.; Basheer, Haneen A.; Monge, Rosa; Sánchez-Álvarez, Pablo; Doblaré, Manuel; Shnyder, Steven D.; Vinader, Victoria; Afarinkia, Kamyar

    2015-01-01

    We report the first application of a microfluidic device to observe chemotactic migration in multicellular spheroids. A microfluidic device was designed comprising a central microchamber and two lateral channels through which reagents can be introduced. Multicellular spheroids were embedded in collagen and introduced to the microchamber. A gradient of fetal bovine serum (FBS) was established across the central chamber by addition of growth media containing serum into one of the lateral channels. We observe that spheroids of oral squamous carcinoma cells OSC–19 invade collectively in the direction of the gradient of FBS. This invasion is more directional and aggressive than that observed for individual cells in the same experimental setup. In contrast to spheroids of OSC–19, U87-MG multicellular spheroids migrate as individual cells. A study of the exposure of spheroids to the chemoattractant shows that the rate of diffusion into the spheroid is slow and thus, the chemoattractant wave engulfs the spheroid before diffusing through it. PMID:26444904

  13. Regulation of Multicellular Spheroids by MAPK and FYN Kinase.

    PubMed

    Lee, Casey; Ramos, Daniel M

    2016-08-01

    Understanding of the biology of oral squamous cell carcinoma (SCC) has not progressed significantly in the past 60 years, with 5-year survival remaining at approximately 50%. The epidemic of Human Papilloma Virus and its associated SCC warrants a renewed emphasis on fully understanding this disease. We previously used the 3-dimensional multicellular spheroid (MCS) model system to evaluate SCC behavior more accurately. In this study, we determined that SCC growth in MCS approximates epithelial to mesenchymal transition. Organization of an MCS requires the full-length β6 integrin subunit and its maintenance requires mitogen-activated protein kinase (MAPK). Limiting FYN kinase activation results in the down-regulation of E-cadherin, β-catenin and an increase in expression of N-cadherin and SNAIL. These results indicate that the microenvironment and growth patterns in an MCS are complex and require MAPK and FYN kinase. PMID:27466485

  14. EMBEDDED MULTICELLULAR SPHEROIDS AS A BIOMIMETIC 3D CANCER MODEL FOR EVALUATING DRUG AND DRUG-DEVICE COMBINATIONS

    PubMed Central

    Charoen, Kristie M.; Fallica, Brian; Colson, Yolonda L.; Zaman, Muhammad H.; Grinstaff, Mark W.

    2014-01-01

    Multicellular aggregates of cells, termed spheroids, are of interest for studying tumor behavior and for evaluating the response of pharmacologically active agents. Spheroids more faithfully reproduce the tumor macrostructure found in vivo compared to classical 2D monolayers. We present a method for embedding spheroids within collagen gels followed by quantitative and qualitative whole spheroid and single cell analyses enabling characterization over the length scales from molecular to macroscopic. Spheroid producing and embedding capabilities are demonstrated for U2OS and MDAMB 231 cell lines, of osteosarcoma and breast adenocarncinoma origin, respectively. Finally, using the MDA-MB-231 tumor model, the chemotherapeutic response between paclitaxel delivery as a bolus dose, as practiced in the clinic, is compared to delivery within an expansile nanoparticle. The expansile nanoparticle delivery route provides a superior outcome and the results mirror those observed in a murine xenograft model. These findings highlight the synergistic beneficial results that may arise from the use of a drug delivery system, and the need to evaluate both drug candidates and delivery systems in the research and pre-clinical screening phases of a new cancer therapy development program. PMID:24360576

  15. Nonlinear 3D Projection Printing of Concave Hydrogel Microstructures for Long-Term Multicellular Spheroid and Embryoid Body Culture

    PubMed Central

    Hribar, K.C; Finlay, D.; Ma, X.; Qu, X.; Ondeck, M. G.; Chung, P. H.; Zanella, F.; Engler, A. J.; Sheikh, F.; Vuori, K.; Chen, S.

    2015-01-01

    Long-term culture and monitoring of individual multicellular spheroids and embryoid bodies (EBs) remains a challenge for in vitro cell propogation. Here, we used a continuous 3D projection printing approach – with an important modification of nonlinear exposure — to generate concave hydrogel microstructures that permit spheroid growth and long-term maintenance, without the need for spheroid transfer. Breast cancer spheroids grown to 10 d in the concave structures showed hypoxic cores and signs of necrosis using immunofluorescent and histochemical staining, key features of the tumor microenvironment in vivo. EBs consisting of induced pluripotent stem cells (iPSCs) grown on the hydrogels demonstrated narrow size distribution and undifferentiated markers at 3 d, followed by signs of differentiation by the presence of cavities and staining of the three germ layers at 10 d. These findings demonstrate a new method for long-term (e.g. beyond spheroid formation at day 2, and with media exchange) 3D cell culture that should be able to assist in cancer spheroid studies as well as embryogenesis and patient-derived disease modeling with iPSC EBs. PMID:25900329

  16. Nonlinear 3D projection printing of concave hydrogel microstructures for long-term multicellular spheroid and embryoid body culture.

    PubMed

    Hribar, K C; Finlay, D; Ma, X; Qu, X; Ondeck, M G; Chung, P H; Zanella, F; Engler, A J; Sheikh, F; Vuori, K; Chen, S C

    2015-06-01

    Long-term culture and monitoring of individual multicellular spheroids and embryoid bodies (EBs) remains a challenge for in vitro cell propagation. Here, we used a continuous 3D projection printing approach - with an important modification of nonlinear exposure - to generate concave hydrogel microstructures that permit spheroid growth and long-term maintenance, without the need for spheroid transfer. Breast cancer spheroids grown to 10 d in the concave structures showed hypoxic cores and signs of necrosis using immunofluorescent and histochemical staining, key features of the tumor microenvironment in vivo. EBs consisting of induced pluripotent stem cells (iPSCs) grown on the hydrogels demonstrated narrow size distribution and undifferentiated markers at 3 d, followed by signs of differentiation by the presence of cavities and staining of the three germ layers at 10 d. These findings demonstrate a new method for long-term (e.g. beyond spheroid formation at day 2, and with media exchange) 3D cell culture that should be able to assist in cancer spheroid studies as well as embryogenesis and patient-derived disease modeling with iPSC EBs. PMID:25900329

  17. Nonlinear 3D projection printing of concave hydrogel microstructures for long-term multicellular spheroid and embryoid body culture.

    PubMed

    Hribar, K C; Finlay, D; Ma, X; Qu, X; Ondeck, M G; Chung, P H; Zanella, F; Engler, A J; Sheikh, F; Vuori, K; Chen, S C

    2015-06-01

    Long-term culture and monitoring of individual multicellular spheroids and embryoid bodies (EBs) remains a challenge for in vitro cell propagation. Here, we used a continuous 3D projection printing approach - with an important modification of nonlinear exposure - to generate concave hydrogel microstructures that permit spheroid growth and long-term maintenance, without the need for spheroid transfer. Breast cancer spheroids grown to 10 d in the concave structures showed hypoxic cores and signs of necrosis using immunofluorescent and histochemical staining, key features of the tumor microenvironment in vivo. EBs consisting of induced pluripotent stem cells (iPSCs) grown on the hydrogels demonstrated narrow size distribution and undifferentiated markers at 3 d, followed by signs of differentiation by the presence of cavities and staining of the three germ layers at 10 d. These findings demonstrate a new method for long-term (e.g. beyond spheroid formation at day 2, and with media exchange) 3D cell culture that should be able to assist in cancer spheroid studies as well as embryogenesis and patient-derived disease modeling with iPSC EBs.

  18. Differential penetration of targeting agents into multicellular spheroids derived from human neuroblastoma

    SciTech Connect

    Mairs, R.J.; Angerson, W.J.; Babich, J.W.; Murray, T. )

    1991-01-01

    The authors have used a multicellular tumour spheroid model for determination of the penetration of various targeting agents of potential use in the treatment of neuroblastoma. Both the radiopharmaceutical meta-iodobenzylguanidine (mIBG) and the {beta} subunit of nerve growth factor ({beta}-NGF) distributed uniformly throughout spheroids, though the latter was poorly concentrated relative to mIBG. In contrast, the anti-neuroectodermal monoclonal antibody. UJ13A bound only to peripheral cell layers with little accumulation in the spheroid interior. Differential penetration of targeting agents may influence the choice of conjugated radionuclide which is likely to achieve maximum therapeutic benefit.

  19. Advances in the formation, use and understanding of multi-cellular spheroids

    PubMed Central

    Achilli, Toni-Marie; Meyer, Julia; Morgan, Jeffrey R

    2015-01-01

    Introduction Developing in vitro models for studying cell biology and cell physiology is of great importance to the fields of biotechnology, cancer research, drug discovery, toxicity testing, as well as the emerging fields of tissue engineering and regenerative medicine. Traditional two dimensional (2D) methods of mammalian cell culture have several limitations and it is increasingly recognized that cells grown in a three dimensional (3D) environment more closely represent normal cellular function due to the increased cell-to-cell interactions, and by mimicking the in vivo architecture of natural organs and tissues. Areas Covered In this review, we discuss the methods to form 3D multi-cellular spheroids, the advantages and limitations of these methods, and assays used to characterize the function of spheroids. The use of spheroids has led to many advances in basic cell sciences, including understanding cancer cell interactions, creating models for drug discovery and cancer metastasis, and they are being investigated as basic units for engineering tissue constructs. As so, this review will focus on contributions made to each of these fields using spheroid models. Expert Opinion Multi-cellular spheroids are rich in biological content and mimic better the in vivo environment than 2D cell culture. New technologies to form and analyze spheroids are rapidly increasing their adoption and expanding their applications. PMID:22784238

  20. Rapid formation of size-controllable multicellular spheroids via 3D acoustic tweezers.

    PubMed

    Chen, Kejie; Wu, Mengxi; Guo, Feng; Li, Peng; Chan, Chung Yu; Mao, Zhangming; Li, Sixing; Ren, Liqiang; Zhang, Rui; Huang, Tony Jun

    2016-07-01

    The multicellular spheroid is an important 3D cell culture model for drug screening, tissue engineering, and fundamental biological research. Although several spheroid formation methods have been reported, the field still lacks high-throughput and simple fabrication methods to accelerate its adoption in drug development industry. Surface acoustic wave (SAW) based cell manipulation methods, which are known to be non-invasive, flexible, and high-throughput, have not been successfully developed for fabricating 3D cell assemblies or spheroids, due to the limited understanding on SAW-based vertical levitation. In this work, we demonstrated the capability of fabricating multicellular spheroids in the 3D acoustic tweezers platform. Our method used drag force from microstreaming to levitate cells in the vertical direction, and used radiation force from Gor'kov potential to aggregate cells in the horizontal plane. After optimizing the device geometry and input power, we demonstrated the rapid and high-throughput nature of our method by continuously fabricating more than 150 size-controllable spheroids and transferring them to Petri dishes every 30 minutes. The spheroids fabricated by our 3D acoustic tweezers can be cultured for a week with good cell viability. We further demonstrated that spheroids fabricated by this method could be used for drug testing. Unlike the 2D monolayer model, HepG2 spheroids fabricated by the 3D acoustic tweezers manifested distinct drug resistance, which matched existing reports. The 3D acoustic tweezers based method can serve as a novel bio-manufacturing tool to fabricate complex 3D cell assembles for biological research, tissue engineering, and drug development. PMID:27327102

  1. Emergent Properties of Tumor Microenvironment in a Real-Life Model of Multicell Tumor Spheroids

    PubMed Central

    Milotti, Edoardo; Chignola, Roberto

    2010-01-01

    Multicellular tumor spheroids are an important in vitro model of the pre-vascular phase of solid tumors, for sizes well below the diagnostic limit: therefore a biophysical model of spheroids has the ability to shed light on the internal workings and organization of tumors at a critical phase of their development. To this end, we have developed a computer program that integrates the behavior of individual cells and their interactions with other cells and the surrounding environment. It is based on a quantitative description of metabolism, growth, proliferation and death of single tumor cells, and on equations that model biochemical and mechanical cell-cell and cell-environment interactions. The program reproduces existing experimental data on spheroids, and yields unique views of their microenvironment. Simulations show complex internal flows and motions of nutrients, metabolites and cells, that are otherwise unobservable with current experimental techniques, and give novel clues on tumor development and strong hints for future therapies. PMID:21152429

  2. Comparative analysis of tumor spheroid generation techniques for differential in vitro drug toxicity

    PubMed Central

    Raghavan, Shreya; Rowley, Katelyn R.; Mehta, Geeta

    2016-01-01

    Multicellular tumor spheroids are powerful in vitro models to perform preclinical chemosensitivity assays. We compare different methodologies to generate tumor spheroids in terms of resultant spheroid morphology, cellular arrangement and chemosensitivity. We used two cancer cell lines (MCF7 and OVCAR8) to generate spheroids using i) hanging drop array plates; ii) liquid overlay on ultra-low attachment plates; iii) liquid overlay on ultra-low attachment plates with rotating mixing (nutator plates). Analysis of spheroid morphometry indicated that cellular compaction was increased in spheroids generated on nutator and hanging drop array plates. Collagen staining also indicated higher compaction and remodeling in tumor spheroids on nutator and hanging drop arrays compared to conventional liquid overlay. Consequently, spheroids generated on nutator or hanging drop plates had increased chemoresistance to cisplatin treatment (20-60% viability) compared to spheroids on ultra low attachment plates (10-20% viability). Lastly, we used a mathematical model to demonstrate minimal changes in oxygen and cisplatin diffusion within experimentally generated spheroids. Our results demonstrate that in vitro methods of tumor spheroid generation result in varied cellular arrangement and chemosensitivity. PMID:26918944

  3. Generation and functional assessment of 3D multicellular spheroids in droplet based microfluidics platform.

    PubMed

    Sabhachandani, P; Motwani, V; Cohen, N; Sarkar, S; Torchilin, V; Konry, T

    2016-02-01

    Here we describe a robust, microfluidic technique to generate and analyze 3D tumor spheroids, which resembles tumor microenvironment and can be used as a more effective preclinical drug testing and screening model. Monodisperse cell-laden alginate droplets were generated in polydimethylsiloxane (PDMS) microfluidic devices that combine T-junction droplet generation and external gelation for spheroid formation. The proposed approach has the capability to incorporate multiple cell types. For the purposes of our study, we generated spheroids with breast cancer cell lines (MCF-7 drug sensitive and resistant) and co-culture spheroids of MCF-7 together with a fibroblast cell line (HS-5). The device has the capability to house 1000 spheroids on chip for drug screening and other functional analysis. Cellular viability of spheroids in the array part of the device was maintained for two weeks by continuous perfusion of complete media into the device. The functional performance of our 3D tumor models and a dose dependent response of standard chemotherapeutic drug, doxorubicin (Dox) and standard drug combination Dox and paclitaxel (PCT) was analyzed on our chip-based platform. Altogether, our work provides a simple and novel, in vitro platform to generate, image and analyze uniform, 3D monodisperse alginate hydrogel tumors for various omic studies and therapeutic efficiency screening, an important translational step before in vivo studies. PMID:26686985

  4. Anti-gastric cancer activity in three-dimensional tumor spheroids of bufadienolides.

    PubMed

    Wang, Jixia; Zhang, Xiuli; Li, Xiaolong; Zhang, Yun; Hou, Tao; Wei, Lai; Qu, Lala; Shi, Liying; Liu, Yanfang; Zou, Lijuan; Liang, Xinmiao

    2016-01-01

    Multicellular spheroids of cancer cells have been increasingly used to screen anti-tumor compounds, owing to their in vivo like microenvironment and structure as well as compatibility to high-throughput/high-content screening. Here we report the potency and efficacy of a family of bufadienolides to inhibit the growth of gastric cancer cell line HGC-27 in three-dimensional (3D) spheroidal models. Examining the morphological and growth patterns of several cell lines in round-bottomed ultra-low attachment microplate suggested that HGC-27 cells formed reproducibly multicellular spheroidal structures. Profiling of 15 natural bufadienolides isolated from toad skin indicated that 8 14-hydroxy bufadienolides displayed inhibitory activity of the growth of HGC-27 spheroids in a dose-dependent manner. Notably, compared to clinical drugs taxol and epirubicin, active bufadienolides were found to penetrate more effectively into the HGC-27 spheroids, but with a narrower effective concentration range and a shorter lasting inhibitory effect. Furthermore, compared to two-dimensional (2D) cell monolayer assays, active bufadienolides exhibited weaker efficacy and different potency in 3D spheroid model, demonstrating the great potential of 3D multicellular cell spheroid models in anti-cancer drug discovery and development. PMID:27098119

  5. Anti-gastric cancer activity in three-dimensional tumor spheroids of bufadienolides

    PubMed Central

    Wang, Jixia; Zhang, Xiuli; Li, Xiaolong; Zhang, Yun; Hou, Tao; Wei, Lai; Qu, Lala; Shi, Liying; Liu, Yanfang; Zou, Lijuan; Liang, Xinmiao

    2016-01-01

    Multicellular spheroids of cancer cells have been increasingly used to screen anti-tumor compounds, owing to their in vivo like microenvironment and structure as well as compatibility to high-throughput/high-content screening. Here we report the potency and efficacy of a family of bufadienolides to inhibit the growth of gastric cancer cell line HGC-27 in three-dimensional (3D) spheroidal models. Examining the morphological and growth patterns of several cell lines in round-bottomed ultra-low attachment microplate suggested that HGC-27 cells formed reproducibly multicellular spheroidal structures. Profiling of 15 natural bufadienolides isolated from toad skin indicated that 8 14-hydroxy bufadienolides displayed inhibitory activity of the growth of HGC-27 spheroids in a dose-dependent manner. Notably, compared to clinical drugs taxol and epirubicin, active bufadienolides were found to penetrate more effectively into the HGC-27 spheroids, but with a narrower effective concentration range and a shorter lasting inhibitory effect. Furthermore, compared to two-dimensional (2D) cell monolayer assays, active bufadienolides exhibited weaker efficacy and different potency in 3D spheroid model, demonstrating the great potential of 3D multicellular cell spheroid models in anti-cancer drug discovery and development. PMID:27098119

  6. miR-509-3p is clinically significant and strongly attenuates cellular migration and multi-cellular spheroids in ovarian cancer

    PubMed Central

    Pedersen, Lykke; Lim, Emilia; Hernandez-Herrera, Anadulce; Rowat, Amy C.; Patil, Sagar L.; Chan, Clara K.; Wen, Yunfei; Zhang, Xinna; Basu-Roy, Upal; Mansukhani, Alka; Chu, Andy; Sipahimalani, Payal; Bowlby, Reanne; Brooks, Denise; Thiessen, Nina; Coarfa, Cristian; Ma, Yussanne; Moore, Richard A.; Schein, Jacquie E.; Mungall, Andrew J.; Liu, Jinsong; Pecot, Chad V.; Sood, Anil K.; Jones, Steven J.M.; Marra, Marco A.; Gunaratne, Preethi H.

    2016-01-01

    Ovarian cancer presents as an aggressive, advanced stage cancer with widespread metastases that depend primarily on multicellular spheroids in the peritoneal fluid. To identify new druggable pathways related to metastatic progression and spheroid formation, we integrated microRNA and mRNA sequencing data from 293 tumors from The Cancer Genome Atlas (TCGA) ovarian cancer cohort. We identified miR-509-3p as a clinically significant microRNA that is more abundant in patients with favorable survival in both the TCGA cohort (P = 2.3E–3), and, by in situ hybridization (ISH), in an independent cohort of 157 tumors (P < 1.0E–3). We found that miR-509-3p attenuated migration and disrupted multi-cellular spheroids in HEYA8, OVCAR8, SKOV3, OVCAR3, OVCAR4 and OVCAR5 cell lines. Consistent with disrupted spheroid formation, in TCGA data miR-509-3p's most strongly anti-correlated predicted targets were enriched in components of the extracellular matrix (ECM). We validated the Hippo pathway effector YAP1 as a direct miR-509-3p target. We showed that siRNA to YAP1 replicated 90% of miR-509-3p-mediated migration attenuation in OVCAR8, which contained high levels of YAP1 protein, but not in the other cell lines, in which levels of this protein were moderate to low. Our data suggest that the miR-509-3p/YAP1 axis may be a new druggable target in cancers with high YAP1, and we propose that therapeutically targeting the miR-509-3p/YAP1/ECM axis may disrupt early steps in multi-cellular spheroid formation, and so inhibit metastasis in epithelial ovarian cancer and potentially in other cancers. PMID:27036018

  7. miR-509-3p is clinically significant and strongly attenuates cellular migration and multi-cellular spheroids in ovarian cancer.

    PubMed

    Pan, Yinghong; Robertson, Gordon; Pedersen, Lykke; Lim, Emilia; Hernandez-Herrera, Anadulce; Rowat, Amy C; Patil, Sagar L; Chan, Clara K; Wen, Yunfei; Zhang, Xinna; Basu-Roy, Upal; Mansukhani, Alka; Chu, Andy; Sipahimalani, Payal; Bowlby, Reanne; Brooks, Denise; Thiessen, Nina; Coarfa, Cristian; Ma, Yussanne; Moore, Richard A; Schein, Jacquie E; Mungall, Andrew J; Liu, Jinsong; Pecot, Chad V; Sood, Anil K; Jones, Steven J M; Marra, Marco A; Gunaratne, Preethi H

    2016-05-01

    Ovarian cancer presents as an aggressive, advanced stage cancer with widespread metastases that depend primarily on multicellular spheroids in the peritoneal fluid. To identify new druggable pathways related to metastatic progression and spheroid formation, we integrated microRNA and mRNA sequencing data from 293 tumors from The Cancer Genome Atlas (TCGA) ovarian cancer cohort. We identified miR-509-3p as a clinically significant microRNA that is more abundant in patients with favorable survival in both the TCGA cohort (P = 2.3E-3), and, by in situ hybridization (ISH), in an independent cohort of 157 tumors (P < 1.0E-3). We found that miR-509-3p attenuated migration and disrupted multi-cellular spheroids in HEYA8, OVCAR8, SKOV3, OVCAR3, OVCAR4 and OVCAR5 cell lines. Consistent with disrupted spheroid formation, in TCGA data miR-509-3p's most strongly anti-correlated predicted targets were enriched in components of the extracellular matrix (ECM). We validated the Hippo pathway effector YAP1 as a direct miR-509-3p target. We showed that siRNA to YAP1 replicated 90% of miR-509-3p-mediated migration attenuation in OVCAR8, which contained high levels of YAP1 protein, but not in the other cell lines, in which levels of this protein were moderate to low. Our data suggest that the miR-509-3p/YAP1 axis may be a new druggable target in cancers with high YAP1, and we propose that therapeutically targeting the miR-509-3p/YAP1/ECM axis may disrupt early steps in multi-cellular spheroid formation, and so inhibit metastasis in epithelial ovarian cancer and potentially in other cancers. PMID:27036018

  8. Quasi-spherical microwells on superhydrophobic substrates for long term culture of multicellular spheroids and high throughput assays.

    PubMed

    Liu, Tianqing; Winter, Marnie; Thierry, Benjamin

    2014-07-01

    Multicellular tumour spheroids closely recapitulate the physiological environment of tumour tissues. However, their implementation in drug screening assays remains limited due to the technological challenges of forming large numbers of high quality spheroids in platforms compatible with high throughput screening. A simple bench-top microfabrication strategy is demonstrated here based on the principle of ice lithography carried out on superhydrophobic substrates to fabricate quasi-spherical microwells (spheriwells). The microwells shapes and dimensions are directly controlled by the hydrophobicity of the substrate and the volume of the water droplets. The prepared concave microwells enable the formation of dense and homogeneous multicellular tumour spheroids. Spheroids formed within spheriwells are trapped within the microwells, which eliminate loss during media manipulation and facilitate long-term on-chip culture. Morphological and phenotypical changes associated with the growth of MCF-7 adenocarcinoma cells in spheriwells were characterised using imaging flow cytometry and revealed the appearance of heterogeneous populations with loss of E-Cadherin expression. The compatibility of the spheriwells with an on-chip MTT assay is demonstrated. The very unusual shape of the spheriwells, prepared using materials and methods routinely used in most research laboratories, provides a straightforward and scalable platform to prepare high quality multicellular tumour spheroids compatible with high throughput biological screening assays. PMID:24797879

  9. Core-shell hydrogel beads with extracellular matrix for tumor spheroid formation

    PubMed Central

    Yu, L.; Grist, S. M.; Nasseri, S. S.; Ni, C.; Cheung, K. C.

    2015-01-01

    Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since they may provide a better model of the tumor than conventional monolayer culture. Moreover, tumor cell interaction with the extracellular matrix can determine cell organization and behavior. In this work, a microfluidic system was used to form cell-laden core-shell beads which incorporate elements of the extracellular matrix and support the formation of multicellular spheroids. The bead core (comprising a mixture of alginate, collagen, and reconstituted basement membrane, with gelation by temperature control) and shell (comprising alginate hydrogel, with gelation by ionic crosslinking) were simultaneously formed through flow focusing using a cooled flow path into the microfluidic chip. During droplet gelation, the alginate acts as a fast-gelling shell which aids in preventing droplet coalescence and in maintaining spherical droplet geometry during the slower gelation of the collagen and reconstituted basement membrane components as the beads warm up. After droplet gelation, the encapsulated MCF-7 cells proliferated to form uniform spheroids when the beads contained all three components: alginate, collagen, and reconstituted basement membrane. The dose-dependent response of the MCF-7 cell tumor spheroids to two anticancer drugs, docetaxel and tamoxifen, was compared to conventional monolayer culture. PMID:25945144

  10. Transitioning from multi-phase to single-phase microfluidics for long-term culture and treatment of multicellular spheroids.

    PubMed

    McMillan, Kay S; Boyd, Marie; Zagnoni, Michele

    2016-09-21

    When compared to methodologies based on low adhesion or hanging drop plates, droplet microfluidics offers several advantages for the formation and culture of multicellular spheroids, such as the potential for higher throughput screening and the use of reduced cell numbers, whilst providing increased stability for plate handling. However, a drawback of the technology is its characteristic compartmentalisation which limits the nutrients available to cells within an emulsion and poses challenges to the exchange of the encapsulated solution, often resulting in short-term cell culture and/or viability issues. The aim of this study was to develop a multi-purpose microfluidic platform that combines the high-throughput characteristics of multi-phase flows with that of ease of perfusion typical of single-phase microfluidics. We developed a versatile system to upscale the formation and long-term culture of multicellular spheroids for testing anticancer treatments, creating an array of fluidically addressable, compact spheroids that could be cultured in either medium or within a gel scaffold. The work provides proof-of-concept results for using this system to test both chemo- and radio-therapeutic protocols using in vitro 3D cancer models.

  11. Transitioning from multi-phase to single-phase microfluidics for long-term culture and treatment of multicellular spheroids.

    PubMed

    McMillan, Kay S; Boyd, Marie; Zagnoni, Michele

    2016-09-21

    When compared to methodologies based on low adhesion or hanging drop plates, droplet microfluidics offers several advantages for the formation and culture of multicellular spheroids, such as the potential for higher throughput screening and the use of reduced cell numbers, whilst providing increased stability for plate handling. However, a drawback of the technology is its characteristic compartmentalisation which limits the nutrients available to cells within an emulsion and poses challenges to the exchange of the encapsulated solution, often resulting in short-term cell culture and/or viability issues. The aim of this study was to develop a multi-purpose microfluidic platform that combines the high-throughput characteristics of multi-phase flows with that of ease of perfusion typical of single-phase microfluidics. We developed a versatile system to upscale the formation and long-term culture of multicellular spheroids for testing anticancer treatments, creating an array of fluidically addressable, compact spheroids that could be cultured in either medium or within a gel scaffold. The work provides proof-of-concept results for using this system to test both chemo- and radio-therapeutic protocols using in vitro 3D cancer models. PMID:27477673

  12. Superparamagnetic iron oxide nanoparticles exert different cytotoxic effects on cells grown in monolayer cell culture versus as multicellular spheroids

    NASA Astrophysics Data System (ADS)

    Theumer, Anja; Gräfe, Christine; Bähring, Franziska; Bergemann, Christian; Hochhaus, Andreas; Clement, Joachim H.

    2015-04-01

    The aim of this study was to investigate the interaction of superparamagnetic iron oxide nanoparticles (SPION) with human blood-brain barrier-forming endothelial cells (HBMEC) in two-dimensional cell monolayers as well as in three-dimensional multicellular spheroids. The precise nanoparticle localisation and the influence of the NP on the cellular viability and the intracellular Akt signalling were studied in detail. Long-term effects of different polymer-coated nanoparticles (neutral fluidMAG-D, anionic fluidMAG-CMX and cationic fluidMAG-PEI) and the corresponding free polymers on cellular viability of HBMEC were investigated by real time cell analysis studies. Nanoparticles exert distinct effects on HBMEC depending on the nanoparticles' surface charge and concentration, duration of incubation and cellular context. The most severe effects were caused by PEI-coated nanoparticles. Concentrations above 25 μg/ml led to increased amounts of dead cells in monolayer culture as well as in multicellular spheroids. On the level of intracellular signalling, context-dependent differences were observed. Monolayer cultures responded on nanoparticle incubation with an increase in Akt phosphorylation whereas spheroids on the whole show a decreased Akt activity. This might be due to the differential penetration and distribution of PEI-coated nanoparticles.

  13. Metabolic Study of Breast MCF-7 Tumor Spheroids after Gamma Irradiation by 1H NMR Spectroscopy and Microimaging

    PubMed Central

    Palma, Alessandra; Grande, Sveva; Luciani, Anna Maria; Mlynárik, Vladimír; Guidoni, Laura; Viti, Vincenza; Rosi, Antonella

    2016-01-01

    Multicellular tumor spheroids are an important model system to investigate the response of tumor cells to radio- and chemotherapy. They share more properties with the original tumor than cells cultured as 2D monolayers do, which helps distinguish the intrinsic properties of monolayer cells from those induced during cell aggregation in 3D spheroids. The paper investigates some metabolic aspects of small tumor spheroids of breast cancer and their originating MCF-7 cells, grown as monolayer, by means of high–resolution (HR) 1H NMR spectroscopy and MR microimaging before and after gamma irradiation. The spectra of spheroids were characterized by higher intensity of mobile lipids, mostly neutral lipids, and glutamine (Gln) signals with respect to their monolayer cells counterpart, mainly owing to the lower oxygen supply in spheroids. Morphological changes of small spheroids after gamma-ray irradiation, such as loss of their regular shape, were observed by MR microimaging. Lipid signal intensity increased after irradiation, as evidenced in both MR localized spectra of the single spheroid and in HR NMR spectra of spheroid suspensions. Furthermore, the intense Gln signal from spectra of irradiated spheroids remained unchanged, while the low Gln signal observed in monolayer cells increased after irradiation. Similar results were observed in cells grown in hypoxic conditions. The different behavior of Gln in 2D monolayers and in 3D spheroids supports the hypothesis that a lower oxygen supply induces both an upregulation of Gln synthetase and a downregulation of glutaminases with the consequent increase in Gln content, as already observed under hypoxic conditions. The data herein indicate that 1H NMR spectroscopy can be a useful tool for monitoring cell response to different constraints. The use of spheroid suspensions seems to be a feasible alternative to localized spectroscopy since similar effects were found after radiation treatment. PMID:27200293

  14. Secretory prostate apoptosis response (Par)-4 sensitizes multicellular spheroids (MCS) of glioblastoma multiforme cells to tamoxifen-induced cell death

    PubMed Central

    Jagtap, Jayashree C.; Parveen, D.; Shah, Reecha D.; Desai, Aarti; Bhosale, Dipali; Chugh, Ashish; Ranade, Deepak; Karnik, Swapnil; Khedkar, Bhushan; Mathur, Aaishwarya; Natesh, Kumar; Chandrika, Goparaju; Shastry, Padma

    2014-01-01

    Glioblastoma multiforme (GBM) is the most malignant form of brain tumor and is associated with resistance to conventional therapy and poor patient survival. Prostate apoptosis response (Par)-4, a tumor suppressor, is expressed as both an intracellular and secretory/extracellular protein. Though secretory Par-4 induces apoptosis in cancer cells, its potential in drug-resistant tumors remains to be fully explored. Multicellular spheroids (MCS) of cancer cells often acquire multi-drug resistance and serve as ideal experimental models. We investigated the role of Par-4 in Tamoxifen (TAM)-induced cell death in MCS of human cell lines and primary cultures of GBM tumors. TCGA and REMBRANT data analysis revealed that low levels of Par-4 correlated with low survival period (21.85 ± 19.30 days) in GBM but not in astrocytomas (59.13 ± 47.26 days) and oligodendrogliomas (58.04 ± 59.80 days) suggesting low PAWR expression as a predictive risk factor in GBM. Consistently, MCS of human cell lines and primary cultures displayed low Par-4 expression, high level of chemo-resistance genes and were resistant to TAM-induced cytotoxicity. In monolayer cells, TAM-induced cytotoxicity was associated with enhanced expression of Par-4 and was alleviated by silencing of Par-4 using specific siRNA. TAM effectively induced secretory Par-4 in conditioned medium (CM) of cells cultured as monolayer but not in MCS. Moreover, MCS were rendered sensitive to TAM-induced cell death by exposure to conditioned medium (CM)-containing Par-4 (derived from TAM-treated monolayer cells). Also TAM reduced the expression of Akt and PKCζ in GBM cells cultured as monolayer but not in MCS. Importantly, combination of TAM with inhibitors to PI3K inhibitor (LY294002) or PKCζ resulted in secretion of Par-4 and cell death in MCS. Since membrane GRP78 is overexpressed in most cancer cells but not normal cells, and secretory Par-4 induces apoptosis by binding to membrane GRP78, secretory Par-4 is an

  15. Transplantation of insulin-secreting multicellular spheroids for the treatment of type 1 diabetes in mice.

    PubMed

    Kusamori, Kosuke; Nishikawa, Makiya; Mizuno, Narumi; Nishikawa, Tomoko; Masuzawa, Akira; Shimizu, Kazunori; Konishi, Satoshi; Takahashi, Yuki; Takakura, Yoshinobu

    2014-01-10

    The efficacy of cell-based therapy depends on the function and survival of transplanted cells, which have been suggested to be enhanced by spheroid formation. However, few attempts at spheroid generation from insulin-secreting cells, which may be used to treat type 1 diabetes, have been reported. We therefore developed spheroids from the mouse insulinoma cell line NIT-1 by using polydimethylsiloxane (PDMS)-based microwells with a coating of poly(N-isopropylacrylamide) (PNIPAAm). The prepared NIT-1 spheroids or dissociated NIT-1 cells were transplanted into the subrenal capsule in streptozotocin-induced diabetic mice. NIT-1 spheroids prepared using the PNIPAAm-coated PDMS-based microwells had a uniformly sized spherical structure with a diameter of 200-300μm. The PNIPAAm coating increased cell survival in the spheroids and the recovery of the spheroids from the microwells. In diabetic mice, the transplanted NIT-1 spheroids reduced blood glucose levels to normal values faster than dissociated NIT-1 cells did. Additionally, survival was higher among NIT-1 cells in spheroids than among dissociated NIT-1 cells 24h after transplantation. These results indicate that insulin-secreting NIT-1 spheroids prepared using PNIPAAm-coated PDMS-based microwells are more effective for the treatment of type 1 diabetes than dissociated cells in suspension. PMID:24184345

  16. Influence of ionizing radiation on oxygen profiles in different types of multicellular spheroids

    SciTech Connect

    Nylen, T.; Acker, H.; Boelling, B.H.; Holterman, G.; Carlsson, J. )

    1989-11-01

    Human glioma (U-118 MG and U-138 MG), human colorectal adenocarcinoma (HT-29), human thyroid carcinoma (HTh 7), and hamster embryonic lung (V79-379A) spheroids were irradiated with either single doses of 16 or 40 Gy or fractionated doses of eight times 5 Gy. Oxygen profiles in the spheroids were measured with microelectrodes at different times following irradiation, and these profiles were then compared with the oxygen profiles measured in parallel cultured nonirradiated spheroids. No significant radiation-induced changes in the oxygen profiles were seen in any of the spheroids within the first few days after irradiation. The glioma spheroids did not show any significant increase in oxygen tension even after longer times; however, they were growth inhibited, and the number of S-phase cells was strongly suppressed. Increases in oxygen tension did occur in the HT-29 and V79-379A spheroids but only appeared more than a week after irradiation, when degeneration had started. Histological changes and decrease in diameter were seen in the spheroids that started to degenerate about 5 days after irradiation. Thus radiation doses in the therapeutic range did not, for the spheroids studied, produce rapid increases in the oxygen tension. When a change occurred, it appeared rather late and was probably a consequence of cell degeneration.

  17. Inferring Growth Control Mechanisms in Growing Multi-cellular Spheroids of NSCLC Cells from Spatial-Temporal Image Data

    PubMed Central

    Müller, Margareta; Vignon-Clementel, Irene E.; Drasdo, Dirk

    2016-01-01

    We develop a quantitative single cell-based mathematical model for multi-cellular tumor spheroids (MCTS) of SK-MES-1 cells, a non-small cell lung cancer (NSCLC) cell line, growing under various nutrient conditions: we confront the simulations performed with this model with data on the growth kinetics and spatial labeling patterns for cell proliferation, extracellular matrix (ECM), cell distribution and cell death. We start with a simple model capturing part of the experimental observations. We then show, by performing a sensitivity analysis at each development stage of the model that its complexity needs to be stepwise increased to account for further experimental growth conditions. We thus ultimately arrive at a model that mimics the MCTS growth under multiple conditions to a great extent. Interestingly, the final model, is a minimal model capable of explaining all data simultaneously in the sense, that the number of mechanisms it contains is sufficient to explain the data and missing out any of its mechanisms did not permit fit between all data and the model within physiological parameter ranges. Nevertheless, compared to earlier models it is quite complex i.e., it includes a wide range of mechanisms discussed in biological literature. In this model, the cells lacking oxygen switch from aerobe to anaerobe glycolysis and produce lactate. Too high concentrations of lactate or too low concentrations of ATP promote cell death. Only if the extracellular matrix density overcomes a certain threshold, cells are able to enter the cell cycle. Dying cells produce a diffusive growth inhibitor. Missing out the spatial information would not permit to infer the mechanisms at work. Our findings suggest that this iterative data integration together with intermediate model sensitivity analysis at each model development stage, provide a promising strategy to infer predictive yet minimal (in the above sense) quantitative models of tumor growth, as prospectively of other tissue

  18. Targeted labeling of an early-stage tumor spheroid in a chorioallantoic membrane model with upconversion nanoparticles

    NASA Astrophysics Data System (ADS)

    Liu, Kai; Holz, Jasmin A.; Ding, Yadan; Liu, Xiaomin; Zhang, Youlin; Tu, Langping; Kong, Xianggui; Priem, Bram; Nadort, Annemarie; Lambrechts, Saskia A. G.; Aalders, Maurice C. G.; Buma, Wybren Jan; Liu, Yichun; Zhang, Hong

    2015-01-01

    In vivo detection of cancer at an early-stage, i.e. smaller than 2 mm, is a challenge in biomedicine. In this work target labeling of an early-stage tumor spheroid (~500 μm) is realized for the first time in a chick embryo chorioallantoic membrane (CAM) model with monoclonal antibody functionalized upconversion nanoparticles (UCNPs-mAb).In vivo detection of cancer at an early-stage, i.e. smaller than 2 mm, is a challenge in biomedicine. In this work target labeling of an early-stage tumor spheroid (~500 μm) is realized for the first time in a chick embryo chorioallantoic membrane (CAM) model with monoclonal antibody functionalized upconversion nanoparticles (UCNPs-mAb). Electronic supplementary information (ESI) available: Details of experimental procedures for the sample preparation and characterization, Chick CAM model, 3-D multicellular tumor spheroids, UCNPs circulating in CAM. See DOI: 10.1039/c4nr05638h

  19. Development of complex-shaped liver multicellular spheroids as a human-based model for nanoparticle toxicity assessment in vitro.

    PubMed

    Dubiak-Szepietowska, Monika; Karczmarczyk, Aleksandra; Jönsson-Niedziółka, Martin; Winckler, Thomas; Feller, Karl-Heinz

    2016-03-01

    The emergence of human-based models is incontestably required for the study of complex physiological pathways and validation of reliable in vitro methods as alternative for in vivo studies in experimental animals for toxicity assessment. With this objective, we have developed and tested three dimensional environments for cells using different types of hydrogels including transglutaminase-cross-linked gelatin, collagen type I, and growth-factor depleted Matrigel. Cells grown in Matrigel exhibited the greatest cell proliferation and spheroid diameter. Moreover, analysis of urea and albumin biosynthesis revealed that the created system allowed the immortalized liver cell line HepG2 to re-establish normal hepatocyte-like properties which were not observed under the conditions of conventional cell cultures. This study presents a scalable technology for production of complex-shaped liver multicellular spheroids as a system which improves the predictive value of cell-based assays for safety and risk assessment. The time- and dose-dependent toxicity of nanoparticles demonstrates a higher cytotoxic effect when HepG2 cells grown as monolayer than embedded in hydrogels. The experimental setup provided evidence that the cell environment has significant influence on cell sensitivity and that liver spheroid is a useful and novel tool to examine nanoparticle dosing effect even at the level of in vitro studies. Therefore, this system can be applied to a wide variety of potentially hostile compounds in basic screening to provide initial warning of adverse effects and trigger subsequent analysis and remedial actions. PMID:26825373

  20. Evaluation of anti-HER2 scFv-conjugated PLGA-PEG nanoparticles on 3D tumor spheroids of BT474 and HCT116 cancer cells

    NASA Astrophysics Data System (ADS)

    Thuy Duong Le, Thi; Pham, Thu Hong; Nghia Nguyen, Trong; Giang Ngo, Thi Hong; Nhung Hoang, Thi My; Huan Le, Quang

    2016-06-01

    Three-dimensional culture cells (spheroids) are one of the multicellular culture models that can be applied to anticancer chemotherapeutic development. Multicellular spheroids more closely mimic in vivo tumor-like patterns of physiologic environment and morphology. In previous research, we designed docetaxel-loaded pegylated poly(D, L-lactide-co-glycolide) nanoparticles conjugated with anti-HER2 single chain antibodies (scFv-Doc-PLGA-PEG) and evaluated them in 2D cell culture. In this study, we continuously evaluate the cellular uptake and cytotoxic effect of scFv-Doc-PLGA-PEG on a 3D tumor spheroid model of BT474 (HER2-overexpressing) and HCT116 (HER2-underexpressing) cancer cells. The results showed that the nanoparticle formulation conjugated with scFv had a significant internalization effect on the spheroids of HER2-overexpressing cancer cells as compared to the spheroids of HER2-underexpressing cancer cells. Therefore, cytotoxic effects of targeted nanoparticles decreased the size and increased necrotic score of HER2-overexpressing tumor spheroids. Thus, these scFv-Doc-PLGA-PEG nanoparticles have potential for active targeting for HER2-overexpressing cancer therapy. In addition, BT474 and HCT116 spheroids can be used as a tumor model for evaluation of targeting therapies.

  1. Ellipsoid Segmentation Model for Analyzing Light-Attenuated 3D Confocal Image Stacks of Fluorescent Multi-Cellular Spheroids

    PubMed Central

    Barbier, Michaël; Jaensch, Steffen; Cornelissen, Frans; Vidic, Suzana; Gjerde, Kjersti; de Hoogt, Ronald; Graeser, Ralph; Gustin, Emmanuel; Chong, Yolanda T.

    2016-01-01

    In oncology, two-dimensional in-vitro culture models are the standard test beds for the discovery and development of cancer treatments, but in the last decades, evidence emerged that such models have low predictive value for clinical efficacy. Therefore they are increasingly complemented by more physiologically relevant 3D models, such as spheroid micro-tumor cultures. If suitable fluorescent labels are applied, confocal 3D image stacks can characterize the structure of such volumetric cultures and, for example, cell proliferation. However, several issues hamper accurate analysis. In particular, signal attenuation within the tissue of the spheroids prevents the acquisition of a complete image for spheroids over 100 micrometers in diameter. And quantitative analysis of large 3D image data sets is challenging, creating a need for methods which can be applied to large-scale experiments and account for impeding factors. We present a robust, computationally inexpensive 2.5D method for the segmentation of spheroid cultures and for counting proliferating cells within them. The spheroids are assumed to be approximately ellipsoid in shape. They are identified from information present in the Maximum Intensity Projection (MIP) and the corresponding height view, also known as Z-buffer. It alerts the user when potential bias-introducing factors cannot be compensated for and includes a compensation for signal attenuation. PMID:27303813

  2. Ellipsoid Segmentation Model for Analyzing Light-Attenuated 3D Confocal Image Stacks of Fluorescent Multi-Cellular Spheroids.

    PubMed

    Barbier, Michaël; Jaensch, Steffen; Cornelissen, Frans; Vidic, Suzana; Gjerde, Kjersti; de Hoogt, Ronald; Graeser, Ralph; Gustin, Emmanuel; Chong, Yolanda T

    2016-01-01

    In oncology, two-dimensional in-vitro culture models are the standard test beds for the discovery and development of cancer treatments, but in the last decades, evidence emerged that such models have low predictive value for clinical efficacy. Therefore they are increasingly complemented by more physiologically relevant 3D models, such as spheroid micro-tumor cultures. If suitable fluorescent labels are applied, confocal 3D image stacks can characterize the structure of such volumetric cultures and, for example, cell proliferation. However, several issues hamper accurate analysis. In particular, signal attenuation within the tissue of the spheroids prevents the acquisition of a complete image for spheroids over 100 micrometers in diameter. And quantitative analysis of large 3D image data sets is challenging, creating a need for methods which can be applied to large-scale experiments and account for impeding factors. We present a robust, computationally inexpensive 2.5D method for the segmentation of spheroid cultures and for counting proliferating cells within them. The spheroids are assumed to be approximately ellipsoid in shape. They are identified from information present in the Maximum Intensity Projection (MIP) and the corresponding height view, also known as Z-buffer. It alerts the user when potential bias-introducing factors cannot be compensated for and includes a compensation for signal attenuation. PMID:27303813

  3. Estimating the cell density and invasive radius of three-dimensional glioblastoma tumor spheroids grown in vitro

    NASA Astrophysics Data System (ADS)

    Stein, Andrew M.; Nowicki, Michal O.; Demuth, Tim; Berens, Michael E.; Lawler, Sean E.; Chiocca, E. Antonio; Sander, Leonard M.

    2007-08-01

    To gain insight into brain tumor invasion, experiments are conducted on multicellular tumor spheroids grown in collagen gel. Typically, a radius of invasion is reported, which is obtained by human measurement. We present a simple, heuristic algorithm for automated invasive radii estimation (AIRE) that uses local fluctuations of the image intensity. We then derive an analytical expression relating the image graininess to the cell density for a model imaging system. The result agrees with the experiment up to a multiplicative constant and thus describes a novel method for estimating the cell density from bright-field images.

  4. In Vitro, Matrix-Free Formation Of Solid Tumor Spheroids

    NASA Technical Reports Server (NTRS)

    Gonda, Steve R.; Marley, Garry M.

    1993-01-01

    Cinostatic bioreactor promotes formation of relatively large solid tumor spheroids exhibiting diameters from 750 to 2,100 micrometers. Process useful in studying efficacy of chemotherapeutic agents and of interactions between cells not constrained by solid matrices. Two versions have been demonstrated; one for anchorage-independent cells and one for anchorage-dependent cells.

  5. Transcriptome profile of the early stages of breast cancer tumoral spheroids

    PubMed Central

    Pacheco-Marín, Rosario; Melendez-Zajgla, Jorge; Castillo-Rojas, Gonzalo; Mandujano-Tinoco, Edna; Garcia-Venzor, Alfredo; Uribe-Carvajal, Salvador; Cabrera-Orefice, Alfredo; Gonzalez-Torres, Carolina; Gaytan-Cervantes, Javier; Mitre-Aguilar, Irma B.; Maldonado, Vilma

    2016-01-01

    Oxygen or nutrient deprivation of early stage tumoral spheroids can be used to reliably mimic the initial growth of primary and metastatic cancer cells. However, cancer cell growth during the initial stages has not been fully explored using a genome-wide approach. Thus, in the present study, we investigated the transcriptome of breast cancer cells during the initial stages of tumoral growth using RNAseq in a model of Multicellular Tumor Spheroids (MTS). Network analyses showed that a metastatic signature was enriched as several adhesion molecules were deregulated, including EPCAM, E-cadherin, integrins and syndecans, which were further supported by an increase in cell migration. Interestingly, we also found that the cancer cells at this stage of growth exhibited a paradoxical hyperactivation of oxidative mitochondrial metabolism. In addition, we found a large number of regulated (long non coding RNA) lncRNAs, several of which were co-regulated with neighboring genes. The regulatory role of some of these lncRNAs on mRNA expression was demonstrated with gain of function assays. This is the first report of an early-stage MTS transcriptome, which not only reveals a complex expression landscape, but points toward an important contribution of long non-coding RNAs in the final phenotype of three-dimensional cellular models. PMID:27021602

  6. Tumor spheroid model for the biologically targeted radiotherapy of neuroblastoma micrometastases

    SciTech Connect

    Walker, K.A.; Mairs, R.; Murray, T.; Hilditch, T.E.; Wheldon, T.E.; Gregor, A.; Hann, I.M. )

    1990-02-01

    Neuroblastoma is a pediatric malignancy with a poor prognosis at least partly attributable to an early pattern of dissemination. New approaches to treatment of micrometastases include targeted radiotherapy using radiolabeled antibodies or molecules which are taken up preferentially by tumor cells. Multicellular tumor spheroids (MTS) resemble micrometastases during the avascular phase of their development. A human neuroblastoma cell line (NBl-G) was grown as MTS and incubated briefly with a radiolabeled monoclonal antibody ({sup 131}I-UJ13A) directed against neuroectodermal antigens. Spheroid response was evaluated in terms of regrowth delay or proportion sterilized. A dose-response relationship was demonstrated in terms of {sup 131}I activity or duration of incubation. Control experiments using unlabeled UJ13A, radiolabeled nonspecific antibody (T2.10), radiolabeled human serum albumin, and radiolabeled sodium iodide showed these to be relatively ineffective compared to {sup 131}I-UJ13A. The cell line NBl-G grown as MTS has also been found to preferentially accumulate the radiolabeled catecholamine precursor molecule m-({sup 131}I)iodobenzylguanidine compared to cell lines derived from other tumor types. NBl-G cells grown as MTS provide a promising laboratory model for targeted radiotherapy of neuroblastoma micrometastases using radiolabeled antibodies or m-iodobenzylguanidine.

  7. Quantitative bioimaging of platinum group elements in tumor spheroids.

    PubMed

    Niehoff, Ann-Christin; Grünebaum, Jonas; Moosmann, Aline; Mulac, Dennis; Söbbing, Judith; Niehaus, Rebecca; Buchholz, Rebecca; Kröger, Sabrina; Wiehe, Arno; Wagner, Sylvia; Sperling, Michael; von Briesen, Hagen; Langer, Klaus; Karst, Uwe

    2016-09-28

    Limited drug penetration into tumor tissue is a significant factor to the effectiveness of cancer therapy. Tumor spheroids, a 3D cell culture model system, can be used to study drug penetration for pharmaceutical development. In this study, a method for quantitative bioimaging of platinum group elements by laser ablation (LA) coupled to inductively coupled plasma mass spectrometry (ICP-MS) is presented. Different matrix-matched standards were used to develop a quantitative LA-ICP-MS method with high spatial resolution. To investigate drug penetration, tumor spheroids were incubated with platinum complexes (Pt(II)acetylacetonate, cisplatin) and the palladium tagged photosensitizer 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (mTHPP). Distribution and accumulation of the pharmaceuticals were determined with the developed method. PMID:27619092

  8. Quantitative bioimaging of platinum group elements in tumor spheroids.

    PubMed

    Niehoff, Ann-Christin; Grünebaum, Jonas; Moosmann, Aline; Mulac, Dennis; Söbbing, Judith; Niehaus, Rebecca; Buchholz, Rebecca; Kröger, Sabrina; Wiehe, Arno; Wagner, Sylvia; Sperling, Michael; von Briesen, Hagen; Langer, Klaus; Karst, Uwe

    2016-09-28

    Limited drug penetration into tumor tissue is a significant factor to the effectiveness of cancer therapy. Tumor spheroids, a 3D cell culture model system, can be used to study drug penetration for pharmaceutical development. In this study, a method for quantitative bioimaging of platinum group elements by laser ablation (LA) coupled to inductively coupled plasma mass spectrometry (ICP-MS) is presented. Different matrix-matched standards were used to develop a quantitative LA-ICP-MS method with high spatial resolution. To investigate drug penetration, tumor spheroids were incubated with platinum complexes (Pt(II)acetylacetonate, cisplatin) and the palladium tagged photosensitizer 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (mTHPP). Distribution and accumulation of the pharmaceuticals were determined with the developed method.

  9. In silico estimates of the free energy rates in growing tumor spheroids

    NASA Astrophysics Data System (ADS)

    Narayanan, H.; Verner, S. N.; Mills, K. L.; Kemkemer, R.; Garikipati, K.

    2010-05-01

    The physics of solid tumor growth can be considered at three distinct size scales: the tumor scale, the cell-extracellular matrix (ECM) scale and the sub-cellular scale. In this paper we consider the tumor scale in the interest of eventually developing a system-level understanding of the progression of cancer. At this scale, cell populations and chemical species are best treated as concentration fields that vary with time and space. The cells have chemo-mechanical interactions with each other and with the ECM, consume glucose and oxygen that are transported through the tumor, and create chemical by-products. We present a continuum mathematical model for the biochemical dynamics and mechanics that govern tumor growth. The biochemical dynamics and mechanics also engender free energy changes that serve as universal measures for comparison of these processes. Within our mathematical framework we therefore consider the free energy inequality, which arises from the first and second laws of thermodynamics. With the model we compute preliminary estimates of the free energy rates of a growing tumor in its pre-vascular stage by using currently available data from single cells and multicellular tumor spheroids.

  10. Survivin isoform Delta Ex3 regulates tumor spheroid formation.

    PubMed

    Espinosa, Magali; Ceballos-Cancino, Gisela; Callaghan, Richard; Maldonado, Vilma; Patiño, Nelly; Ruíz, Víctor; Meléndez-Zajgla, Jorge

    2012-05-01

    Survivin is an important member of the Inhibitor of Apoptosis Proteins (IAPs) family and has essential roles in apoptosis and cell cycle progression. This gene is commonly upregulated in human cancer and provides an exciting diagnostic and therapeutic target. Survivin is expressed as several isoforms that are generated by alternative splicing, and some of these present antagonistic activities. Currently, information regarding the regulation of these isoforms is lacking. In this study, we sought to analyze survivin Delta Ex3 expression in a three-dimensional model of avascular tumors and its overexpression effects in processes such as proliferation, clonogenicity and apoptosis. We found a positive correlation between spheroid growth and survivin Delta Ex3 expression during the exponential phase. We demonstrated that this isoform not only decreased apoptosis but also inhibited tumor spheroid formation by decreasing proliferation and clonogenic survival. These results point toward a dual and antagonistic effect of this spliced survivin isoform in cancer development.

  11. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions

    SciTech Connect

    Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan; Krebs, Alice; Christian, Sven; Prinz, Florian; Osterland, Marc; Golfier, Sven; Räse, Sebastian; Ansari, Nariman; Esner, Milan; Bickle, Marc; Pampaloni, Francesco; Mattheyer, Christian; Stelzer, Ernst H.; Parczyk, Karsten; Prechtl, Stefan; Steigemann, Patrick

    2014-04-15

    Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. - Highlights: • Establishment of a novel method for 3D cell culture based high-content screening. • First reported high

  12. 96-well format-based microfluidic platform for parallel interconnection of multiple multicellular spheroids.

    PubMed

    Kim, Jin-Young; Fluri, David A; Kelm, Jens M; Hierlemann, Andreas; Frey, Olivier

    2015-06-01

    In this article, we present a microfluidic platform, compatible with conventional 96-well formats, that enables facile and parallelized culturing and testing of spherical microtissues in a standard incubator. The platform can accommodate multiple microtissues (up to 66) of different cell types, formed externally by using the hanging-drop method, and enables microtissue interconnection through microfluidic channels for continuous media perfusion or dosage of substances. The platform contains 11 separate channels, and each channel has six tissue compartments. Primary rat liver tissues were cultured over 8 days, and multiple tumor tissues (HCT116) were exposed to various concentrations of 5-fluorouracil for platform characterization.

  13. Tensile Forces Originating from Cancer Spheroids Facilitate Tumor Invasion.

    PubMed

    Kopanska, Katarzyna S; Alcheikh, Yara; Staneva, Ralitza; Vignjevic, Danijela; Betz, Timo

    2016-01-01

    The mechanical properties of tumors and the tumor environment provide important information for the progression and characterization of cancer. Tumors are surrounded by an extracellular matrix (ECM) dominated by collagen I. The geometrical and mechanical properties of the ECM play an important role for the initial step in the formation of metastasis, presented by the migration of malignant cells towards new settlements as well as the vascular and lymphatic system. The extent of this cell invasion into the ECM is a key medical marker for cancer prognosis. In vivo studies reveal an increased stiffness and different architecture of tumor tissue when compared to its healthy counterparts. The observed parallel collagen organization on the tumor border and radial arrangement at the invasion zone has raised the question about the mechanisms organizing these structures. Here we study the effect of contractile forces originated from model tumor spheroids embedded in a biomimetic collagen I matrix. We show that contractile forces act immediately after seeding and deform the ECM, thus leading to tensile radial forces within the matrix. Relaxation of this tension via cutting the collagen does reduce invasion, showing a mechanical relation between the tensile state of the ECM and invasion. In turn, these results suggest that tensile forces in the ECM facilitate invasion. Furthermore, simultaneous contraction of the ECM and tumor growth leads to the condensation and reorientation of the collagen at the spheroid's surface. We propose a tension-based model to explain the collagen organization and the onset of invasion by forces originating from the tumor. PMID:27271249

  14. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions.

    PubMed

    Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan; Krebs, Alice; Christian, Sven; Prinz, Florian; Osterland, Marc; Golfier, Sven; Räse, Sebastian; Ansari, Nariman; Esner, Milan; Bickle, Marc; Pampaloni, Francesco; Mattheyer, Christian; Stelzer, Ernst H; Parczyk, Karsten; Prechtl, Stefan; Steigemann, Patrick

    2014-04-15

    Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions.

  15. Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment.

    PubMed

    Harney, Allison S; Wang, Yarong; Condeelis, John S; Entenberg, David

    2016-01-01

    In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment. PMID:27341448

  16. Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

    PubMed Central

    Harney, Allison S.; Wang, Yarong; Condeelis, John S.; Entenberg, David

    2016-01-01

    In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment. PMID:27341448

  17. Colorectal cancer-derived tumor spheroids retain the characteristics of original tumors.

    PubMed

    Lee, Sun-Hwa; Hong, Jun Hwa; Park, Hwan Ki; Park, Jun Seok; Kim, Bo-Kyung; Lee, Jung-Yi; Jeong, Ji Yun; Yoon, Ghil Suk; Inoue, Masahiro; Choi, Gyu-Seog; Lee, In-Kyu

    2015-10-10

    Primary cultures of cancer cells are useful for developing personalized medicine. In this study, we characterized three lines of three-dimensional (3D) tumor spheroids established directly from tumor tissues of patients with colorectal cancers (CRCs). Each line mainly included EpCAM-positive cells and cells expressing putative cancer stem cell markers such as CD133, CD44, CD24, ALDH1, and LGR5. These characteristic stem cell markers remained identically for months in vitro. Short tandem repeat genotyping suggested that genetic fingerprints of these tumor spheroids were similar to those of the original tumor tissues from which they were derived. Mutational analysis showed that each line had the same mutation profile for APC, KRAS, MLH1, serine-threonine kinase 11, and TP53 as its parental tumor tissue. One line harboring an activating KRAS mutation was resistant to cetuximab while the remaining two lines harboring wild-type KRAS showed different responses to cetuximab. Immunohistochemical analysis showed that xenograft tumors derived from these lines retained the histopathological and mutational patterns of their parental tumors. Collectively, these results clearly showed that 3D tumor spheroids directly generated from tumor tissues of patients with CRCs preserved the characteristics of their parental tumor tissues and could be used for developing personalized medicines for CRCs.

  18. Label-free mitosis detection in tumor spheroids using tissue dynamics imaging

    NASA Astrophysics Data System (ADS)

    An, Ran; Jeong, Kwan; Turek, John; Nolte, David

    2012-03-01

    The detection of cellular mitosis inside three-dimensional living tissue at depths up to 1 mm has been beyond the detection limits of conventional microscopies. In this paper, we demonstrate the use of motility contrast imaging and fluctuation spectroscopy to detect motional signatures that we attribute to mitotic events within groups of 100 cells in multicellular tumor spheroids. Motility contrast imaging is a coherence-domain speckle-imaging technique that uses low-coherence off-axis holography as a coherence gate to localize dynamic light scattering from selected depths inside tissue. Fluctuation spectroscopy is performed on a pervoxel basis to generate micro-spectrograms that display frequency content vs. time. Mitosis, especially in Telophase and Cytokinesis, is a relatively fast and high-amplitude phenomenon that should display energetic features within the micro-spectrograms. By choosing an appropriate frequency range and threshold, we detect energetic events with a density and rate that are comparable to the expected mitotic fraction in the UMR cell line. By studying these mitotic events in tumors of two different sizes, we show that micro-spectrograms contain characteristically different information content than macro-spectrograms (averaged over many voxels) in which the mitotic signatures (which are overall a low-probability event) are averaged out. The detection of mitotic fraction in thick living tissue has important consequences for the use of tissue-based assays for drug discovery.

  19. Development of three-dimensional lung multicellular spheroids in air- and liquid-interface culture for the evaluation of anticancer therapeutics.

    PubMed

    Meenach, Samantha A; Tsoras, Alexandra N; McGarry, Ronald C; Mansour, Heidi M; Hilt, J Zach; Anderson, Kimberly W

    2016-04-01

    Three-dimensional (3D) lung multicellular spheroids (MCS) in liquid-covered culture (LCC) and air-interface culture (AIC) conditions have both been developed for the evaluation of aerosol anticancer therapeutics in solution and aerosols, respectively. The MCS were formed by seeding lung cancer cells on top of collagen where they formed spheroids due to the prevalence of cell-to-cell interactions. LCC MCS were exposed to paclitaxel (PTX) in media whereas AIC MCS were exposed to dry powder PEGylated phospholipid aerosol microparticles containing paclitaxel. The difference in viability for 2D versus 3D culture for both LCC and AIC was evaluated along with the effects of the particles on lung epithelium via transepithelial electrical resistance (TEER) measurements. For LCC and AIC conditions, the 3D spheroids were more resistant to treatment with higher IC50 values for A549 and H358 cell lines. TEER results initially indicated a decrease in resistance upon drug or particle exposure, however, these values increased over the course of several days indicating the ability of the cells to recover. Overall, these studies offer a comprehensive in vitro evaluation of aerosol particles used in the treatment of lung cancer while introducing a new method for culturing lung cancer MCS in both LCC and AIC conditions.

  20. Development of three-dimensional lung multicellular spheroids in air- and liquid-interface culture for the evaluation of anticancer therapeutics

    PubMed Central

    MEENACH, SAMANTHA A.; TSORAS, ALEXANDRA N.; McGARRY, RONALD C.; MANSOUR, HEIDI M.; HILT, J. ZACH; ANDERSON, KIMBERLY W.

    2016-01-01

    Three-dimensional (3D) lung multicellular spheroids (MCS) in liquid-covered culture (LCC) and air-interface culture (AIC) conditions have both been developed for the evaluation of aerosol anticancer therapeutics in solution and aerosols, respectively. The MCS were formed by seeding lung cancer cells on top of collagen where they formed spheroids due to the prevalence of cell-to-cell interactions. LCC MCS were exposed to paclitaxel (PTX) in media whereas AIC MCS were exposed to dry powder PEGylated phospholipid aerosol microparticles containing paclitaxel. The difference in viability for 2D versus 3D culture for both LCC and AIC was evaluated along with the effects of the particles on lung epithelium via transepithelial electrical resistance (TEER) measurements. For LCC and AIC conditions, the 3D spheroids were more resistant to treatment with higher IC50 values for A549 and H358 cell lines. TEER results initially indicated a decrease in resistance upon drug or particle exposure, however, these values increased over the course of several days indicating the ability of the cells to recover. Overall, these studies offer a comprehensive in vitro evaluation of aerosol particles used in the treatment of lung cancer while introducing a new method for culturing lung cancer MCS in both LCC and AIC conditions. PMID:26846376

  1. Tensile Forces Originating from Cancer Spheroids Facilitate Tumor Invasion

    PubMed Central

    Kopanska, Katarzyna S.; Alcheikh, Yara; Staneva, Ralitza; Vignjevic, Danijela; Betz, Timo

    2016-01-01

    The mechanical properties of tumors and the tumor environment provide important information for the progression and characterization of cancer. Tumors are surrounded by an extracellular matrix (ECM) dominated by collagen I. The geometrical and mechanical properties of the ECM play an important role for the initial step in the formation of metastasis, presented by the migration of malignant cells towards new settlements as well as the vascular and lymphatic system. The extent of this cell invasion into the ECM is a key medical marker for cancer prognosis. In vivo studies reveal an increased stiffness and different architecture of tumor tissue when compared to its healthy counterparts. The observed parallel collagen organization on the tumor border and radial arrangement at the invasion zone has raised the question about the mechanisms organizing these structures. Here we study the effect of contractile forces originated from model tumor spheroids embedded in a biomimetic collagen I matrix. We show that contractile forces act immediately after seeding and deform the ECM, thus leading to tensile radial forces within the matrix. Relaxation of this tension via cutting the collagen does reduce invasion, showing a mechanical relation between the tensile state of the ECM and invasion. In turn, these results suggest that tensile forces in the ECM facilitate invasion. Furthermore, simultaneous contraction of the ECM and tumor growth leads to the condensation and reorientation of the collagen at the spheroid’s surface. We propose a tension-based model to explain the collagen organization and the onset of invasion by forces originating from the tumor. PMID:27271249

  2. The RBE-LET relationship for rodent intestinal crypt cell survival, testes weight loss, and multicellular spheroid cell survival after heavy-ion irradiation

    NASA Technical Reports Server (NTRS)

    Rodriguez, A.; Alpen, E. L.; Powers-Risius, P.

    1992-01-01

    This report presents data for survival of mouse intestinal crypt cells, mouse testes weight loss as an indicator of survival of spermatogonial stem cells, and survival of rat 9L spheroid cells after irradiation in the plateau region of unmodified particle beams ranging in mass from 4He to 139La. The LET values range from 1.6 to 953 keV/microns. These studies examine the RBE-LET relationship for two normal tissues and for an in vitro tissue model, multicellular spheroids. When the RBE values are plotted as a function of LET, the resulting curve is characterized by a region in which RBE increases with LET, a peak RBE at an LET value of 100 keV/microns, and a region of decreasing RBE at LETs greater than 100 keV/microns. Inactivation cross sections (sigma) for these three biological systems have been calculated from the exponential terminal slope of the dose-response relationship for each ion. For this determination the dose is expressed as particle fluence and the parameter sigma indicates effect per particle. A plot of sigma versus LET shows that the curve for testes weight loss is shifted to the left, indicating greater radiosensitivity at lower LETs than for crypt cell and spheroid cell survival. The curves for cross section versus LET for all three model systems show similar characteristics with a relatively linear portion below 100 keV/microns and a region of lessened slope in the LET range above 100 keV/microns for testes and spheroids. The data indicate that the effectiveness per particle increases as a function of LET and, to a limited extent, Z, at LET values greater than 100 keV/microns. Previously published results for spread Bragg peaks are also summarized, and they suggest that RBE is dependent on both the LET and the Z of the particle.

  3. Human ovarian cancer multicellular spheroids: a model for testing antiproliferation activity of Devil's club (Oplopanax horridus) and anticancer agents.

    PubMed

    Tai, Joseph; Cheung, Susan S C; Hasman, David

    2014-06-01

    This study was conducted to employ an ovarian cancer Ovcar 10 three-dimensional model to assess the antiproliferation activity of the medicinal plant Devil's club, Oplopanax horridus, and its active compound, alone and in combination, with chemotherapeutic agents compared to Ovcar 10 two-dimensional cells grown as monolayer cells. Ovcar 10 three-dimensional spheroids were prepared with a rotary cell culture system. Cell counting kit-8 assessed the antiproliferation activity. Apoptosis-related gene expression in three-dimensional spheroids and two- dimensional cells was analyzed with an apoptosis antibody array. Flow cytometry was used to analyze the cell cycle. Ovcar 10 cells formed compact three-dimensional spheroids after 5 days of culture in a rotary culture system. Ovcar 10 three-dimensional spheroids were significantly more resistant to killing by Devil's club extract, its active compound alone, gemcitabine, and paclitaxel, but not cisplatin compared to two-dimensional cells, with IC50 levels closer to that observed in vivo. Devil's club extract and its active compound alone significantly enhanced the antiproliferation activity of cisplatin and gemcitabine at some concentrations, but did not affect the activity of paclitaxel. A number of apoptosis-related genes were differentially expressed in three-dimensional spheroids, two-dimensional cells, and cells treated with Devil's club extract compared to untreated controls. In three-dimensional spheroids, the proportion of cells in the G2/M phase was slightly increased and the S phase was slightly decreased compared to two-dimensional cells. Ovcar 10 cells in three-dimensional spheroids altered the expression of multiple apoptosis-related genes, which may have contributed to the increased resistance of the cells to some drugs. PMID:24922275

  4. Efficacy of camptothecin and polymer-conjugated camptothecin in tumor spheroids and solid tumors.

    PubMed

    Ying, Victoria; Haverstick, Kraig; Page, Rodney L; Saltzman, W Mark

    2007-01-01

    Camptothecin (CPT) is an anti-cancer drug with low solubility in aqueous solutions, which limits its efficacy during chemotherapy. To bypass this problem, CPT was conjugated to poly(ethylene glycol) (PEG) to make CPT more hydrophilic: CM-PEG-CPT (carboxylmethylpoly(ethlyene glycol)-camptothecin), CM-PEG-GLY-CPT (carboxylmethyl-poly(ethlyene glycol)-glycine-camptothecin) and CM-PEG-SAR-CPT (carboxylmethyl-poly(ethlyene glycol)-sarcosine camptothecin) were synthesized. These conjugates differed in the amino-acid linker, which altered the hydrolysis rate of CPT from CPT-PEG. We tested the hypothesis that CPT conjugates were more effective than unconjugated CPT in effectiveness upon direct delivery to solid tumors using two systems: in vitro tumor spheroids suspended in collagen gels and in vivo solid tumors in rats. CPT was effective in spheroids, but not in flank tumors. However, when CPT was conjugated, there was improvement in the treatment of spheroids and, to a lesser extent, tumors in rats. There was no difference in therapeutic effects among the various conjugates. We conclude that conjugation of CPT to PEG enhances CPT solubility and improves effectiveness of delivery to tumors.

  5. Three-dimensional (3D) tumor spheroid invasion assay.

    PubMed

    Vinci, Maria; Box, Carol; Eccles, Suzanne A

    2015-01-01

    Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck - SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis. PMID:25993495

  6. Drug cytotoxicity and signaling pathway analysis with three-dimensional tumor spheroids in a microwell-based microfluidic chip for drug screening.

    PubMed

    Chen, Yongli; Gao, Dan; Liu, Hongxia; Lin, Shuo; Jiang, Yuyang

    2015-10-22

    Currently, there has been a growing need for developing in vitro models to better reflect organism response to chemotherapy at tissue level. For this reason, a microfluidic platform was developed for mimicking physiological microenvironment of solid tumor with multicellular tumor spheroids (MTS) for anticancer drug screening. Importantly, the power of this system over traditional systems is that it is simple to operate and high integration in a more physiologically relevant context. As a proof of concept, long-term MTS cultures with uniform structure were realized on the microfluidic based platform. The response of doxorubicin and paclitaxel on different types of spheroids were simultaneously performed by in situ Live/Dead fluorescence stain to provide spatial distribution of dead cells as well as cytotoxicity information. In addition, the established platform combined with microplate reader was capable to determine the cytotoxicity of different sized MTS, showing a more powerful tool than cell staining examination at the end-point of assay. The HCT116 spheroids were then lysed on chip followed by signaling transduction pathway analysis. To our knowledge, the on chip drug screening study is the first to address the drug susceptibility testing and the offline detailed drug signaling pathway analysis combination on one system. Thus, this novel microfluidic platform provides a useful tool for drug screening with tumor spheroids, which is crucial for drug discovery and development. PMID:26526913

  7. The effect of co-delivery of paclitaxel and curcumin by transferrin-targeted PEG-PE-based mixed micelles on resistant ovarian cancer in 3-D spheroids and in vivo tumors.

    PubMed

    Sarisozen, Can; Abouzeid, Abraham H; Torchilin, Vladimir P

    2014-10-01

    Multicellular 3D cancer cell culture (spheroids) resemble to in vivo tumors in terms of shape, cell morphology, growth kinetics, gene expression and drug response. However, these characteristics cause very limited drug penetration into deeper parts of the spheroids. In this study, we used multi drug resistant (MDR) ovarian cancer cell spheroid and in vivo tumor models to evaluate the co-delivery of paclitaxel (PCL) and a potent NF-κB inhibitor curcumin (CUR). PCL and CUR were co-loaded into the polyethylene glycol-phosphatidyl ethanolamine (PEG-PE) based polymeric micelles modified with transferrin (TF) as the targeting ligand. Cytotoxicity, cellular association and accumulation into the deeper layers were investigated in the spheroids and compared with the monolayer cell culture. Comparing to non-targeted micelles, flow cytometry and confocal imaging proved significantly deeper and higher micelle penetration into the spheroids with TF-targeting. Both in monolayers and in spheroids, PCL cytotoxicity was significantly increased when co-delivered with CUR in non-targeted micelles or as single agent in TF-targeted micelles, whereas TF-modification of co-loaded micelles did not further enhance the cytotoxicity. In vivo tumor inhibition studies showed good correlation with the 3D cell culture experiments, which suggests the current spheroid model can be used as an intermediate model for the evaluation of co-delivery of anticancer compounds in targeted micelles. PMID:25016976

  8. The effect of co-delivery of paclitaxel and curcumin by transferrin-targeted PEG-PE-based mixed micelles on resistant ovarian cancer in 3-D spheroids and in vivo tumors

    PubMed Central

    Sarisozen, Can; Abouzeid, Abraham H.; Torchilin, Vladimir P.

    2014-01-01

    Multicellular 3D cancer cell culture (spheroids) resemble to in vivo tumors in terms of shape, cell morphology, growth kinetics, gene expression and drug response. However, these characteristics cause very limited drug penetration into deeper parts of the spheroids. In this study, we used multi drug resistant (MDR) ovarian cancer cell spheroid and in vivo tumor models to evaluate the co-delivery of paclitaxel (PCL) and a potent NF-κB inhibitor curcumin (CUR). PCL and CUR were co-loaded into the polyethylene glycol-phosphatidyl ethanolamine (PEG-PE) based polymeric micelles modified with Transferrin (TF) as the targeting ligand. Cytotoxicity, cellular association and accumulation into the deeper layers were investigated in the spheroids and compared with the monolayer cell culture. Comparing to non-targeted micelles, flow cytometry and confocal imaging proved significantly deeper and higher micelle penetration into the spheroids with TF-targeting. Both in monolayers and spheroids, PCL cytotoxicity was significantly increased when co-delivered with CUR in non-targeted micelles or as single agent in TF-targeted micelles, whereas TF-modification of co-loaded micelles did not further enhance the cytotoxicity. In vivo tumor inhibition studies showed good correlation with the 3D cell culture experiments, which suggests the current spheroid model can be used as an intermediate model for evaluation of co-delivery of anticancer compounds in targeted micelles. PMID:25016976

  9. Co-Culture of Tumor Spheroids and Fibroblasts in a Collagen Matrix-Incorporated Microfluidic Chip Mimics Reciprocal Activation in Solid Tumor Microenvironment

    PubMed Central

    Jeong, Su-Yeong; Lee, Ji-Hyun; Shin, Yoojin; Chung, Seok; Kuh, Hyo-Jeong

    2016-01-01

    Multicellular 3D culture and interaction with stromal components are considered essential elements in establishing a ‘more clinically relevant’ tumor model. Matrix-embedded 3D cultures using a microfluidic chip platform can recapitulate the microscale interaction within tumor microenvironments. As a major component of tumor microenvironment, cancer-associated fibroblasts (CAFs) play a role in cancer progression and drug resistance. Here, we present a microfluidic chip-based tumor tissue culture model that integrates 3D tumor spheroids (TSs) with CAF in proximity within a hydrogel scaffold. HT-29 human colorectal carcinoma cells grew into 3D TSs and the growth was stimulated when co-cultured with fibroblasts as shown by 1.5-folds increase of % changes in diameter over 5 days. TS cultured for 6 days showed a reduced expression of Ki-67 along with increased expression of fibronectin when co-cultured with fibroblasts compared to mono-cultured TSs. Fibroblasts were activated under co-culture conditions, as demonstrated by increases in α-SMA expression and migratory activity. When exposed to paclitaxel, a survival advantage was observed in TSs co-cultured with activated fibroblasts. Overall, we demonstrated the reciprocal interaction between TSs and fibroblasts in our 7-channel microfluidic chip. The co-culture of 3D TS-CAF in a collagen matrix-incorporated microfluidic chip may be useful to study the tumor microenvironment and for evaluation of drug screening and evaluation. PMID:27391808

  10. Identifications of novel mechanisms in breast cancer cells involving duct-like multicellular spheroid formation after exposure to the Random Positioning Machine.

    PubMed

    Kopp, Sascha; Slumstrup, Lasse; Corydon, Thomas J; Sahana, Jayashree; Aleshcheva, Ganna; Islam, Tawhidul; Magnusson, Nils E; Wehland, Markus; Bauer, Johann; Infanger, Manfred; Grimm, Daniela

    2016-01-01

    Many cell types form three-dimensional aggregates (MCS; multicellular spheroids), when they are cultured under microgravity. MCS often resemble the organ, from which the cells have been derived. In this study we investigated human MCF-7 breast cancer cells after a 2 h-, 4 h-, 16 h-, 24 h- and 5d-exposure to a Random Positioning Machine (RPM) simulating microgravity. At 24 h few small compact MCS were detectable, whereas after 5d many MCS were floating in the supernatant above the cells, remaining adherently (AD). The MCS resembled the ducts formed in vivo by human epithelial breast cells. In order to clarify the underlying mechanisms, we harvested MCS and AD cells separately from each RPM-culture and measured the expression of 29 selected genes with a known involvement in MCS formation. qPCR analyses indicated that cytoskeletal genes were unaltered in short-term samples. IL8, VEGFA, and FLT1 were upregulated in 2 h/4 h AD-cultures. The ACTB, TUBB, EZR, RDX, FN1, VEGFA, FLK1 Casp9, Casp3, PRKCA mRNAs were downregulated in 5d-MCS-samples. ESR1 was upregulated in AD, and PGR1 in both phenotypes after 5d. A pathway analysis revealed that the corresponding gene products are involved in organization and regulation of the cell shape, in cell tip formation and membrane to membrane docking.

  11. Identifications of novel mechanisms in breast cancer cells involving duct-like multicellular spheroid formation after exposure to the Random Positioning Machine.

    PubMed

    Kopp, Sascha; Slumstrup, Lasse; Corydon, Thomas J; Sahana, Jayashree; Aleshcheva, Ganna; Islam, Tawhidul; Magnusson, Nils E; Wehland, Markus; Bauer, Johann; Infanger, Manfred; Grimm, Daniela

    2016-01-01

    Many cell types form three-dimensional aggregates (MCS; multicellular spheroids), when they are cultured under microgravity. MCS often resemble the organ, from which the cells have been derived. In this study we investigated human MCF-7 breast cancer cells after a 2 h-, 4 h-, 16 h-, 24 h- and 5d-exposure to a Random Positioning Machine (RPM) simulating microgravity. At 24 h few small compact MCS were detectable, whereas after 5d many MCS were floating in the supernatant above the cells, remaining adherently (AD). The MCS resembled the ducts formed in vivo by human epithelial breast cells. In order to clarify the underlying mechanisms, we harvested MCS and AD cells separately from each RPM-culture and measured the expression of 29 selected genes with a known involvement in MCS formation. qPCR analyses indicated that cytoskeletal genes were unaltered in short-term samples. IL8, VEGFA, and FLT1 were upregulated in 2 h/4 h AD-cultures. The ACTB, TUBB, EZR, RDX, FN1, VEGFA, FLK1 Casp9, Casp3, PRKCA mRNAs were downregulated in 5d-MCS-samples. ESR1 was upregulated in AD, and PGR1 in both phenotypes after 5d. A pathway analysis revealed that the corresponding gene products are involved in organization and regulation of the cell shape, in cell tip formation and membrane to membrane docking. PMID:27230828

  12. Identifications of novel mechanisms in breast cancer cells involving duct-like multicellular spheroid formation after exposure to the Random Positioning Machine

    PubMed Central

    Kopp, Sascha; Slumstrup, Lasse; Corydon, Thomas J.; Sahana, Jayashree; Aleshcheva, Ganna; Islam, Tawhidul; Magnusson, Nils E.; Wehland, Markus; Bauer, Johann; Infanger, Manfred; Grimm, Daniela

    2016-01-01

    Many cell types form three-dimensional aggregates (MCS; multicellular spheroids), when they are cultured under microgravity. MCS often resemble the organ, from which the cells have been derived. In this study we investigated human MCF-7 breast cancer cells after a 2 h-, 4 h-, 16 h-, 24 h- and 5d-exposure to a Random Positioning Machine (RPM) simulating microgravity. At 24 h few small compact MCS were detectable, whereas after 5d many MCS were floating in the supernatant above the cells, remaining adherently (AD). The MCS resembled the ducts formed in vivo by human epithelial breast cells. In order to clarify the underlying mechanisms, we harvested MCS and AD cells separately from each RPM-culture and measured the expression of 29 selected genes with a known involvement in MCS formation. qPCR analyses indicated that cytoskeletal genes were unaltered in short-term samples. IL8, VEGFA, and FLT1 were upregulated in 2 h/4 h AD-cultures. The ACTB, TUBB, EZR, RDX, FN1, VEGFA, FLK1 Casp9, Casp3, PRKCA mRNAs were downregulated in 5d-MCS-samples. ESR1 was upregulated in AD, and PGR1 in both phenotypes after 5d. A pathway analysis revealed that the corresponding gene products are involved in organization and regulation of the cell shape, in cell tip formation and membrane to membrane docking. PMID:27230828

  13. A simple, low-cost staining method for rapid-throughput analysis of tumor spheroids

    PubMed Central

    Eckerdt, Frank; Alvarez, Angel; Bell, Jonathan; Arvanitis, Constadina; Iqbal, Asneha; Arslan, Ahmet D.; Hu, Bo; Cheng, Shi-Yuan; Goldman, Stewart; Platanias, Leonidas C.

    2016-01-01

    Tumor spheroids are becoming an important tool for the investigation of cancer stem cell (CSC) function in tumors; thus, low-cost and high-throughput methods for drug screening of tumor spheroids are needed. Using neurospheres as non-adherent three-dimensional (3-D) cultures, we developed a simple, low-cost acridine orange (AO)–based method that allows for rapid analysis of live neurospheres by fluorescence microscopy in a 96-well format. This assay measures the cross-section area of a spheroid, which corresponds to cell viability. Our novel method allows rapid screening of a panel of anti-proliferative drugs to assess inhibitory effects on the growth of cancer stem cells in 3-D cultures. PMID:26757811

  14. Scaffold-free formation of a millimeter-scale multicellular spheroid with an internal cavity from magnetically levitated 3T3 cells that ingested iron oxide-containing microspheres.

    PubMed

    Lee, Joon Ho; Hur, Won

    2014-05-01

    This report describes fabrication of a millimeter-scale three-dimensional (3D) multicellular structure with a central cavity based on magnetic levitation of 3T3 cells that had ingested Fe3 O4 -containing microcapsules. Magnetically levitated cells initially formed a disc-shaped cell cluster at the air-medium interface and transformed into a spheroid (up to 2.8 mm in diameter) after 10-day incubation under a magnet. Hematoxylin-and-eosin-stained section revealed that an eosinophilic shell of cells enclosed a pale-staining core of the spheroid. Mitotic or elongated and aligned cells were found at the outer periphery of the shell, while Fe3 O4 deposits were distributed in the inner part of the shell. Surgical dissection indicated that the spheroid had a hollow interior filled with a fluid-state cell suspension. Accordingly, it was demonstrated that magnetically levitated 3T3 cells organized themselves into a tissue-like spheroid, resulting in core cell death. The spheroid can be used as a 3D tissue model and as building blocks that fused to form a more complicated structure. PMID:24254251

  15. Improved Methods to Generate Spheroid Cultures from Tumor Cells, Tumor Cells & Fibroblasts or Tumor-Fragments: Microenvironment, Microvesicles and MiRNA

    PubMed Central

    Lao, Zheng; Kelly, Catherine J.; Yang, Xiang-Yang; Jenkins, W. Timothy; Toorens, Erik; Ganguly, Tapan; Evans, Sydney M.; Koch, Cameron J.

    2015-01-01

    Diagnostic and prognostic indicators are key components to achieve the goal of personalized cancer therapy. Two distinct approaches to this goal include predicting response by genetic analysis and direct testing of possible therapies using cultures derived from biopsy specimens. Optimally, the latter method requires a rapid assessment, but growing xenograft tumors or developing patient-derived cell lines can involve a great deal of time and expense. Furthermore, tumor cells have much different responses when grown in 2D versus 3D tissue environments. Using a modification of existing methods, we show that it is possible to make tumor-fragment (TF) spheroids in only 2–3 days. TF spheroids appear to closely model characteristics of the original tumor and may be used to assess critical therapy-modulating features of the microenvironment such as hypoxia. A similar method allows the reproducible development of spheroids from mixed tumor cells and fibroblasts (mixed-cell spheroids). Prior literature reports have shown highly variable development and properties of mixed-cell spheroids and this has hampered the detailed study of how individual tumor-cell components interact. In this study, we illustrate this approach and describe similarities and differences using two tumor models (U87 glioma and SQ20B squamous-cell carcinoma) with supporting data from additional cell lines. We show that U87 and SQ20B spheroids predict a key microenvironmental factor in tumors (hypoxia) and that SQ20B cells and spheroids generate similar numbers of microvesicles. We also present pilot data for miRNA expression under conditions of cells, tumors, and TF spheroids. PMID:26208323

  16. Drug testing and flow cytometry analysis on a large number of uniform sized tumor spheroids using a microfluidic device

    PubMed Central

    Patra, Bishnubrata; Peng, Chien-Chung; Liao, Wei-Hao; Lee, Chau-Hwang; Tung, Yi-Chung

    2016-01-01

    Three-dimensional (3D) tumor spheroid possesses great potential as an in vitro model to improve predictive capacity for pre-clinical drug testing. In this paper, we combine advantages of flow cytometry and microfluidics to perform drug testing and analysis on a large number (5000) of uniform sized tumor spheroids. The spheroids are formed, cultured, and treated with drugs inside a microfluidic device. The spheroids can then be harvested from the device without tedious operation. Due to the ample cell numbers, the spheroids can be dissociated into single cells for flow cytometry analysis. Flow cytometry provides statistical information in single cell resolution that makes it feasible to better investigate drug functions on the cells in more in vivo-like 3D formation. In the experiments, human hepatocellular carcinoma cells (HepG2) are exploited to form tumor spheroids within the microfluidic device, and three anti-cancer drugs: Cisplatin, Resveratrol, and Tirapazamine (TPZ), and their combinations are tested on the tumor spheroids with two different sizes. The experimental results suggest the cell culture format (2D monolayer vs. 3D spheroid) and spheroid size play critical roles in drug responses, and also demonstrate the advantages of bridging the two techniques in pharmaceutical drug screening applications. PMID:26877244

  17. Drug testing and flow cytometry analysis on a large number of uniform sized tumor spheroids using a microfluidic device

    NASA Astrophysics Data System (ADS)

    Patra, Bishnubrata; Peng, Chien-Chung; Liao, Wei-Hao; Lee, Chau-Hwang; Tung, Yi-Chung

    2016-02-01

    Three-dimensional (3D) tumor spheroid possesses great potential as an in vitro model to improve predictive capacity for pre-clinical drug testing. In this paper, we combine advantages of flow cytometry and microfluidics to perform drug testing and analysis on a large number (5000) of uniform sized tumor spheroids. The spheroids are formed, cultured, and treated with drugs inside a microfluidic device. The spheroids can then be harvested from the device without tedious operation. Due to the ample cell numbers, the spheroids can be dissociated into single cells for flow cytometry analysis. Flow cytometry provides statistical information in single cell resolution that makes it feasible to better investigate drug functions on the cells in more in vivo-like 3D formation. In the experiments, human hepatocellular carcinoma cells (HepG2) are exploited to form tumor spheroids within the microfluidic device, and three anti-cancer drugs: Cisplatin, Resveratrol, and Tirapazamine (TPZ), and their combinations are tested on the tumor spheroids with two different sizes. The experimental results suggest the cell culture format (2D monolayer vs. 3D spheroid) and spheroid size play critical roles in drug responses, and also demonstrate the advantages of bridging the two techniques in pharmaceutical drug screening applications.

  18. Quantitative Spatiotemporal Analysis of Antibody Fragment Diffusion and Endocytic Consumption in Tumor Spheroids

    PubMed Central

    Thurber, Greg M.; Wittrup, K. Dane

    2010-01-01

    Antibody-based cancer treatment depends upon distribution of the targeting macromolecule throughout tumor tissue, and spatial heterogeneity could significantly limit efficacy in many cases. Antibody distribution in tumor tissue is a function of drug dosage, antigen concentration, binding affinity, antigen internalization, drug extravasation from blood vessels, diffusion in the tumor extracellular matrix, and systemic clearance rates. We have isolated the effects of a subset of these variables by live-cell microscopic imaging of single-chain antibody fragments against carcinoembryonic antigen in LS174T tumor spheroids. The measured rates of scFv penetration and retention were compared with theoretical predictions based on simple scaling criteria. The theory predicts that antibody dose must be large enough to drive a sufficient diffusive flux of antibody to overcome cellular internalization, and exposure time must be long enough to allow penetration to the spheroid center. The experimental results in spheroids are quantitatively consistent with these predictions. Therefore, simple scaling criteria can be applied to accurately predict antibody and antibody fragment penetration distance in tumor tissue. PMID:18451160

  19. High-throughput image analysis of tumor spheroids: a user-friendly software application to measure the size of spheroids automatically and accurately.

    PubMed

    Chen, Wenjin; Wong, Chung; Vosburgh, Evan; Levine, Arnold J; Foran, David J; Xu, Eugenia Y

    2014-07-08

    The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application - SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min on a minimally configured laptop, or around 1 min on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary "Manual Initialize" and "Hand Draw" tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model

  20. Quantitative 1H MRI and MRS Microscopy of Individual V79 Lung Tumor Spheroids

    NASA Astrophysics Data System (ADS)

    Minard, Kevin R.; Guo, Xiuling; Wind, Robert A.

    1998-08-01

    In this Communication1H MRI and MRS microscopy experiments of individual V79 lung tumor spheroids with diameters between 550 and 650 μm are reported. The results have been used to determine theT1,T2, andDvalues as well as the concentrations of water, total choline, creatine/phosphocreatine, and mobile lipids in the viable rims and in the necrotic centers.

  1. Optimization of Aqueous Biphasic Tumor Spheroid Microtechnology for Anti-Cancer Drug Testing in 3D Culture

    PubMed Central

    Lemmo, Stephanie; Atefi, Ehsan; Luker, Gary D.; Tavana, Hossein

    2014-01-01

    Tumor spheroids are three-dimensional clusters of cancer cells that exhibit characteristics of poorly perfused tumors and hence present a relevant model for testing the efficacy of anti-cancer compounds. The use of spheroids for drug screening is hindered by technological complexities for high throughput generation of consistent size spheroids individually addressable by drug compounds. Here we present and optimize a simple spheroid technology based on the use of an aqueous two-phase system. Cancer cells confined in a drop of the denser aqueous dextran phase are robotically dispensed into a microwell containing the immersion aqueous polyethylene glycol phase. Cells remain within the drop and form a viable spheroid, without a need for any external stimuli. The size of resulting spheroids is sensitive to volume variations of dispensed drops from the air displacement pipetting head of a commercial liquid handling robot. Therefore, we parametrically optimize the process of dispensing of dextran phase drops. For a given cell density, this optimization reproducibly generates consistent size spheroids in standard 96-well plates. In addition, we evaluate the use of a commercial biochemical assay to examine cellular viability of cancer cell spheroids. Spheroids show a dose-dependent response to cisplatin similar to a monolayer culture. However unlike their two-dimensional counterpart, spheroids exhibit resistance to paclitaxel treatment. This technology, which uses only commercially-available reagents and equipment, can potentially expedite anti-cancer drug discovery. Although the use of robotics makes the ATPS spheroid technology particularly useful for drug screening applications, this approach is compatible with simpler liquid handling techniques such as manual micropipetting and offers a straightforward method of 3D cell culture in research laboratories. PMID:25221631

  2. Hypoxia Responsive, Tumor Penetrating Lipid Nanoparticles for Delivery of Chemotherapeutics to Pancreatic Cancer Cell Spheroids.

    PubMed

    Kulkarni, Prajakta; Haldar, Manas K; Katti, Preeya; Dawes, Courtney; You, Seungyong; Choi, Yongki; Mallik, Sanku

    2016-08-17

    Solid tumors are often poorly irrigated due to structurally compromised microcirculation. Uncontrolled multiplication of cancer cells, insufficient blood flow, and the lack of enough oxygen and nutrients lead to the development of hypoxic regions in the tumor tissues. As the partial pressure of oxygen drops below the necessary level (10 psi), the cancer cells modulate their genetic makeup to survive. Hypoxia triggers tumor progression by enhancing angiogenesis, cancer stem cell production, remodeling of the extracellular matrix, and epigenetic changes in the cancer cells. However, the hypoxic regions are usually located deep in the tumors and are usually inaccessible to the intravenously injected drug carrier or the drug. Considering the designs of the reported nanoparticles, it is likely that the drug is delivered to the peripheral tumor tissues, close to the blood vessels. In this study, we prepared lipid nanoparticles (LNs) comprising the synthesized hypoxia-responsive lipid and a peptide-lipid conjugate. We observed that the resultant LNs penetrated to the hypoxic regions of the tumors. Under low oxygen partial pressure, the hypoxia-responsive lipid undergoes reduction, destabilizing the lipid membrane, and releasing encapsulated drugs from the nanoparticles. We demonstrated the results employing spheroidal cultures of the pancreatic cancer cells BxPC-3. We observed that the peptide-decorated, drug encapsulated LNs reduced the viability of pancreatic cancer cells of the spheroids to 35% under hypoxic conditions. PMID:27391789

  3. Cisplatin Resistant Spheroids Model Clinically Relevant Survival Mechanisms in Ovarian Tumors

    PubMed Central

    Miller, Daniel H.; Medina, Jamie E.; Hamilton, Joshua W.; Messerli, Mark A.; Brodsky, Alexander S.

    2016-01-01

    The majority of ovarian tumors eventually recur in a drug resistant form. Using cisplatin sensitive and resistant cell lines assembled into 3D spheroids we profiled gene expression and identified candidate mechanisms and biological pathways associated with cisplatin resistance. OVCAR-8 human ovarian carcinoma cells were exposed to sub-lethal concentrations of cisplatin to create a matched cisplatin-resistant cell line, OVCAR-8R. Genome-wide gene expression profiling of sensitive and resistant ovarian cancer spheroids identified 3,331 significantly differentially expressed probesets coding for 3,139 distinct protein-coding genes (Fc >2, FDR < 0.05) (S2 Table). Despite significant expression changes in some transporters including MDR1, cisplatin resistance was not associated with differences in intracellular cisplatin concentration. Cisplatin resistant cells were significantly enriched for a mesenchymal gene expression signature. OVCAR-8R resistance derived gene sets were significantly more biased to patients with shorter survival. From the most differentially expressed genes, we derived a 17-gene expression signature that identifies ovarian cancer patients with shorter overall survival in three independent datasets. We propose that the use of cisplatin resistant cell lines in 3D spheroid models is a viable approach to gain insight into resistance mechanisms relevant to ovarian tumors in patients. Our data support the emerging concept that ovarian cancers can acquire drug resistance through an epithelial-to-mesenchymal transition. PMID:26986722

  4. Differential Superiority of Heavy Charged-Particle Irradiation to X-Rays: Studies on Biological Effectiveness and Side Effect Mechanisms in Multicellular Tumor and Normal Tissue Models

    PubMed Central

    Walenta, Stefan; Mueller-Klieser, Wolfgang

    2016-01-01

    This review is focused on the radiobiology of carbon ions compared to X-rays using multicellular models of tumors and normal mucosa. The first part summarizes basic radiobiological effects, as observed in cancer cells. The second, more clinically oriented part of the review, deals with radiation-induced cell migration and mucositis. Multicellular spheroids from V79 hamster cells were irradiated with X-rays or carbon ions under ambient or restricted oxygen supply conditions. Reliable oxygen enhancement ratios could be derived to be 2.9, 2.8, and 1.4 for irradiation with photons, 12C+6 in the plateau region, and 12C+6 in the Bragg peak, respectively. Similarly, a relative biological effectiveness of 4.3 and 2.1 for ambient pO2 and hypoxia was obtained, respectively. The high effectiveness of carbon ions was reflected by an enhanced accumulation of cells in G2/M and a dose-dependent massive induction of apoptosis. These data clearly show that heavy charged particles are more efficient in sterilizing tumor cells than conventional irradiation even under hypoxic conditions. Clinically relevant doses (3 Gy) of X-rays induced an increase in migratory activity of U87 but not of LN229 or HCT116 tumor cells. Such an increase in cell motility following irradiation in situ could be the source of recurrence. In contrast, carbon ion treatment was associated with a dose-dependent decrease in migration with all cell lines and under all conditions investigated. The radiation-induced loss of cell motility was correlated, in most cases, with corresponding changes in β1 integrin expression. The photon-induced increase in cell migration was paralleled by an elevated phosphorylation status of the epidermal growth factor receptor and AKT-ERK1/2 pathway. Such a hyperphosphorylation did not occur during 12C+6 irradiation under all conditions registered. Comparing the gene toxicity of X-rays with that of particles using the γH2AX technique in organotypic cultures of the oral mucosa, the

  5. Differential Superiority of Heavy Charged-Particle Irradiation to X-Rays: Studies on Biological Effectiveness and Side Effect Mechanisms in Multicellular Tumor and Normal Tissue Models.

    PubMed

    Walenta, Stefan; Mueller-Klieser, Wolfgang

    2016-01-01

    This review is focused on the radiobiology of carbon ions compared to X-rays using multicellular models of tumors and normal mucosa. The first part summarizes basic radiobiological effects, as observed in cancer cells. The second, more clinically oriented part of the review, deals with radiation-induced cell migration and mucositis. Multicellular spheroids from V79 hamster cells were irradiated with X-rays or carbon ions under ambient or restricted oxygen supply conditions. Reliable oxygen enhancement ratios could be derived to be 2.9, 2.8, and 1.4 for irradiation with photons, (12)C(+6) in the plateau region, and (12)C(+6) in the Bragg peak, respectively. Similarly, a relative biological effectiveness of 4.3 and 2.1 for ambient pO2 and hypoxia was obtained, respectively. The high effectiveness of carbon ions was reflected by an enhanced accumulation of cells in G2/M and a dose-dependent massive induction of apoptosis. These data clearly show that heavy charged particles are more efficient in sterilizing tumor cells than conventional irradiation even under hypoxic conditions. Clinically relevant doses (3 Gy) of X-rays induced an increase in migratory activity of U87 but not of LN229 or HCT116 tumor cells. Such an increase in cell motility following irradiation in situ could be the source of recurrence. In contrast, carbon ion treatment was associated with a dose-dependent decrease in migration with all cell lines and under all conditions investigated. The radiation-induced loss of cell motility was correlated, in most cases, with corresponding changes in β1 integrin expression. The photon-induced increase in cell migration was paralleled by an elevated phosphorylation status of the epidermal growth factor receptor and AKT-ERK1/2 pathway. Such a hyperphosphorylation did not occur during (12)C(+6) irradiation under all conditions registered. Comparing the gene toxicity of X-rays with that of particles using the γH2AX technique in organotypic cultures of the oral

  6. Differential Superiority of Heavy Charged-Particle Irradiation to X-Rays: Studies on Biological Effectiveness and Side Effect Mechanisms in Multicellular Tumor and Normal Tissue Models.

    PubMed

    Walenta, Stefan; Mueller-Klieser, Wolfgang

    2016-01-01

    This review is focused on the radiobiology of carbon ions compared to X-rays using multicellular models of tumors and normal mucosa. The first part summarizes basic radiobiological effects, as observed in cancer cells. The second, more clinically oriented part of the review, deals with radiation-induced cell migration and mucositis. Multicellular spheroids from V79 hamster cells were irradiated with X-rays or carbon ions under ambient or restricted oxygen supply conditions. Reliable oxygen enhancement ratios could be derived to be 2.9, 2.8, and 1.4 for irradiation with photons, (12)C(+6) in the plateau region, and (12)C(+6) in the Bragg peak, respectively. Similarly, a relative biological effectiveness of 4.3 and 2.1 for ambient pO2 and hypoxia was obtained, respectively. The high effectiveness of carbon ions was reflected by an enhanced accumulation of cells in G2/M and a dose-dependent massive induction of apoptosis. These data clearly show that heavy charged particles are more efficient in sterilizing tumor cells than conventional irradiation even under hypoxic conditions. Clinically relevant doses (3 Gy) of X-rays induced an increase in migratory activity of U87 but not of LN229 or HCT116 tumor cells. Such an increase in cell motility following irradiation in situ could be the source of recurrence. In contrast, carbon ion treatment was associated with a dose-dependent decrease in migration with all cell lines and under all conditions investigated. The radiation-induced loss of cell motility was correlated, in most cases, with corresponding changes in β1 integrin expression. The photon-induced increase in cell migration was paralleled by an elevated phosphorylation status of the epidermal growth factor receptor and AKT-ERK1/2 pathway. Such a hyperphosphorylation did not occur during (12)C(+6) irradiation under all conditions registered. Comparing the gene toxicity of X-rays with that of particles using the γH2AX technique in organotypic cultures of the oral

  7. Assessing the immunomodulatory role of heteroglycan in a tumor spheroid and macrophage co-culture model system.

    PubMed

    Devi, K Sanjana P; Mishra, Debasish; Roy, Bibhas; Ghosh, Sudip K; Maiti, Tapas K

    2015-01-01

    The therapeutic benefits of glycans have garnered much attention over the last few decades with most studies being reported in 2D cultures or in animal models. The present work is therefore aimed to assess the effects of an immunomodulatory heteroglycan in a 3D milieu. Briefly, HT29 tumor spheroids were incubated with THP-1 macrophages at 1:1 ratio in a culture medium supplemented with immune stimulants such as heteroglycans or LPS. Spheroidal distortion, migration of tumor cells from the periphery of the spheroids and 46% of higher macrophage invasiveness was noted in heteroglycan-treated co-cultures with respect to control cultures. Histological sections of the treated co-cultures revealed the presence of high apoptotic tumor cells in the spheroidal periphery. CD11c and CD68 staining further suggested the predominance of macrophages in the vicinity of the apoptotic tumor cells. Such an in vitro created tissue system may thereby confirm the effectiveness of heteroglycan in activating the immune cells to exhibit tumor cytotoxic properties. PMID:25965450

  8. Assessing the immunomodulatory role of heteroglycan in a tumor spheroid and macrophage co-culture model system.

    PubMed

    Devi, K Sanjana P; Mishra, Debasish; Roy, Bibhas; Ghosh, Sudip K; Maiti, Tapas K

    2015-01-01

    The therapeutic benefits of glycans have garnered much attention over the last few decades with most studies being reported in 2D cultures or in animal models. The present work is therefore aimed to assess the effects of an immunomodulatory heteroglycan in a 3D milieu. Briefly, HT29 tumor spheroids were incubated with THP-1 macrophages at 1:1 ratio in a culture medium supplemented with immune stimulants such as heteroglycans or LPS. Spheroidal distortion, migration of tumor cells from the periphery of the spheroids and 46% of higher macrophage invasiveness was noted in heteroglycan-treated co-cultures with respect to control cultures. Histological sections of the treated co-cultures revealed the presence of high apoptotic tumor cells in the spheroidal periphery. CD11c and CD68 staining further suggested the predominance of macrophages in the vicinity of the apoptotic tumor cells. Such an in vitro created tissue system may thereby confirm the effectiveness of heteroglycan in activating the immune cells to exhibit tumor cytotoxic properties.

  9. Monoclonal antibodies directed against surface molecules of multicell spheroids

    NASA Technical Reports Server (NTRS)

    Martinez, Andrew O.

    1993-01-01

    The objective of this project is to generate a library of monoclonal antibodies (MAbs) to surface molecules of mammalian tumor and transformed cells grown as multicell spheroids (MCS). These MCS are highly organized, three dimensional multicellular structures which exhibit many characteristics of in vivo organized tissues not found in conventional monolayer or suspension culture; therefore, MCS make better in vitro model systems to study the interactions of mammalian cells. Additionally, they provide a functional assay for surface adhesion molecules.

  10. Cell proliferation kinetics and radiation response in 9L tumor spheroids

    SciTech Connect

    Sweigert, S.E.

    1984-05-01

    Cell kinetic parameters, including population doubling-time, cell cycle time, and growth fraction, were measured in 9L gliosarcoma spheroids. These parameters were studied as the spheroids grew from 50 ..mu..m to over 900 ..mu..m in diameter. Experiments relating the cell kinetic parameters to the radiation response of 9L spheroids were also carried out. The major findings were that the average cell cycle time (T/sub c/), is considerably longer in large spheroids than in exponentially-growing monolayers, the radiosensitivity of noncycling (but still viable) cells in spheroids is not significantly different from that of cycling spheroid cells, and the radiation-induced division delay is approximately twice as long in spheroid cells as in monolayer cells given equal radiation doses. The cell loss factor for spheroids of various sizes was calculated, by using the measured kinetic parameters in the basic equations for growth of a cell population. 157 references, 6 figures, 3 tables.

  11. Scalable Differentiation of Human iPSCs in a Multicellular Spheroid-based 3D Culture into Hepatocyte-like Cells through Direct Wnt/β-catenin Pathway Inhibition

    PubMed Central

    Pettinato, Giuseppe; Ramanathan, Rajesh; Fisher, Robert A; Mangino, Martin J.; Zhang, Ning; Wen, Xuejun

    2016-01-01

    Treatment of acute liver failure by cell transplantation is hindered by a shortage of human hepatocytes. Current protocols for hepatic differentiation of human induced pluripotent stem cells (hiPSCs) result in low yields, cellular heterogeneity, and limited scalability. In the present study, we have developed a novel multicellular spheroid-based hepatic differentiation protocol starting from embryoid bodies of hiPSCs (hiPSC-EBs) for robust mass production of human hepatocyte-like cells (HLCs) using two novel inhibitors of the Wnt pathway. The resultant hiPSC-EB-HLCs expressed liver-specific genes, secreted hepatic proteins such as Albumin, Alpha Fetoprotein, and Fibrinogen, metabolized ammonia, and displayed cytochrome P450 activities and functional activities typical of mature primary hepatocytes, such as LDL storage and uptake, ICG uptake and release, and glycogen storage. Cell transplantation of hiPSC-EB-HLC in a rat model of acute liver failure significantly prolonged the mean survival time and resolved the liver injury when compared to the no-transplantation control animals. The transplanted hiPSC-EB-HLCs secreted human albumin into the host plasma throughout the examination period (2 weeks). Transplantation successfully bridged the animals through the critical period for survival after acute liver failure, providing promising clues of integration and full in vivo functionality of these cells after treatment with WIF-1 and DKK-1. PMID:27616299

  12. Scalable Differentiation of Human iPSCs in a Multicellular Spheroid-based 3D Culture into Hepatocyte-like Cells through Direct Wnt/β-catenin Pathway Inhibition

    PubMed Central

    Pettinato, Giuseppe; Ramanathan, Rajesh; Fisher, Robert A; Mangino, Martin J.; Zhang, Ning; Wen, Xuejun

    2016-01-01

    Treatment of acute liver failure by cell transplantation is hindered by a shortage of human hepatocytes. Current protocols for hepatic differentiation of human induced pluripotent stem cells (hiPSCs) result in low yields, cellular heterogeneity, and limited scalability. In the present study, we have developed a novel multicellular spheroid-based hepatic differentiation protocol starting from embryoid bodies of hiPSCs (hiPSC-EBs) for robust mass production of human hepatocyte-like cells (HLCs) using two novel inhibitors of the Wnt pathway. The resultant hiPSC-EB-HLCs expressed liver-specific genes, secreted hepatic proteins such as Albumin, Alpha Fetoprotein, and Fibrinogen, metabolized ammonia, and displayed cytochrome P450 activities and functional activities typical of mature primary hepatocytes, such as LDL storage and uptake, ICG uptake and release, and glycogen storage. Cell transplantation of hiPSC-EB-HLC in a rat model of acute liver failure significantly prolonged the mean survival time and resolved the liver injury when compared to the no-transplantation control animals. The transplanted hiPSC-EB-HLCs secreted human albumin into the host plasma throughout the examination period (2 weeks). Transplantation successfully bridged the animals through the critical period for survival after acute liver failure, providing promising clues of integration and full in vivo functionality of these cells after treatment with WIF-1 and DKK-1. PMID:27616299

  13. Scalable Differentiation of Human iPSCs in a Multicellular Spheroid-based 3D Culture into Hepatocyte-like Cells through Direct Wnt/β-catenin Pathway Inhibition.

    PubMed

    Pettinato, Giuseppe; Ramanathan, Rajesh; Fisher, Robert A; Mangino, Martin J; Zhang, Ning; Wen, Xuejun

    2016-01-01

    Treatment of acute liver failure by cell transplantation is hindered by a shortage of human hepatocytes. Current protocols for hepatic differentiation of human induced pluripotent stem cells (hiPSCs) result in low yields, cellular heterogeneity, and limited scalability. In the present study, we have developed a novel multicellular spheroid-based hepatic differentiation protocol starting from embryoid bodies of hiPSCs (hiPSC-EBs) for robust mass production of human hepatocyte-like cells (HLCs) using two novel inhibitors of the Wnt pathway. The resultant hiPSC-EB-HLCs expressed liver-specific genes, secreted hepatic proteins such as Albumin, Alpha Fetoprotein, and Fibrinogen, metabolized ammonia, and displayed cytochrome P450 activities and functional activities typical of mature primary hepatocytes, such as LDL storage and uptake, ICG uptake and release, and glycogen storage. Cell transplantation of hiPSC-EB-HLC in a rat model of acute liver failure significantly prolonged the mean survival time and resolved the liver injury when compared to the no-transplantation control animals. The transplanted hiPSC-EB-HLCs secreted human albumin into the host plasma throughout the examination period (2 weeks). Transplantation successfully bridged the animals through the critical period for survival after acute liver failure, providing promising clues of integration and full in vivo functionality of these cells after treatment with WIF-1 and DKK-1. PMID:27616299

  14. Cancer stem cell differentiation: TGFβ1 and versican may trigger molecules for the organization of tumor spheroids.

    PubMed

    Oktem, G; Sercan, O; Guven, U; Uslu, R; Uysal, A; Goksel, G; Ayla, S; Bilir, A

    2014-08-01

    Cancer stem cells (CSCs) have the ability to self-renew similar to normal stem cells. This process is linked with metastasis and resistance to chemotherapy and radiotherapy. In the present study, we constructed an in vitro differentiation model for CSCs. CSCs isolated and proliferated for one passage were maintained as monolayers or spheroid-forming cells with serum included media for differentiation process. Differentiation of adhesion molecules and cellular ultrastructural properties were investigated and compared in both monolayer and spheroid cultures. CD133+/CD44+ cancer-initiating cells were isolated from DU-145 human prostate cancer cell line monolayer cultures and propagated as tumor spheroids and compared with the remaining heterogeneous cancer cell bulk population. Microarray-based gene expression analysis was applied to determine genes with differential expression and protein expression levels of candidates were analyzed by immunohistochemistry. Electron microscopy showed detailed analysis of morphology. TGFβ1 was found to be significantly upregulated in monolayer CSCs. High expression levels of VCAN, COL7A1, ITGβ3, MMP16, RPL13A, COL4A2 and TIMP1 and low expression levels of THBS1, MMP1 and MMP14 were detected when CSCs were maintained as serum-grown prostate CSC spheroids. Immunohistochemistry supported increased immunoreactivity of TGFβ1 in monolayer cultures and VCAN in spheroids. CSCs were found to possess multipotential differentiation capabilities through upregulation and/or downregulation of their markers. TGFβ1 is a triggering molecule, it stimulates versican, Col7A1, ITGβ3 and, most importantly, the upregulation of versican was only detected in CSCs. Our data support a model where CSCs must be engaged by one or more signaling cascades to differentiate and initiate tumor formation. This mechanism occurs with intracellular and extracellular signals and it is possible that CSCc themselves may be a source for extracellular signaling. These

  15. Evaluation of Consistency in Spheroid Invasion Assays

    PubMed Central

    Cisneros Castillo, Liliana R.; Oancea, Andrei-Dumitru; Stüllein, Christian; Régnier-Vigouroux, Anne

    2016-01-01

    Multicellular tumor spheroids embedded in a matrix represent invaluable tools to analyze cell invasion. Spheroid sizes and invasiveness are the main observables easily measurable to evaluate effects of biological or pharmaceutical manipulations on invasion. They largely account for these 3-D platforms variability, leading to flaws in data interpretation. No method has been established yet that characterizes this variability and guarantees a reliable use of 3-D platforms. Spheroid initial/end sizes and invasiveness were systematically analyzed and compared in spheroids of U87MG cells generated by three different methods and embedded at different times in a collagen matrix. A normality test was used to characterize size distribution. We introduced the linearity-over-yield analysis as a novel mathematical tool to assess end sizes and invasion reproducibility. We further provide a proof of concept by applying these tools to the analysis of a treatment known to be effective beforehand. We demonstrate that implementation of these statistical and mathematical tools warranted a confident quantification and interpretation of in 3-D conducted assays. We propose these tools could be incorporated in a guideline for generation and use of 3-D platforms. PMID:27334575

  16. Emerging patterns in tumor systems: simulating the dynamics of multicellular clusters with an agent-based spatial agglomeration model.

    PubMed

    Mansury, Yuri; Kimura, Mark; Lobo, Jose; Deisboeck, Thomas S

    2002-12-01

    Brain cancer cells invade early on surrounding parenchyma, which makes it impossible to surgically remove all tumor cells and thus significantly worsens the prognosis of the patient. Specific structural elements such as multicellular clusters have been seen in experimental settings to emerge within the invasive cell system and are believed to express the systems' guidance toward nutritive sites in a heterogeneous environment. Based on these observations, we developed a novel agent-based model of spatio-temporal search and agglomeration to investigate the dynamics of cell motility and aggregation with the assumption that tumors behave as complex dynamic self-organizing biosystems. In this model, virtual cells migrate because they are attracted by higher nutrient concentrations and to avoid overpopulated areas with high levels of toxic metabolites. A specific feature of our model is the capability of cells to search both globally and locally. This concept is applied to simulate cell-surface receptor-mediated information processing of tumor cells such that a cell searching for a more growth-permissive place "learns" the information content of a brain tissue region within a two-dimensional lattice in two stages, processing first the global and then the local input. In both stages, differences in microenvironment characteristics define distinctions in energy expenditure for a moving cell and thus influence cell migration, proliferation, agglomeration, and cell death. Numerical results of our model show a phase transition leading to the emergence of two distinct spatio-temporal patterns depending on the dominant search mechanism. If global search is dominant, the result is a small number of large clusters exhibiting rapid spatial expansion but shorter lifetime of the tumor system. By contrast, if local search is dominant, the trade-off is many small clusters with longer lifetime but much slower velocity of expansion. Furthermore, in the case of such dominant local search

  17. Co-culture of 3D tumor spheroids with fibroblasts as a model for epithelial–mesenchymal transition in vitro

    SciTech Connect

    Kim, Sun-Ah; Lee, Eun Kyung; Kuh, Hyo-Jeong

    2015-07-15

    Epithelial–mesenchymal transition (EMT) acts as a facilitator of metastatic dissemination in the invasive margin of malignant tumors where active tumor–stromal crosstalks take place. Co-cultures of cancer cells with cancer-associated fibroblasts (CAFs) are often used as in vitro models of EMT. We established a tumor–fibroblast proximity co-culture using HT-29 tumor spheroids (TSs) with CCD-18co fibroblasts. When co-cultured with TSs, CCD-18co appeared activated, and proliferative activity as well as cell migration increased. Expression of fibronectin increased whereas laminin and type I collagen decreased in TSs co-cultured with fibroblasts compared to TSs alone, closely resembling the margin of in vivo xenograft tissue. Active TGFβ1 in culture media significantly increased in TS co-cultures but not in 2D co-cultures of cancer cells–fibroblasts, indicating that 3D context-associated factors from TSs may be crucial to crosstalks between cancer cells and fibroblasts. We also observed in TSs co-cultured with fibroblasts increased expression of α-SMA, EGFR and CTGF; reduced expression of membranous β-catenin and E-cadherin, together suggesting an EMT-like changes similar to a marginal region of xenograft tissue in vivo. Overall, our in vitro TS–fibroblast proximity co-culture mimics the EMT-state of the invasive margin of in vivo tumors in early metastasis. - Highlights: • An adjacent co-culture of tumor spheroids and fibroblasts is presented as EMT model. • Activation of fibroblasts and increased cell migration were shown in co-culture. • Expression of EMT-related factors in co-culture was similar to that in tumor tissue. • Crosstalk between spheroids and fibroblasts was demonstrated by secretome analysis.

  18. Generation of a tumor spheroid in a microgravity environment as a 3D model of melanoma.

    PubMed

    Marrero, Bernadette; Messina, Jane L; Heller, Richard

    2009-10-01

    An in vitro 3D model was developed utilizing a synthetic microgravity environment to facilitate studying the cell interactions. 2D monolayer cell culture models have been successfully used to understand various cellular reactions that occur in vivo. There are some limitations to the 2D model that are apparent when compared to cells grown in a 3D matrix. For example, some proteins that are not expressed in a 2D model are found up-regulated in the 3D matrix. In this paper, we discuss techniques used to develop the first known large, free-floating 3D tissue model used to establish tumor spheroids. The bioreactor system known as the High Aspect Ratio Vessel (HARVs) was used to provide a microgravity environment. The HARVs promoted aggregation of keratinocytes (HaCaT) that formed a construct that served as scaffolding for the growth of mouse melanoma. Although there is an emphasis on building a 3D model with the proper extracellular matrix and stroma, we were able to develop a model that excluded the use of matrigel. Immunohistochemistry and apoptosis assays provided evidence that this 3D model supports B16.F10 cell growth, proliferation, and synthesis of extracellular matrix. Immunofluorescence showed that melanoma cells interact with one another displaying observable cellular morphological changes. The goal of engineering a 3D tissue model is to collect new information about cancer development and develop new potential treatment regimens that can be translated to in vivo models while reducing the use of laboratory animals. PMID:19533253

  19. Multi-parametric imaging of tumor spheroids with ultra-bright and tunable nanoparticle O2 probes

    NASA Astrophysics Data System (ADS)

    Dmitriev, Ruslan I.; Borisov, Sergey M.; Jenkins, James; Papkovsky, Dmitri B.

    2015-03-01

    Multi-modal probes allow for flexible choice of imaging equipment when performing quenched-phosphorescence O2 measurements: one- or two-photon, PLIM or intensity-based ratiometric read-outs. Spectral and temporal (e.g. FLIMPLIM) discrimination can be used to image O2 together with pH, Ca2+, mitochondrial membrane potential, cell death markers or cell/organelle specific markers. However, the main challenge of existing nanoparticle probes is their limited diffusion across thick (> 20-50 μm) 3D cell models such as tumor spheroids. Here, we present new class of polymeric nanoparticle probes having tunable size, charge, cell-penetrating ability, and reporter dyes. Being spectrally similar to the recently described MM2, PA2 and other O2 probes, they are 5-10 times brighter, demonstrate improved ratiometric response and their surface chemistry can be easily modified. With cultures of 2D and 3D cell models (fibroblasts, PC12 aggregates, HCT116 human colon cancer spheroids) we found cell-specific staining by these probes. However, the efficient staining of model of interest can be tuned by changing number of positive and negative surface groups at nanoparticle, to allow most efficient loading. We also demonstrate how real-time monitoring of oxygenation can be used to select optimal spheroid production with low variability in size and high cell viability.

  20. Photobleaching and phototoxicity of KillerRed in tumor spheroids induced by continuous wave and pulsed laser illumination.

    PubMed

    Kuznetsova, Daria S; Shirmanova, Marina V; Dudenkova, Varvara V; Subochev, Pavel V; Turchin, Ilya V; Zagaynova, Elena V; Lukyanov, Sergey A; Shakhov, Boris E; Kamensky, Vladislav A

    2015-11-01

    The purpose of this study was to evaluate photobleaching of the genetically encoded photosensitizer KillerRed in tumor spheroids upon pulsed and continuous wave (CW) laser irradiation and to analyze the mechanisms of cancer cell death after the treatment. We observed the light-dose dependent mechanism of KillerRed photobleaching over a wide range of fluence rates. Loss of fluorescence was limited to 80% at light doses of 150 J/cm(2) and more. Based on the bleaching curves, six PDT regimes were applied for irradiation using CW and pulsed regimes at a power density of 160 mW/cm(2) and light doses of 140 J/cm(2) , 170 J/cm(2) and 200 J/cm(2). Irradiation of KillerRed-expressing spheroids in the pulsed mode (pulse duration 15 ns, pulse repetition rate 10 Hz) induced predominantly apoptotic cell death, while in the case of CW mode the cancer cells underwent necrosis. In general, these results improve our understanding of photobleaching mechanisms in GFP-like proteins and show the importance of appropriate selection of treatment mode for PDT with KillerRed. Representative fluorescence image of two KillerRed-expressing spheroids before and immediately after CW irradiation.

  1. 3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained

    PubMed Central

    Zanoni, Michele; Piccinini, Filippo; Arienti, Chiara; Zamagni, Alice; Santi, Spartaco; Polico, Rolando; Bevilacqua, Alessandro; Tesei, Anna

    2016-01-01

    The potential of a spheroid tumor model composed of cells in different proliferative and metabolic states for the development of new anticancer strategies has been amply demonstrated. However, there is little or no information in the literature on the problems of reproducibility of data originating from experiments using 3D models. Our analyses, carried out using a novel open source software capable of performing an automatic image analysis of 3D tumor colonies, showed that a number of morphology parameters affect the response of large spheroids to treatment. In particular, we found that both spheroid volume and shape may be a source of variability. We also compared some commercially available viability assays specifically designed for 3D models. In conclusion, our data indicate the need for a pre-selection of tumor spheroids of homogeneous volume and shape to reduce data variability to a minimum before use in a cytotoxicity test. In addition, we identified and validated a cytotoxicity test capable of providing meaningful data on the damage induced in large tumor spheroids of up to diameter in 650 μm by different kinds of treatments. PMID:26752500

  2. An in vitro assessment of liposomal topotecan simulating metronomic chemotherapy in combination with radiation in tumor-endothelial spheroids

    PubMed Central

    Jyoti, Amar; Fugit, Kyle D.; Sethi, Pallavi; McGarry, Ronald C.; Anderson, Bradley D.; Upreti, Meenakshi

    2015-01-01

    Low dose metronomic chemotherapy (LDMC) refers to prolonged administration of low dose chemotherapy designed to minimize toxicity and target the tumor endothelium, causing tumor growth inhibition. Topotecan (TPT) when administered at its maximum tolerated dose (MTD) is often associated with systemic hematological toxicities. Liposomal encapsulation of TPT enhances efficacy by shielding it from systemic clearance, allowing greater uptake and extended tissue exposure in tumors. Extended release of TPT from liposomal formulations also has the potential to mimic metronomic therapies with fewer treatments. Here we investigate potential toxicities of equivalent doses of free and actively loaded liposomal TPT (LTPT) and compare them to a fractionated low dose regimen of free TPT in tumor-endothelial spheroids (TES) with/without radiation exposure for a prolonged period of 10 days. Using confocal microscopy, TPT fluorescence was monitored to determine the accumulation of drug within TES. These studies showed TES, being more reflective of the in vivo tumor microenvironment, were more sensitive to LTPT in comparison to free TPT with radiation. More importantly, the response of TES to low-dose metronomic TPT with radiation was comparable to similar treatment with LTPT. This TES study suggests nanoparticle formulations designed for extended release of drug can simulate LDMC in vivo. PMID:26468877

  3. Fully Automated One-Step Production of Functional 3D Tumor Spheroids for High-Content Screening.

    PubMed

    Monjaret, François; Fernandes, Mathieu; Duchemin-Pelletier, Eve; Argento, Amelie; Degot, Sébastien; Young, Joanne

    2016-04-01

    Adoption of spheroids within high-content screening (HCS) has lagged behind high-throughput screening (HTS) due to issues with running complex assays on large three-dimensional (3D) structures.To enable multiplexed imaging and analysis of spheroids, different cancer cell lines were grown in 3D on micropatterned 96-well plates with automated production of nine uniform spheroids per well. Spheroids achieve diameters of up to 600 µm, and reproducibility was experimentally validated (interwell and interplate CV(diameter) <5%). Biphoton imaging confirmed that micropatterned spheroids exhibit characteristic cell heterogeneity with distinct microregions. Furthermore, central necrosis appears at a consistent spheroid size, suggesting standardized growth.Using three reference compounds (fluorouracil, irinotecan, and staurosporine), we validated HT-29 micropatterned spheroids on an HCS platform, benchmarking against hanging-drop spheroids. Spheroid formation and imaging in a single plate accelerate assay workflow, and fixed positioning prevents structures from overlapping or sticking to the well wall, augmenting image processing reliability. Furthermore, multiple spheroids per well increase the statistical confidence sufficiently to discriminate compound mechanisms of action and generate EC50 values for endpoints of cell death, architectural change, and size within a single-pass read. Higher quality data and a more efficient HCS work chain should encourage integration of micropatterned spheroid models within fundamental research and drug discovery applications.

  4. Three-Dimensional Spheroid Cell Culture Model for Target Identification Utilizing High-Throughput RNAi Screens.

    PubMed

    Iles, LaKesla R; Bartholomeusz, Geoffrey A

    2016-01-01

    The intrinsic limitations of 2D monolayer cell culture models have prompted the development of 3D cell culture model systems for in vitro studies. Multicellular tumor spheroid (MCTS) models closely simulate the pathophysiological milieu of solid tumors and are providing new insights into tumor biology as well as differentiation, tissue organization, and homeostasis. They are straightforward to apply in high-throughput screens and there is a great need for the development of reliable and robust 3D spheroid-based assays for high-throughput RNAi screening for target identification and cell signaling studies highlighting their potential in cancer research and treatment. In this chapter we describe a stringent standard operating procedure for the use of MCTS for high-throughput RNAi screens. PMID:27581289

  5. Restructuring dynamics of DU 145 and LNCaP prostate cancer spheroids.

    PubMed

    Song, Hong; Jain, Shamik K; Enmon, Richard M; O'Connor, Kim C

    2004-01-01

    Neoplastic cells acquire multidrug resistance as they assemble into multicellular spheroids. Image analysis and Monte Carlo simulation provided an insight into the adhesion and motility events during spheroid restructuring in liquid-overlay culture of DU 145 and LNCaP human prostate cancer cells. Irregularly shaped, two-dimensional aggregates restructured through incremental cell movements into three-dimensional spheroids. Of the two cultures examined, restructuring was more pronounced for DU 145 aggregates. Motile DU 145 cells formed spheroids with a minimum cell overlay of 30% for 25-mers as estimated by simulation versus 5% for adhesive LNCaP cells in aggregates of the same size. Over 72 h, the texture ratio increased from 0.55 +/- 0.05 for DU 145 aggregates with projected areas exceeding 2000 microm2 to a value approaching 0.75 +/- 0.02 (P < 0.05). For LNCaP aggregates of comparable size, the increase in texture ratio was more modest, less than 15% during the same time period (P < 0.05). Combined, these data suggest that motility events govern the overall rate of spheroid restructuring. This information has application to the chemosensitization of solid tumors and kinetic modeling of spheroid production. PMID:15723561

  6. Real-time monitoring of cisplatin cytotoxicity on three-dimensional spheroid tumor cells

    PubMed Central

    Baek, NamHuk; Seo, Ok Won; Lee, Jaehwa; Hulme, John; An, Seong Soo A

    2016-01-01

    Three-dimensional (3D) cell cultivation is a powerful technique for monitoring and understanding diverse cellular mechanisms in developmental cancer and neuronal biology, tissue engineering, and drug development. 3D systems could relate better to in vivo models than two-dimensional (2D) cultures. Several factors, such as cell type, survival rate, proliferation rate, and gene and protein expression patterns, determine whether a particular cell line can be adapted to a 3D system. The 3D system may overcome some of the limitations of 2D cultures in terms of cell–cell communication and cell networks, which are essential for understanding differentiation, structural organization, shape, and extended connections with other cells or organs. Here, the effect of the anticancer drug cisplatin, also known as cis-diamminedichloroplatinum (II) or CDDP, on adenosine triphosphate (ATP) generation was investigated using 3D spheroid-forming cells and real-time monitoring for 7 days. First, 12 cell lines were screened for their ability to form 3D spheroids: prostate (DU145), testis (F9), embryonic fibroblast (NIH-3T3), muscle (C2C12), embryonic kidney (293T), neuroblastoma (SH-SY5Y), adenocarcinomic alveolar basal epithelial cell (A549), cervical cancer (HeLa), HeLa contaminant (HEp2), pituitary epithelial-like cell (GH3), embryonic cell (PA317), and osteosarcoma (U-2OS) cells. Of these, eight cell lines were selected: NIH-3T3, C2C12, 293T, SH-SY5Y, A549, HeLa, PA317, and U-2OS; and five underwent real-time monitoring of CDDP cytotoxicity: HeLa, A549, 293T, SH-SY5Y, and U-2OS. ATP generation was blocked 1 day after addition of 50 μM CDDP, but cytotoxicity in HeLa, A549, SH-SY5Y, and U-2OS cells could be visualized only 4 days after treatment. In 293T cells, CDDP failed to kill entirely the culture and ATP generation was only partially blocked after 1 day. This suggests potential CDDP resistance of 293T cells or metabolic clearance of the drug. Real-time monitoring and ATP

  7. Real-time monitoring of cisplatin cytotoxicity on three-dimensional spheroid tumor cells.

    PubMed

    Baek, NamHuk; Seo, Ok Won; Lee, Jaehwa; Hulme, John; An, Seong Soo A

    2016-01-01

    Three-dimensional (3D) cell cultivation is a powerful technique for monitoring and understanding diverse cellular mechanisms in developmental cancer and neuronal biology, tissue engineering, and drug development. 3D systems could relate better to in vivo models than two-dimensional (2D) cultures. Several factors, such as cell type, survival rate, proliferation rate, and gene and protein expression patterns, determine whether a particular cell line can be adapted to a 3D system. The 3D system may overcome some of the limitations of 2D cultures in terms of cell-cell communication and cell networks, which are essential for understanding differentiation, structural organization, shape, and extended connections with other cells or organs. Here, the effect of the anticancer drug cisplatin, also known as cis-diamminedichloroplatinum (II) or CDDP, on adenosine triphosphate (ATP) generation was investigated using 3D spheroid-forming cells and real-time monitoring for 7 days. First, 12 cell lines were screened for their ability to form 3D spheroids: prostate (DU145), testis (F9), embryonic fibroblast (NIH-3T3), muscle (C2C12), embryonic kidney (293T), neuroblastoma (SH-SY5Y), adenocarcinomic alveolar basal epithelial cell (A549), cervical cancer (HeLa), HeLa contaminant (HEp2), pituitary epithelial-like cell (GH3), embryonic cell (PA317), and osteosarcoma (U-2OS) cells. Of these, eight cell lines were selected: NIH-3T3, C2C12, 293T, SH-SY5Y, A549, HeLa, PA317, and U-2OS; and five underwent real-time monitoring of CDDP cytotoxicity: HeLa, A549, 293T, SH-SY5Y, and U-2OS. ATP generation was blocked 1 day after addition of 50 μM CDDP, but cytotoxicity in HeLa, A549, SH-SY5Y, and U-2OS cells could be visualized only 4 days after treatment. In 293T cells, CDDP failed to kill entirely the culture and ATP generation was only partially blocked after 1 day. This suggests potential CDDP resistance of 293T cells or metabolic clearance of the drug. Real-time monitoring and ATP

  8. II. Capsular vaso-mimicry formed by transgenic mammary tumor spheroids implanted ectopically into mouse dorsal skin fold: implications for cellular mechanisms of metastasis

    PubMed Central

    Witkiewicz, Halina

    2013-01-01

    Most cancer patients die of metastatic disease, not primary tumors, while biological mechanisms leading to metastases remain unclear and effective therapies are missing. Using a mouse dorsal skin chamber model we had observed that tumor growth and vasculature formation could be influenced by the way in vitro cultured (avascular) spheroids of N202 breast tumor cells were implanted; co-implantation of lactating breast tissue created stimulating microenvironment, whereas the absence of the graft resulted in temporary tumor dormancy. This report addressed the issue of cellular mechanisms of the vasculogenic switch that ended the dormancy. In situ ultrastructural analysis revealed that the tumors survived in ectopic microenvironment until some of host and tumor stem cells evolved independently into cells initiating the vasculogenic switch. The tumor cells that survived and proliferated under hypoxic conditions for three weeks were supported by erythrogenic autophagy of others. However, the host microenvironment first responded as it would to non-immunogenic foreign bodies, i.e., by encapsulating the tumor spheroids with collagen-producing fibroblasts. That led to a form of vaso-mimicry consisting of tumor cells amid tumor-derived erythrosomes (synonym of erythrocytes), megakaryocytes and platelets, and encapsulating them all, the host fibroblasts. Such capsular vaso-mimicry could potentially facilitate metastasis by fusing with morphologically similar lymphatic vessels or veins. Once incorporated into the host circulatory system, tumor cells could be carried away passively by blood flow, regardless of their genetic heterogeneity. The fake vascular segment would have permeability properties different from genuine vascular endothelium. The capsular vaso-mimicry was different from vasculogenic mimicry earlier observed in metastases-associated malignant tumors where channels formed by tumor cells were said to contain circulating blood. Structures similar to the vasculogenic

  9. Phenylboronic Acid-Mediated Tumor Targeting of Chitosan Nanoparticles

    PubMed Central

    Wang, Xin; Tang, Huang; Wang, Chongzhi; Zhang, Jialiang; Wu, Wei; Jiang, Xiqun

    2016-01-01

    The phenylboronic acid-conjugated chitosan nanoparticles were prepared by particle surface modification. The size, zeta potential and morphology of the nanoparticles were characterized by dynamic light scattering, zeta potential measurement and transmission electron microscopy. The cellular uptake, tumor penetration, biodistribution and antitumor activity of the nanoparticles were evaluated by using monolayer cell model, 3-D multicellular spheroid model and H22 tumor-bearing mice. The incorporation of phenylboronic acid group into chitosan nanoparticles impart a surface charge-reversible characteristic to the nanoparticles. In vitro evaluation using 2-D and 3-D cell models showed that phenylboronic acid-decorated nanoparticles were more easily internalized by tumor cells compared to non-decorated chitosan nanoparticles, and could deliver more drug into tumor cells due to the active targeting effect of boronic acid group. Furthermore, the phenylboronic acid-decorated nanoparticles displayed a deeper penetration and persistent accumulation in the multicellular spheroids, resulting in better inhibition growth to multicellular spheroids than non-decorated nanoparticles. Tumor penetration, drug distribution and near infrared fluorescence imaging revealed that phenylboronic acid-decorated nanoparticles could penetrate deeper and accumulate more in tumor area than non-decorated ones. In vivo antitumor examination demonstrated that the phenylboronic acid-decorated nanoparticles have superior efficacy in restricting tumor growth and prolonging the survival time of tumor-bearing mice than free drug and drug-loaded chitosan nanoparticles. PMID:27375786

  10. Spatial distribution of elements in the spheroids by prostate tumor cells using synchrotron radiation x-ray fluorescence

    NASA Astrophysics Data System (ADS)

    Leitão, Roberta G.; Santos, Carlos Antônio N.; Junior, Antônio Palumbo; Souza, Pedro A. V. R.; Canellas, Catarine G. L.; Anjos, Marcelino J.; Nasciutti, Luiz E.; Lopes, Ricardo T.

    2012-05-01

    The formation of three-dimensional cell microspheres such as spheroids has attracted attention as a useful culture technique. In this study, we investigated the trace elemental distribution (mapping) in spheroids derived from tissue prostate cancer (PCa). The measurements were performed in standard geometry of 45° incidence, exciting with a white beam and using an optical capillary with 20 μm diameter collimation in the XRF beam line at the Synchrotron Light National Laboratory (Campinas, Brazil). The results showed that most elements analyzed presented non-uniform distribution. P, S and Cl showed similar elemental distribution in all the samples analyzed. K, Ca, Fe, and Cu showed different elemental distribution for the spheroids analyzed. Zinc presented more intense distributions in the spheroid central region for all spheroids analyzed.

  11. Spatial distribution of elements in the spheroids by prostate tumor cells using synchrotron radiation x-ray fluorescence

    SciTech Connect

    Leitao, Roberta G.; Santos, Carlos Antonio N.; Junior, Antonio Palumbo; Souza, Pedro A. V. R.; Canellas, Catarine G. L.; Anjos, Marcelino J.; Nasciutti, Luiz E.; Lopes, Ricardo T.

    2012-05-17

    The formation of three-dimensional cell microspheres such as spheroids has attracted attention as a useful culture technique. In this study, we investigated the trace elemental distribution (mapping) in spheroids derived from tissue prostate cancer (PCa). The measurements were performed in standard geometry of 45 deg. incidence, exciting with a white beam and using an optical capillary with 20 {mu}m diameter collimation in the XRF beam line at the Synchrotron Light National Laboratory (Campinas, Brazil). The results showed that most elements analyzed presented non-uniform distribution. P, S and Cl showed similar elemental distribution in all the samples analyzed. K, Ca, Fe, and Cu showed different elemental distribution for the spheroids analyzed. Zinc presented more intense distributions in the spheroid central region for all spheroids analyzed.

  12. Beta-Catenin Regulated ALDH1A1 is a Target in Ovarian Cancer Spheroids

    PubMed Central

    Condello, Salvatore; Morgan, Cynthia A.; Nagdas, Sarbajeet; Cao, Liyun; Turek, John; Hurley, Thomas D.; Matei, Daniela

    2014-01-01

    Cancer cells form three dimensional (3D) multicellular aggregates (or spheroids) under non-adherent culture conditions. In ovarian cancer (OC), spheroids serve as a vehicle for cancer cell dissemination in the peritoneal cavity, protecting cells from environmental stress-induced anoikis. To identify new targetable molecules in OC spheroids, we investigated gene expression profiles and networks upregulated in three dimensional (3D) versus traditional monolayer culture conditions. We identified ALDH1A1, a cancer stem cell marker as being overexpressed in OC spheroids and directly connected to key elements of the β-catenin pathway. B-catenin function and ALDH1A1 expression were increased in OC spheroids vs. monolayers and in successive spheroid generations, suggesting that 3D aggregates are enriched in cells with stem cell characteristics. B-catenin knockdown decreased ALDH1A1 expression levels and β-catenin coimmunoprecipitated with the ALDH1A1 promoter, suggesting that ALDH1A1 is a direct β-catenin target. Both siRNA mediated β-catenin knockdown and A37, a novel ALDH1A1 small molecule enzymatic inhibitor described here for the first time, disrupted OC spheroid formation and cell viability (p<0.001). B-catenin knockdown blocked tumor growth and peritoneal metastasis in an OC xenograft model. These data strongly support the role of β-catenin regulated ALDH1A1 in the maintenance of OC spheroids and propose new ALDH1A1 inhibitors targeting this cell population. PMID:24954508

  13. Looking into Living Cell Systems: Planar Waveguide Microfluidic NMR Detector for in Vitro Metabolomics of Tumor Spheroids.

    PubMed

    Kalfe, Ayten; Telfah, Ahmad; Lambert, Jörg; Hergenröder, Roland

    2015-07-21

    The complex cell metabolism and its link to oncogenic signaling pathways have received huge interest within the last few years. But the lack of advanced analytical tools for the investigation of living cell metabolism is still a challenge to be faced. Therefore, we designed and fabricated a novel miniaturized microslot NMR detector with on-board heater integrated with a microfluidic device as NMR sample holder. For the first time, a tumor spheroid of 500 μm diameter and consisting of 9000 cells has been studied noninvasively and online for 24 h. The dynamic processes of production and degradation of 23 intra- and extracellular metabolites were monitored. Remarkably high concentrations of lactate and alanine were observed, being an indicator for a shift from oxidative to glycolytic metabolism. In summary, this methodical development has proven to be a successful analytical tool for the elucidation of cellular functions and their corresponding biochemical pathways. Additionally, the planar geometry of the microslot NMR detector allows the hyphenation with versatile lab-on-a chip (LOC) technology. This opens a new window for metabolomics studies on living cells and can be implemented into new application fields in biotechnology and life sciences.

  14. Organotypic Culture of Breast Tumor Explants as a Multicellular System for the Screening of Natural Compounds with Antineoplastic Potential

    PubMed Central

    Carranza-Torres, Irma Edith; Guzmán-Delgado, Nancy Elena; Coronado-Martínez, Consuelo; Bañuelos-García, José Inocente; Viveros-Valdez, Ezequiel; Morán-Martínez, Javier; Carranza-Rosales, Pilar

    2015-01-01

    Breast cancer is the leading cause of death in women worldwide. The search for novel compounds with antitumor activity, with less adverse effects and higher efficacy, and the development of methods to evaluate their toxicity is an area of intense research. In this study we implemented the preparation and culture of breast tumor explants, which were obtained from precision-cut breast tumor slices. In order to validate the model we are proposing to screen antineoplastic effect of natural compounds, we selected caffeic acid, ursolic acid, and rosmarinic acid. Using the Krumdieck tissue slicer, precision-cut tissue slices were prepared from breast cancer samples; from these slices, 4 mm explants were obtained and incubated with the selected compounds. Viability was assessed by Alamar Blue assay, LDH release, and histopathological criteria. Results showed that the viability of the explants cultured in the presence of paclitaxel (positive control) decreased significantly (P < 0.05); however, tumor samples responded differently to each compound. When the explants were coincubated with paclitaxel and compounds, a synergic effect was observed. This study shows that ex vivo culture of breast cancer explants offers a suitable alternative model for evaluating natural or synthetic compounds with antitumor properties within the complex microenvironment of the tumor. PMID:26075250

  15. Cetuximab Reconstitutes Pro-Inflammatory Cytokine Secretions and Tumor-Infiltrating Capabilities of sMICA-Inhibited NK Cells in HNSCC Tumor Spheroids

    PubMed Central

    Klöss, Stephan; Chambron, Nicole; Gardlowski, Tanja; Weil, Sandra; Koch, Joachim; Esser, Ruth; Pogge von Strandmann, Elke; Morgan, Michael A.; Arseniev, Lubomir; Seitz, Oliver; Köhl, Ulrike

    2015-01-01

    Immunosuppressive factors, such as soluble major histocompatibility complex class I chain-related peptide A (sMICA) and transforming growth factor beta 1 (TGF-β1), are involved in tumor immune escape mechanisms (TIEMs) exhibited by head and neck squamous cell carcinomas (HNSCCs) and may represent opportunities for therapeutic intervention. In order to overcome TIEMs, we investigated the antibody-dependent cellular cytotoxicity (ADCC), cytokine release and retargeted tumor infiltration of sMICA-inhibited patient NK cells expressing Fcγ receptor IIIa (FcγRIIIa, CD16a) in the presence of cetuximab, an anti-epidermal growth factor receptor (HER1) monoclonal antibody (mAb). Compared to healthy controls, relapsed HNSCC patients (n = 5), not currently in treatment revealed decreased levels of circulating regulatory NK cell subsets in relation to increased cytotoxic NK cell subpopulations. Elevated sMICA and TGF-β1 plasma levels correlated with diminished TNFα and IFN-γ release and decreased NKG2D (natural killer group 2 member D)-dependent killing of HNSCC cells by NK cells. Incubation of IL-2-activated patient NK cells with patient plasma containing elevated sMICA or sMICA analogs (shed MICA and recombinant MICA) significantly impaired NKG2D-mediated killing by down-regulation of NKG2D surface expression. Of note, CD16 surface expression levels, pro-apoptotic and activation markers, and viability of patient and healthy donor NK cell subpopulations were not affected by this treatment. Accordingly, cetuximab restored killing activity of sMICA-inhibited patient NK cells against cetuximab-coated primary HNSCC cells via ADCC in a dose-dependent manner. Rapid reconstitution of anti-tumor recognition and enhanced tumor infiltration of treated NK cells was monitored by 24 h co-incubation of HNSCC tumor spheroids with cetuximab (1 μg/ml) and was characterized by increased IFN-γ and TNFα secretion. This data show that the impaired NK cell-dependent tumor

  16. Spectroscopic imaging system for high-throughput viability assessment of ovarian spheroids or microdissected tumor tissues (MDTs) in a microfluidic chip

    NASA Astrophysics Data System (ADS)

    St-Georges-Robillard, A.; Masse, M.; Kendall-Dupont, J.; Strupler, M.; Patra, B.; Jermyn, M.; Mes-Masson, A.-M.; Leblond, F.; Gervais, T.

    2016-02-01

    There is a growing effort in the biomicrosystems community to develop a personalized treatment response assay for cancer patients using primary cells, patient-derived spheroids, or live tissues on-chip. Recently, our group has developed a technique to cut tumors in 350 μm diameter microtissues and keep them alive on-chip, enabling multiplexed in vitro drug assays on primary tumor tissue. Two-photon microscopy, confocal microscopy and flow cytometry are the current standard to assay tissue chemosensitivity on-chip. While these techniques provide microscopic and molecular information, they are not adapted for high-throughput analysis of microtissues. We present a spectroscopic imaging system that allows rapid quantitative measurements of multiple fluorescent viability markers simultaneously by using a liquid crystal tunable filter to record fluorescence and transmittance spectra. As a proof of concept, 24 spheroids composed of ovarian cancer cell line OV90 were formed in a microfluidic chip, stained with two live cell markers (CellTrackerTM Green and Orange), and imaged. Fluorescence images acquired were normalized to the acquisition time and gain of the camera, dark noise was removed, spectral calibration was applied, and spatial uniformity was corrected. Spectral un-mixing was applied to separate each fluorophore's contribution. We have demonstrated that rapid and simultaneous viability measurements on multiple spheroids can be achieved, which will have a significant impact on the prediction of a tumor's response to multiple treatment options. This technique may be applied as well in drug discovery to assess the potential of a drug candidate directly on human primary tissue.

  17. Novel Morphologic and Genetic Analysis of Cancer Cells in a 3D Microenvironment Identifies STAT3 as a Regulator of Tumor Permeability Barrier Function.

    PubMed

    Park, Min Chul; Jeong, Hyobin; Son, Sung Hwa; Kim, YounHa; Han, Daeyoung; Goughnour, Peter C; Kang, Taehee; Kwon, Nam Hoon; Moon, Hyo Eun; Paek, Sun Ha; Hwang, Daehee; Seol, Ho Jun; Nam, Do-Hyun; Kim, Sunghoon

    2016-03-01

    Tumor permeability is a critical determinant of drug delivery and sensitivity, but systematic methods to identify factors that perform permeability barrier functions in the tumor microenvironment are not yet available. Multicellular tumor spheroids have become tractable in vitro models to study the impact of a three-dimensional (3D) environment on cellular behavior. In this study, we characterized the spheroid-forming potential of cancer cells and correlated the resulting spheroid morphologies with genetic information to identify conserved cellular processes associated with spheroid structure. Spheroids generated from 100 different cancer cell lines were classified into four distinct groups based on morphology. In particular, round and compact spheroids exhibited highly hypoxic inner cores and permeability barriers against anticancer drugs. Through systematic and correlative analysis, we reveal JAK-STAT signaling as one of the signature pathways activated in round spheroids. Accordingly, STAT3 inhibition in spheroids generated from the established cancer cells and primary glioblastoma patient-derived cells altered the rounded morphology and increased drug sensitivity. Furthermore, combined administration of the STAT3 inhibitor and 5-fluorouracil to a mouse xenograft model markedly reduced tumor growth compared with monotherapy. Collectively, our findings demonstrate the ability to integrate 3D culture and genetic profiling to determine the factors underlying the integrity of the permeability barrier in the tumor microenvironment, and may help to identify and exploit novel mechanisms of drug resistance. PMID:26676754

  18. Novel Morphologic and Genetic Analysis of Cancer Cells in a 3D Microenvironment Identifies STAT3 as a Regulator of Tumor Permeability Barrier Function.

    PubMed

    Park, Min Chul; Jeong, Hyobin; Son, Sung Hwa; Kim, YounHa; Han, Daeyoung; Goughnour, Peter C; Kang, Taehee; Kwon, Nam Hoon; Moon, Hyo Eun; Paek, Sun Ha; Hwang, Daehee; Seol, Ho Jun; Nam, Do-Hyun; Kim, Sunghoon

    2016-03-01

    Tumor permeability is a critical determinant of drug delivery and sensitivity, but systematic methods to identify factors that perform permeability barrier functions in the tumor microenvironment are not yet available. Multicellular tumor spheroids have become tractable in vitro models to study the impact of a three-dimensional (3D) environment on cellular behavior. In this study, we characterized the spheroid-forming potential of cancer cells and correlated the resulting spheroid morphologies with genetic information to identify conserved cellular processes associated with spheroid structure. Spheroids generated from 100 different cancer cell lines were classified into four distinct groups based on morphology. In particular, round and compact spheroids exhibited highly hypoxic inner cores and permeability barriers against anticancer drugs. Through systematic and correlative analysis, we reveal JAK-STAT signaling as one of the signature pathways activated in round spheroids. Accordingly, STAT3 inhibition in spheroids generated from the established cancer cells and primary glioblastoma patient-derived cells altered the rounded morphology and increased drug sensitivity. Furthermore, combined administration of the STAT3 inhibitor and 5-fluorouracil to a mouse xenograft model markedly reduced tumor growth compared with monotherapy. Collectively, our findings demonstrate the ability to integrate 3D culture and genetic profiling to determine the factors underlying the integrity of the permeability barrier in the tumor microenvironment, and may help to identify and exploit novel mechanisms of drug resistance.

  19. Stabilizing multicellularity through ratcheting.

    PubMed

    Libby, Eric; Conlin, Peter L; Kerr, Ben; Ratcliff, William C

    2016-08-19

    The evolutionary transition to multicellularity probably began with the formation of simple undifferentiated cellular groups. Such groups evolve readily in diverse lineages of extant unicellular taxa, suggesting that there are few genetic barriers to this first key step. This may act as a double-edged sword: labile transitions between unicellular and multicellular states may facilitate the evolution of simple multicellularity, but reversion to a unicellular state may inhibit the evolution of increased complexity. In this paper, we examine how multicellular adaptations can act as evolutionary 'ratchets', limiting the potential for reversion to unicellularity. We consider a nascent multicellular lineage growing in an environment that varies between favouring multicellularity and favouring unicellularity. The first type of ratcheting mutations increase cell-level fitness in a multicellular context but are costly in a single-celled context, reducing the fitness of revertants. The second type of ratcheting mutations directly decrease the probability that a mutation will result in reversion (either as a pleiotropic consequence or via direct modification of switch rates). We show that both types of ratcheting mutations act to stabilize the multicellular state. We also identify synergistic effects between the two types of ratcheting mutations in which the presence of one creates the selective conditions favouring the other. Ratcheting mutations may play a key role in diverse evolutionary transitions in individuality, sustaining selection on the new higher-level organism by constraining evolutionary reversion.This article is part of the themed issue 'The major synthetic evolutionary transitions'. PMID:27431522

  20. Stabilizing multicellularity through ratcheting.

    PubMed

    Libby, Eric; Conlin, Peter L; Kerr, Ben; Ratcliff, William C

    2016-08-19

    The evolutionary transition to multicellularity probably began with the formation of simple undifferentiated cellular groups. Such groups evolve readily in diverse lineages of extant unicellular taxa, suggesting that there are few genetic barriers to this first key step. This may act as a double-edged sword: labile transitions between unicellular and multicellular states may facilitate the evolution of simple multicellularity, but reversion to a unicellular state may inhibit the evolution of increased complexity. In this paper, we examine how multicellular adaptations can act as evolutionary 'ratchets', limiting the potential for reversion to unicellularity. We consider a nascent multicellular lineage growing in an environment that varies between favouring multicellularity and favouring unicellularity. The first type of ratcheting mutations increase cell-level fitness in a multicellular context but are costly in a single-celled context, reducing the fitness of revertants. The second type of ratcheting mutations directly decrease the probability that a mutation will result in reversion (either as a pleiotropic consequence or via direct modification of switch rates). We show that both types of ratcheting mutations act to stabilize the multicellular state. We also identify synergistic effects between the two types of ratcheting mutations in which the presence of one creates the selective conditions favouring the other. Ratcheting mutations may play a key role in diverse evolutionary transitions in individuality, sustaining selection on the new higher-level organism by constraining evolutionary reversion.This article is part of the themed issue 'The major synthetic evolutionary transitions'.

  1. Stabilizing multicellularity through ratcheting

    PubMed Central

    Libby, Eric; Conlin, Peter L.; Kerr, Ben; Ratcliff, William C.

    2016-01-01

    The evolutionary transition to multicellularity probably began with the formation of simple undifferentiated cellular groups. Such groups evolve readily in diverse lineages of extant unicellular taxa, suggesting that there are few genetic barriers to this first key step. This may act as a double-edged sword: labile transitions between unicellular and multicellular states may facilitate the evolution of simple multicellularity, but reversion to a unicellular state may inhibit the evolution of increased complexity. In this paper, we examine how multicellular adaptations can act as evolutionary ‘ratchets’, limiting the potential for reversion to unicellularity. We consider a nascent multicellular lineage growing in an environment that varies between favouring multicellularity and favouring unicellularity. The first type of ratcheting mutations increase cell-level fitness in a multicellular context but are costly in a single-celled context, reducing the fitness of revertants. The second type of ratcheting mutations directly decrease the probability that a mutation will result in reversion (either as a pleiotropic consequence or via direct modification of switch rates). We show that both types of ratcheting mutations act to stabilize the multicellular state. We also identify synergistic effects between the two types of ratcheting mutations in which the presence of one creates the selective conditions favouring the other. Ratcheting mutations may play a key role in diverse evolutionary transitions in individuality, sustaining selection on the new higher-level organism by constraining evolutionary reversion. This article is part of the themed issue ‘The major synthetic evolutionary transitions’. PMID:27431522

  2. Digital microfluidics for automated hanging drop cell spheroid culture.

    PubMed

    Aijian, Andrew P; Garrell, Robin L

    2015-06-01

    Cell spheroids are multicellular aggregates, grown in vitro, that mimic the three-dimensional morphology of physiological tissues. Although there are numerous benefits to using spheroids in cell-based assays, the adoption of spheroids in routine biomedical research has been limited, in part, by the tedious workflow associated with spheroid formation and analysis. Here we describe a digital microfluidic platform that has been developed to automate liquid-handling protocols for the formation, maintenance, and analysis of multicellular spheroids in hanging drop culture. We show that droplets of liquid can be added to and extracted from through-holes, or "wells," and fabricated in the bottom plate of a digital microfluidic device, enabling the formation and assaying of hanging drops. Using this digital microfluidic platform, spheroids of mouse mesenchymal stem cells were formed and maintained in situ for 72 h, exhibiting good viability (>90%) and size uniformity (% coefficient of variation <10% intraexperiment, <20% interexperiment). A proof-of-principle drug screen was performed on human colorectal adenocarcinoma spheroids to demonstrate the ability to recapitulate physiologically relevant phenomena such as insulin-induced drug resistance. With automatable and flexible liquid handling, and a wide range of in situ sample preparation and analysis capabilities, the digital microfluidic platform provides a viable tool for automating cell spheroid culture and analysis.

  3. I. Embryonal vasculature formation recapitulated in transgenic mammary tumor spheroids implanted pseudo-orthotopicly into mouse dorsal skin fold: the organoblasts concept

    PubMed Central

    Witkiewicz, Halina

    2013-01-01

    Inadequate understanding of cancer biology is a problem. This work focused on cellular mechanisms of tumor vascularization. According to earlier studies, the tumor vasculature derives from host endothelial cells (angiogenesis) or their precursors of bone marrow origin circulating in the blood (neo-vasculogenesis) unlike in embryos. In this study, we observed the neo-vasculature form in multiple ways from local precursor cells. Recapitulation of primitive as well as advanced embryonal stages of vasculature formation followed co-implantation of avascular ( in vitro cultured) N202 breast tumor spheroids and homologous tissue grafts into mouse dorsal skin chambers. Ultrastructural and immunocytochemical analysis of tissue sections exposed the interactions between the tumor and the graft tissue stem cells. It revealed details of vasculature morphogenesis not seen before in either tumors or embryos. A gradual increase in complexity of the vascular morphogenesis at the tumor site reflected a range of steps in ontogenic evolution of the differentiating cells. Malignant- and surgical injury repair-related tissue growth prompted local cells to initiate extramedullar erythropoiesis and vascular patterning. The new findings included: interdependence between the extramedullar hematopoiesis and assembly of new vessels (both from the locally differentiating precursors); nucleo-cytoplasmic conversion (karyolysis) as the mechanism of erythroblast enucleation; the role of megakaryocytes and platelets in vascular pattern formation before emergence of endothelial cells; lineage relationships between hematopoietic and endothelial cells; the role of extracellular calmyrin in tissue morphogenesis; and calmyrite, a new ultrastructural entity associated with anaerobic energy metabolism. The central role of the extramedullar erythropoiesis in the formation of new vasculature (blood and vessels) emerged here as part of the tissue building process including the lymphatic system and nerves

  4. Inhibition of 3-D tumor spheroids by timed-released hydrophilic and hydrophobic drugs from multilayered polymeric microparticles.

    PubMed

    Lee, Wei Li; Guo, Wei Mei; Ho, Vincent H B; Saha, Amitaksha; Chong, Han Chung; Tan, Nguan Soon; Widjaja, Effendi; Tan, Ern Yu; Loo, Say Chye Joachim

    2014-10-15

    First-line cancer chemotherapy necessitates high parenteral dosage and repeated dosing of a combination of drugs over a prolonged period. Current commercially available chemotherapeutic agents, such as Doxil and Taxol, are only capable of delivering single drug in a bolus dose. The aim of this study is to develop dual-drug-loaded, multilayered microparticles and to investigate their antitumor efficacy compared with single-drug-loaded particles. Results show hydrophilic doxorubicin HCl (DOX) and hydrophobic paclitaxel (PTX) localized in the poly(dl-lactic-co-glycolic acid, 50:50) (PLGA) shell and in the poly(l-lactic acid) (PLLA) core, respectively. The introduction of poly[(1,6-bis-carboxyphenoxy) hexane] (PCPH) into PLGA/PLLA microparticles causes PTX to be localized in the PLLA and PCPH mid-layers, whereas DOX is found in both the PLGA shell and core. PLGA/PLLA/PCPH microparticles with denser shells allow better control of DOX release. A delayed release of PTX is observed with the addition of PCPH. Three-dimensional MCF-7 spheroid studies demonstrate that controlled co-delivery of DOX and PTX from multilayered microparticles produces a greater reduction in spheroid growth rate compared with single-drug-loaded particles. This study provides mechanistic insights into how distinctive structure of multilayered microparticles can be designed to modulate the release profiles of anticancer drugs, and how co-delivery can potentially provide better antitumor response.

  5. Tumor Bioengineering Using a Transglutaminase Crosslinked Hydrogel

    PubMed Central

    Fang, Josephine Y.; Tan, Shih-Jye; Yang, Zhi; Tayag, Charisse; Han, Bo

    2014-01-01

    Development of a physiologically relevant 3D model system for cancer research and drug development is a current challenge. We have adopted a 3D culture system based on a transglutaminase-crosslinked gelatin gel (Col-Tgel) to mimic the tumor 3D microenvironment. The system has several unique advantages over other alternatives including presenting cell-matrix interaction sites from collagen-derived peptides, geometry-initiated multicellular tumor spheroids, and metabolic gradients in the tumor microenvironment. Also it provides a controllable wide spectrum of gel stiffness for mechanical signals, and technical compatibility with imaging based screening due to its transparent properties. In addition, the Col-Tgel provides a cure-in-situ delivery vehicle for tumor xenograft formation in animals enhancing tumor cell uptake rate. Overall, this distinctive 3D system could offer a platform to more accurately mimic in vivo situations to study tumor formation and progression both in vitro and in vivo. PMID:25133673

  6. Recreating the Tumor Microenvironment in a Bilayer, Hyaluronic Acid Hydrogel Construct for the Growth of Prostate Cancer Spheroids

    PubMed Central

    Xu, Xian; Gurski, Lisa A.; Zhang, Chu; Harrington, Daniel A.; Farach-Carson, Mary C.; Jia, Xinqiao

    2012-01-01

    Cancer cells cultured in physiologically relevant, three-dimensional (3D) matrices can recapture many essential features of native tumor tissues. In this study, a hyaluronic acid (HA)-based bilayer hydrogel system that not only supports the tumoroid formation from LNCaP prostate cancer (PCa) cells, but also simulates their reciprocal interactions with the tumor-associated stroma was developed and characterized. HA hydrogels were prepared by mixing solutions of HA precursors functionalized with acrylate groups (HA-AC) and reactive thiols (HA-SH) under physiological conditions. The resultant viscoelastic gels have an average elastic modulus of 234 ± 30 Pa and can be degraded readily by hyaluronidase. The orthogonal and cytocompatible nature of the crosslinking chemistry permits facile incorporation of cytokine-releasing particles and PCa cells. In our bilayer hydrogel construct, the top layer contains heparin (HP)-decorated, HA-based hydrogel particles (HGPs) capable of releasing heparin-binding epidermal growth factor-like growth factor (HB-EGF) in a sustained manner at a rate of 2.5wt%/day cumulatively. LNCaP cells embedded in the bottom layer receive the growth factor signals from the top, and in response form enlarging tumoroids with an average diameter of 85 μm by day 7. Cells in 3D hydrogels assemble into spherical tumoroids, form close cellular contacts through E-cadherin, and show cortical organization of F-actin, whereas those plated as 2D monolayers adopt a spread-out morphology. Compared to cells cultured on 2D, the engineered tumoroids significantly increased the expression of two pro-angiogenic factors, vascular endothelial growth factor-165 (VEGF165) and interleukin-8 (IL-8), both at mRNA and protein levels. Overall, the HA model system provides a useful platform for the study of tumor cell responses to growth factors and for screening of anticancer drugs targeting these pathways. PMID:22999468

  7. Games of multicellularity.

    PubMed

    Kaveh, Kamran; Veller, Carl; Nowak, Martin A

    2016-08-21

    Evolutionary game dynamics are often studied in the context of different population structures. Here we propose a new population structure that is inspired by simple multicellular life forms. In our model, cells reproduce but can stay together after reproduction. They reach complexes of a certain size, n, before producing single cells again. The cells within a complex derive payoff from an evolutionary game by interacting with each other. The reproductive rate of cells is proportional to their payoff. We consider all two-strategy games. We study deterministic evolutionary dynamics with mutations, and derive exact conditions for selection to favor one strategy over another. Our main result has the same symmetry as the well-known sigma condition, which has been proven for stochastic game dynamics and weak selection. For a maximum complex size of n=2 our result holds for any intensity of selection. For n≥3 it holds for weak selection. As specific examples we study the prisoner's dilemma and hawk-dove games. Our model advances theoretical work on multicellularity by allowing for frequency-dependent interactions within groups. PMID:27179461

  8. The effect of motexafin gadolinium on ALA photodynamic therapy in glioma spheroids

    NASA Astrophysics Data System (ADS)

    Mathews, Marlon S.; Sanchez, Rogelio; Sun, Chung-Ho; Madsen, Steen J.; Hirschberg, Henry

    2008-02-01

    Following surgical removal of malignant brain tumors 80% of all cases develop tumor recurrence within 2 cm of the resected margin. The aim of postoperative therapy is therefore elimination of nests of tumor cells remaining in the margins of the resection cavity. Light attenuation in tissue makes it difficult for adequate light fluences to reach depths of 1-2 cm in the resection margin making it difficult for standard intraoperative photodynamic therapy (PDT) to accomplish this goal. Thus additional agents are required that either increase the efficacy of low fluence PDT or inhibit cellular repair, to enhance effectiveness of PDT in the tumor resection cavity. Motexafin gadolinium (MGd) is one such agent previously reported to enhance the cytotoxic potential of radiation therapy, as well as several chemotherapeutic agents by causing redox stress to cancerous cells. MGd is well tolerated with tumor specific uptake in clinical studies. The authors evaluated MGd as a potential PDT enhancing agent at low light fluences using an in vitro model. Multicellular Glioma spheroids (MGS) of approximately 300 micron diameter, obtained from ACBT cell lines were subjected to acute PDT treatments at 6J, 12J, and 18J light fluences. Growth was determined by measuring diameters in two axes. At four weeks a dose dependent inhibition of spheroid growth was seen in 33%, 55%, and 83% of the MGS at 6J, 12J, and 18J respectively, while inhibition followed by a partial reversal of growth was seen in 17%, 33%, and 17% respectively. This study provides a rationale for the use of this drug as a PDT enhancer in the management of brain tumors.

  9. Investigating the effects of combined photodynamic and anti-angiogenic therapies using a three-dimensional in-vivo brain tumor system

    NASA Astrophysics Data System (ADS)

    De Magalhães, Nzola; Liaw, Lih-Huei L.; Li, Linda; Liogys, Angela; Madsen, Steen J.; Hirschberg, Henry; Tromberg, Bruce J.

    2006-02-01

    An in-vivo tumor model composed of multicellular human glioma spheroids implanted on a shell-less chorioallantoic membrane (CAM), has been developed. Following removal of a portion of the ectodermal epithelium layer of the CAM, human glioma spheroids were implanted on day 7 of embryonic development. Tumor invasion, rapid growth and vasculature formation were observed 7 days post implantation. Single tumor cell migration towards blood vessels, angiogenesis and satellite tumor growth were also evident. The human tumor/CAM model is being used to examine the effects of combined ALA PDT and anti-angiogenic agents. The shell-less CAM is well suited for topical, i.p. and i.v. photosensitizer and/or drug application.

  10. Analysis of Gene Expression in 3D Spheroids Highlights a Survival Role for ASS1 in Mesothelioma

    PubMed Central

    Barbone, Dario; Van Dam, Loes; Follo, Carlo; Jithesh, Puthen V.; Zhang, Shu-Dong; Richards, William G.; Bueno, Raphael; Fennell, Dean A.; Broaddus, V. Courtney

    2016-01-01

    To investigate the underlying causes of chemoresistance in malignant pleural mesothelioma, we have studied mesothelioma cell lines as 3D spheroids, which acquire increased chemoresistance compared to 2D monolayers. We asked whether the gene expression of 3D spheroids would reveal mechanisms of resistance. To address this, we measured gene expression of three mesothelioma cell lines, M28, REN and VAMT, grown as 2D monolayers and 3D spheroids. A total of 209 genes were differentially expressed in common by the three cell lines in 3D (138 upregulated and 71 downregulated), although a clear resistance pathway was not apparent. We then compared the list of 3D genes with two publicly available datasets of gene expression of 56 pleural mesotheliomas compared to normal tissues. Interestingly, only three genes were increased in both 3D spheroids and human tumors: argininosuccinate synthase 1 (ASS1), annexin A4 (ANXA4) and major vault protein (MVP); of these, ASS1 was the only consistently upregulated of the three genes by qRT-PCR. To measure ASS1 protein expression, we stained 2 sets of tissue microarrays (TMA): one with 88 pleural mesothelioma samples and the other with additional 88 pleural mesotheliomas paired with matched normal tissues. Of the 176 tumors represented on the two TMAs, ASS1 was expressed in 87 (50%; staining greater than 1 up to 3+). For the paired samples, ASS1 expression in mesothelioma was significantly greater than in the normal tissues. Reduction of ASS1 expression by siRNA significantly sensitized mesothelioma spheroids to the pro-apoptotic effects of bortezomib and of cisplatin plus pemetrexed. Although mesothelioma is considered by many to be an ASS1-deficient tumor, our results show that ASS1 is elevated at the mRNA and protein levels in mesothelioma 3D spheroids and in human pleural mesotheliomas. We also have uncovered a survival role for ASS1, which may be amenable to targeting to undermine mesothelioma multicellular resistance. PMID:26982031

  11. Abnormal hepatic copper accumulation of spheroid composed of liver cells from LEC rats in vitro.

    PubMed

    Ueno, K; Yoshizawa, M; Satoh, T; Yoneda, S; Ohmichi, M; Yamazaki, M; Mori, Y; Suzuki, K T

    1995-11-01

    The LEC rat is a mutant strain displaying hereditary hepatitis, and shows abnormal accumulation of copper (Cu) similar to that occurring in Wilson's disease. We prepared a multicellular spheroid composed of LEC rat liver cells to investigate the mechanism for abnormal accumulation of Cu. These multicellular spheroids were prepared by detaching the monolayer on the collagen-conjugated thermo-responsive polymer coated culture dish at a temperature below the critical solution temperature and culturing on the non-adhesive substratum. Long-term cultured spheroids of LEC rat liver cells as well as SD rat liver cells were attempted. Non-parenchymal cells obtained by collagenase perfusion from the LEC liver were fewer than those from the SD liver. Cells from the LEC rat, over 11 weeks of age, did not form a cell sheet; however, a mixture of parenchymal cells from LEC rats over aged 11 weeks and non-parenchymal cells from SD rats of any age yielded intact spheroids. We examined the toxicity, the accumulation and distribution of Cu in spheroids. The accumulation of Cu in LEC spheroids was higher than that in SD spheroids. Results suggest that spheroids consisting of LEC liver cells are useful as an alternative model to in vivo tests to investigate the mechanism for abnormal accumulation of Cu in liver.

  12. Monoclonal antibodies directed against surface molecules of multicell spheroids

    NASA Technical Reports Server (NTRS)

    Martinez, Andrew O.

    1994-01-01

    The objective of this project is to generate a library of monoclonial antibodies (MAbs) directed against surface molecules of tumor and transformed cells grown as multicell spheroids (MCS). These MCS are highly organized, 3-dimensional multicellular structures which exhibit many characteristics of in vivo organized tissues which are not found in conventional monolayer or suspension culture. In brief, MCS combine the relevance or organized tissues with in vitro methodology making the MCS a good model system to study the interactions of mammalian cells, and thereby provide a functional assay for surface adhesion molecules. This project also involves investigations of cell-cell interactions in a gravity-based environment. It will provide an important base of scientific information for future comparative studies on the effects of hypergravity and simulated microgravity environments on cell-cell interactions. This project also has the potential to yield important materials (e.g. cellular products) which may be useful for the diagnosis and/or treatment of certain human diseases. Moreover, this project supports the training of one undergraduate and one graduate student; thus, it will also assist in developing a pool of future scientists with research experience in gravitational biology research.

  13. Monoclonal antibodies directed against surface molecules of multicell spheroids

    NASA Technical Reports Server (NTRS)

    Martinez, Andrew O.

    1994-01-01

    The objective of this project is to generate a library of monoclonal antibodies (MAbs) directed against surface molecules of tumor and transformed cells grown as multicell spheroids (MCS). These MCS are highly organized, 3-dimensional multicellular structures which exhibit many characteristics of in vivo organized tissues not found in conventional monolayer or suspension culture. Therefore MCS make better in vitro model systems to study the interactions of mammalian cells, and provide a functional assay for surface adhesion molecules. This project also involves investigations of cell-cell interactions in a gravity-based environment. It will provide a base of scientific information necessary to expand the focus of the project in future years to microgravity and hypergravity-based environments. This project also has the potential to yield important materials (e.g., cellular products) which may prove useful in the diagnosis and/or treatment of certain human diseases. Moreover, this project supports the training of both undergraduate and graduate students; thus, it will assist in developing a pool of future scientists with research experience in an area (gravitational biology) of interest to NASA.

  14. Spherical Cancer Models in Tumor Biology1

    PubMed Central

    Weiswald, Louis-Bastien; Bellet, Dominique; Dangles-Marie, Virginie

    2015-01-01

    Three-dimensional (3D) in vitro models have been used in cancer research as an intermediate model between in vitro cancer cell line cultures and in vivo tumor. Spherical cancer models represent major 3D in vitro models that have been described over the past 4 decades. These models have gained popularity in cancer stem cell research using tumorospheres. Thus, it is crucial to define and clarify the different spherical cancer models thus far described. Here, we focus on in vitro multicellular spheres used in cancer research. All these spherelike structures are characterized by their well-rounded shape, the presence of cancer cells, and their capacity to be maintained as free-floating cultures. We propose a rational classification of the four most commonly used spherical cancer models in cancer research based on culture methods for obtaining them and on subsequent differences in sphere biology: the multicellular tumor spheroid model, first described in the early 70s and obtained by culture of cancer cell lines under nonadherent conditions; tumorospheres, a model of cancer stem cell expansion established in a serum-free medium supplemented with growth factors; tissue-derived tumor spheres and organotypic multicellular spheroids, obtained by tumor tissue mechanical dissociation and cutting. In addition, we describe their applications to and interest in cancer research; in particular, we describe their contribution to chemoresistance, radioresistance, tumorigenicity, and invasion and migration studies. Although these models share a common 3D conformation, each displays its own intrinsic properties. Therefore, the most relevant spherical cancer model must be carefully selected, as a function of the study aim and cancer type. PMID:25622895

  15. Biological soliton in multicellular movement

    NASA Astrophysics Data System (ADS)

    Kuwayama, Hidekazu; Ishida, Shuji

    2013-07-01

    Solitons have been observed in various physical phenomena. Here, we show that the distinct characteristics of solitons are present in the mass cell movement of non-chemotactic mutants of the cellular slime mould Dictyostelium discoideum. During starvation, D. discoideum forms multicellular structures that differentiate into spore or stalk cells and, eventually, a fruiting body. Non-chemotactic mutant cells do not form multicellular structures; however, they do undergo mass cell movement in the form of a pulsatile soliton-like structure (SLS). We also found that SLS induction is mediated by adhesive cell-cell interactions. These observations provide novel insights into the mechanisms of biological solitons in multicellular movement.

  16. Communication theory and multicellular biology.

    PubMed

    Mian, I S; Rose, C

    2011-04-01

    In this Perspective, we propose that communication theory--a field of mathematics concerned with the problems of signal transmission, reception and processing--provides a new quantitative lens for investigating multicellular biology, ancient and modern. What underpins the cohesive organisation and collective behaviour of multicellular ecosystems such as microbial colonies and communities (microbiomes) and multicellular organisms such as plants and animals, whether built of simple tissue layers (sponges) or of complex differentiated cells arranged in tissues and organs (members of the 35 or so phyla of the subkingdom Metazoa)? How do mammalian tissues and organs develop, maintain their architecture, become subverted in disease, and decline with age? How did single-celled organisms coalesce to produce many-celled forms that evolved and diversified into the varied multicellular organisms in existence today? Some answers can be found in the blueprints or recipes encoded in (epi)genomes, yet others lie in the generic physical properties of biological matter such as the ability of cell aggregates to attain a certain complexity in size, shape, and pattern. We suggest that Lasswell's maxim "Who says what to whom in what channel with what effect" provides a foundation for understanding not only the emergence and evolution of multicellularity, but also the assembly and sculpting of multicellular ecosystems and many-celled structures, whether of natural or human-engineered origin. We explore how the abstraction of communication theory as an organising principle for multicellular biology could be realised. We highlight the inherent ability of communication theory to be blind to molecular and/or genetic mechanisms. We describe selected applications that analyse the physics of communication and use energy efficiency as a central tenet. Whilst communication theory has and could contribute to understanding a myriad of problems in biology, investigations of multicellular biology

  17. Kinetic approach and estimation of the parameters of cellular interaction between the immunity system and a tumor.

    PubMed

    Kuznetsov, V A; Zhivoglyadov, V P; Stepanova, L A

    1993-01-01

    A method is suggested to estimate multi component dynamic systems, which permits, with the help of the computer-calculated kinetic curves, to obtain information about the possible mechanisms of the system component interaction. The method is based on the structural and parametrical identification of mathematical models presented in the form of a system of nonlinear differential equations, using a multi-criterial approach. Using experimental data of studies on growth kinetics and regression of multicellular tumor EMT6 line spheroids in the mouse allogenic system and the immune system cell accumulation in spheroids a mathematical model has been developed of the cellular interaction process in a spheroid. It has been stated that the rate of macrophage and neutrophil accumulation in a spheroid depends on the amount of tumor cells and is determined by the hyperbolic law (as analogous to the Michaelis-Menthen kinetics), while the accumulation of immune lymphocytes in a tumor is determined besides that by the three-cellular cooperation of lymphocytes, macrophages and tumor cells. According to the model, the inhibition of the process of neutrophil and lymphocyte (but not of macrophages) accumulation is realized through the auto-suppression mechanism. The numerical values of the process parameters, which characterise the rates of accumulation, cellular death in a tumor and of local cellular interactions intensity are obtained. PMID:8239905

  18. Development of Three-Dimensional Multicellular Tissue-Like Constructs for Mutational Analysis Using Macroporous Microcarriers

    NASA Technical Reports Server (NTRS)

    Jordan, Jacqueline A.; Fraga, Denise N.; Gonda, Steve R.

    2002-01-01

    A three-dimensional (3-D), tissue-like model was developed for the genotoxic assessment of space environment. In previous experiments, we found that culturing mammalian cells in a NASA-designed bioreactor, using Cytodex-3 beads as a scaffold, generated 3-D multicellular spheroids. In an effort to generate scaffold-free spheroids, we developed a new 3-D tissue-like model by coculturing fibroblast and epithelial cell in a NASA bioreactor using macroporous Cultispher-S(TradeMark) microcarriers. Big Blue(Registered Trademark) Rat 2(Lambda) fibroblasts, genetically engineered to contain multiple copies (>60 copies/cell) of the Lac I target gene, were cocultured with radio-sensitive human epithelial cells, H184F5. Over an 8-day period, samples were periodically examined by microscopy and histology to confirm cell attachment, growth, and viability. Immunohistochemistry and western analysis were used to evaluate the expression of specific cytoskeletal and adhesion proteins. Key cell culture parameters (glucose, pH, and lactate concentrations) were monitored daily. Controls were two-dimensional mono layers of fibroblast or epithelial cells cultured in T-flasks. Analysis of 3-D spheroids from the bioreactor suggests fibroblast cells attached to and completely covered the bead surface and inner channels by day 3 in the bioreactor. Treatment of the 3-day spheroids with dispase II dissolved the Cultisphers(TradeMark) and produced multicellular, bead-less constructs. Immunohistochemistry confirmed the presence of vi.mentin, cytokeratin and E-cadherin in treated spheroids. Examination of the dispase II treated spheroids with transmission electron microscopy (TEM) also showed the presence of desmosomes. These results suggest that the controlled enzymatic degradation of an artificial matrix in the low shear environment of the NASA-designed bioreactor can produce 3-D tissue-like spheroids. 2

  19. Protein families in multicellular organisms.

    PubMed

    Copley, R R; Schultz, J; Ponting, C P; Bork, P

    1999-06-01

    The complete sequence of the nematode worm Caenorhabditis elegans contains the genetic machinery that is required to undertake the core biological processes of single cells. However, the genome also encodes proteins that are associated with multicellularity, as well as others that are lineage-specific expansions of phylogenetically widespread families and yet more that are absent in non-nematodes. Ongoing analysis is beginning to illuminate the similarities and differences among human proteins and proteins that are encoded by the genomes of the multicellular worm and the unicellular yeast, and will be essential in determining the reliability of transferring experimental data among phylogenetically distant species. PMID:10361098

  20. Magnetic manipulation and spatial patterning of multi-cellular stem cell aggregates†

    PubMed Central

    Bratt-Leal, Andrés M.; Kepple, Kirsten L.; Carpenedo, Richard L.; Cooke, Marissa T.; McDevitt, Todd C.

    2015-01-01

    The controlled assembly and organization of multi-cellular systems to mimic complex tissue structures is critical to the engineering of tissues for therapeutic and diagnostic applications. Recent advances in micro-scale technologies to control multi-cellular aggregate formation typically require chemical modification of the interface between cells and materials and lack multi-scale flexibility. Here we demonstrate that simple physical entrapment of magnetic microparticles within the extracellular space of stem cells spheroids during initial formation enables scaffold-free immobilization, translocation and directed assembly of multi-cellular aggregates across multiple length and time scales, even under dynamic suspension culture conditions. The response of aggregates to externally applied magnetic fields was a direct function of microparticle incorporation, allowing for rapid and transient control of the extracellular environment as well as separation of heterogeneous populations. In addition, spatial patterning of heterogeneous spheroid populations as well as individual multi-cellular aggregates was readily achieved by imposing temporary magnetic fields. Overall, this approach provides novel routes to examine stem cell differentiation and tissue morphogenesis with applications that encompass the creation of new model systems for developmental biology, scaffold-free tissue engineering strategies and scalable bioprocessing technologies. PMID:22076329

  1. Method to measure the radio and chemosensitivity of human spheroids

    SciTech Connect

    Carlsson, J.; Nederman, T.

    1983-01-01

    A method based on the spontaneous outgrowth of cells from spheroids was tested. Different outgrowth patterns were seen depending on the types of spheroids and on the radiation or drug doses. The method allowed dose-effect relations to be determined. Spheroid survival was defined as when the outgrowing monolayers contained at least thousand cells within five weeks. The method was used as an alternative to cloning of isolated single cells. The glioma and osteosarcoma spheroids could not be disintegrated to single cell suspensions since they resisted enzymatic and mechanical treatments for cell separation. Detection of differences in radio and chemosensitivity between different types of spheroids of human origin might be valuable for the understanding of the large variations in therapeutical response often seen between different types of tumors.

  2. Stiffening of Human Mesenchymal Stem Cell Spheroid Microenvironments Induced by Incorporation of Gelatin Microparticles

    PubMed Central

    Baraniak, Priya R.; Cooke, Marissa T.; Saeed, Rabbia; Kinney, Melissa A.; Fridley, Krista M.; McDevitt, Todd C.

    2012-01-01

    Culturing multipotent adult mesenchymal stem cells as 3D aggregates augments their differentiation potential and paracrine activity. One caveat of stem cell spheroids, though, can be the limited diffusional transport barriers posed by the inherent 3D structure of the multicellular aggregates. In order to circumvent such limitations, polymeric microparticles have been incorporated into stem cell aggregates as a means to locally control the biochemical and physical properties of the 3D microenvironment. However, the introduction of biomaterials to the 3D stem cell microenvironment could alter the mechanical forces sensed by cells within aggregates, which in turn could impact various cell behaviors and overall spheroid mechanics. Therefore, the objective of this study was to determine the acute effects of biomaterial incorporation within mesenchymal stem cell spheroids on aggregate structure and mechanical properties. The results of this study demonstrate that although gelatin microparticle incorporation results in similar multi-cellular organization within human mesenchymal stem cell spheroids, the introduction of gelatin materials significantly impacts spheroid mechanical properties. The marked differences in spheroid mechanics induced by microparticle incorporation may hold major implications for in vitro directed differentiation strategies and offer a novel route to engineer the mechanical properties of tissue constructs ex vivo. PMID:22658155

  3. Light Scattering by Spheroids

    NASA Astrophysics Data System (ADS)

    Xie, Ya-Ming; Ji, Xia

    Nowadays, with the development of technology, particles with size at nanoscale have been synthesized in experiments. It is noticed that anisotropy is an unavoidable problem in the production of nanospheres. Besides, nonspherical nanoparticles have also been extensively used in experiments. Comparing with spherical model, spheroidal model can give a better description for the characteristics of nonspherical particles. Thus the study of analytical solution for light scattering by spheroidal particles has practical implications. By expanding incident, scattered, and transmitted electromagnetic fields in terms of appropriate vector spheroidal wave functions, an analytic solution is obtained to the problem of light scattering by spheroids. Unknown field expansion coefficients can be determined with the combination of boundary conditions and rotational-translational addition theorems for vector spheroidal wave functions. Based on the theoretical derivation, a Fortran code has been developed to calculate the extinction cross section and field distribution, whose results agree well with those obtain by FDTD simulation. This research is supported by the National Natural Science Foundation of China No. 91230203.

  4. How 5000 independent rowers coordinate their strokes in order to row into the sunlight: Phototaxis in the multicellular green alga Volvox

    PubMed Central

    2010-01-01

    Background The evolution of multicellular motile organisms from unicellular ancestors required the utilization of previously evolved tactic behavior in a multicellular context. Volvocine green algae are uniquely suited for studying tactic responses during the transition to multicellularity because they range in complexity from unicellular to multicellular genera. Phototactic responses are essential for these flagellates because they need to orientate themselves to receive sufficient light for photosynthesis, but how does a multicellular organism accomplish phototaxis without any known direct communication among cells? Several aspects of the photoresponse have previously been analyzed in volvocine algae, particularly in the unicellular alga Chlamydomonas. Results In this study, the phototactic behavior in the spheroidal, multicellular volvocine green alga Volvox rousseletii (Volvocales, Chlorophyta) was analyzed. In response to light stimuli, not only did the flagella waveform and beat frequency change, but the effective stroke was reversed. Moreover, there was a photoresponse gradient from the anterior to the posterior pole of the spheroid, and only cells of the anterior hemisphere showed an effective response. The latter caused a reverse of the fluid flow that was confined to the anterior hemisphere. The responsiveness to light is consistent with an anterior-to-posterior size gradient of eyespots. At the posterior pole, the eyespots are tiny or absent, making the corresponding cells appear to be blind. Pulsed light stimulation of an immobilized spheroid was used to simulate the light fluctuation experienced by a rotating spheroid during phototaxis. The results demonstrated that in free-swimming spheroids, only those cells of the anterior hemisphere that face toward the light source reverse the beating direction in the presence of illumination; this behavior results in phototactic turning. Moreover, positive phototaxis is facilitated by gravitational forces. Under

  5. Reproducibility of Uniform Spheroid Formation in 384-Well Plates: The Effect of Medium Evaporation.

    PubMed

    Das, Viswanath; Fürst, Tomáš; Gurská, Soňa; Džubák, Petr; Hajdúch, Marián

    2016-10-01

    Spheroid cultures of cancer cells reproduce the spatial dimension-induced in vivo tumor traits more effectively than the conventional two-dimensional cell cultures. With growing interest in spheroids for high-throughput screening (HTS) assays, there is an increasing demand for cost-effective miniaturization of reproducible spheroids in microtiter plates (MPs). However, well-to-well variability in spheroid size, shape, and growth is a frequently encountered problem with almost every culture method that has prevented the transfer of spheroids to the HTS platform. This variability partly arises due to increased susceptibility of MPs to edge effects and evaporation-induced changes in the growth of spheroids. In this study, we examined the effect of evaporation on the reproducibility of spheroids of tumor and nontumor cell lines in 384-well plates, and show that culture conditions that prevent evaporation-induced medium loss result in the formation of uniform spheroids across the plate. Additionally, we also present a few technical improvements to increase the scalability of the liquid-overlay spheroid culturing technique in MPs, together with a simple software routine for the quantification of spheroid size. We believe that these cost-effective improvements will aid in further improvement of spheroid cultures for HTS drug discovery.

  6. Effects of photodynamic therapy on human glioma spheroids

    NASA Astrophysics Data System (ADS)

    Madsen, Steen J.; Sun, Chung-Ho; Chu, Eugene A.; Hirschberg, Henry; Tromberg, Bruce J.

    1999-07-01

    The poor prognosis for patients with malignant brain neoplasm has led to a search for better treatment modalities. Although gliomas are considered to be disseminated tumors in the brain, most recur at the site of the previous tumor resection. Improved local control would thus be of clear benefit. The utility of photodynamic therapy (PDT) in the treatment of brain neoplasms is investigated using a human glioma spheroid model. Specifically, the effects of PDT on human glioma spheroids are investigated using PhotofrinTM and 56-aminolevulinic acid (ALA). The effects of various irradiation schemes were monitored using a simple growth assay. A growth delay was observed at an optical fluence of approximately 35 J cm-2 for spheroids incubated in Photofrin. Spheroids incubated in ALA were unaffected by the PDT treatment regimens examined in this study. This was most likely a result of inadequate photosensitizer concentration.

  7. On The Evolution of Bacterial Multicellularity

    PubMed Central

    Lyons, Nicholas A.; Kolter, Roberto

    2015-01-01

    Multicellularity is one of the most prevalent evolutionary innovations and nowhere is this more apparent than in the bacterial world, which contains many examples of multicellular organisms in a surprising array of forms. Due to their experimental accessibility and the large and diverse genomic data available, bacteria enable us to probe fundamental aspects of the origins of multicellularity. Here we discuss examples of multicellular behaviors in bacteria, the selective pressures that may have led to their evolution, possible origins and intermediate stages, and whether the ubiquity of apparently convergent multicellular forms argues for its inevitability. PMID:25597443

  8. Extracellular matrix composition and rigidity regulate invasive behavior and response to PDT in 3D pancreatic tumor models

    NASA Astrophysics Data System (ADS)

    Cramer, Gwendolyn; El-Hamidi, Hamid; Jafari, Seyedehrojin; Jones, Dustin P.; Celli, Jonathan P.

    2016-03-01

    The composition and mechanical compliance of the extracellular matrix (ECM) have been shown to serve as regulators of tumor growth and invasive behavior. These effects may be particularly relevant in tumors of the pancreas, noted for a profound desmoplastic reaction and an abundance of stroma rich in ECM. In view of recent progress in the clinical implementation of photodynamic therapy (PDT) for pancreatic tumors, in this report we examine how ECM composition and rheological properties impact upon invasive behavior and response to PDT in 3D multicellular pancreatic tumor spheroids in ECM environments with characterized rheological properties. Tumor spheroids were cultured initially in attachment-free conditions to form millimeter-sized spheroids that were transplanted into reconstituted ECM microenvironments (Matrigel and Type I Collagen) that were characterized using bulk oscillatory shear rheology. Analysis of growth behavior shows that the soft collagen ECM promoted growth and extensive invasion and this microenvironment was used in subsequent assessment of PDT and chemotherapy response. Evaluation of treatment response revealed that primary tumor nodule growth is inhibited more effectively with PDT, while verteporfin PDT response is significantly enhanced in the ECM-infiltrating populations that are non-responsive to oxaliplatin chemotherapy. This finding is potentially significant, suggesting the potential for PDT to target these clinically problematic invasive populations that are associated with aggressive metastatic progression and chemoresistance. Experiments to further validate and identify the mechanistic basis of this observation are ongoing.

  9. Monoclonal antibodies directed against surface molecules of multicell spheroids

    NASA Technical Reports Server (NTRS)

    Martinez, Andrew O.

    1993-01-01

    The objective of this project is to generate a library of monoclonal antibodies (MAb's) to surface molecules involved in the cell-cell interactions of mammalian cells grown as multicell spheroids (MCS). MCS are highly organized 3-dimensional multicellular structures which exhibit many characteristics in vivo tissues not found in conventional monolayer or suspension culture. They also provide a functional assay for surface adhesion molecules. In brief, MCS combine the relevance of organized tissues with the accuracy of in vitro methodology. Further, one can manipulate these MCS experimentally to discern important information about their biology.

  10. Directed Self-Assembly of Large Scaffold-free Multicellular Honeycomb Structures

    PubMed Central

    Tejavibulya, Nalin; Youssef, Jacquelyn; Bao, Brian; Ferruccio, Toni-Marie; Morgan, Jeffrey R.

    2011-01-01

    A significant challenge to the field of biofabrication is the rapid construction of large three dimensional (3D) living tissues and organs. Multi-cellular spheroids have been used as building blocks. In this paper, we create large multi-cellular honeycomb building blocks using directed self-assembly, whereby cell-to-cell adhesion, in the context of the shape and obstacles of a micromold, drives the formation of a 3D structure. Computer aided design, rapid prototyping and replica molding were used to fabricate honeycomb-shaped micro-molds. Nonadhesive hydrogels cast from these micro-molds were equilibrated in cell culture medium and seeded with two types of mammalian cells. The cells settled into the honeycomb recess, were unable to attach to the nonadhesive hydrogel and so cell-to-cell adhesion drove the self-assembly of a large multicellular honeycomb within 24 hours. Distinct morphological changes occurred to the honeycomb and its cells indicating the presence of significant cell-mediated tension. Unlike the spheroid, whose size is constrained by a critical diffusion distance needed to maintain cell viability, the overall size of the honeycomb is not limited. The rapid production of the honeycomb building unit, with its multiple rings of high density cells and open lumen spaces, offers interesting new possibilities for biofabrication strategies. PMID:21828905

  11. Constraint Based Modeling Going Multicellular

    PubMed Central

    Martins Conde, Patricia do Rosario; Sauter, Thomas; Pfau, Thomas

    2016-01-01

    Constraint based modeling has seen applications in many microorganisms. For example, there are now established methods to determine potential genetic modifications and external interventions to increase the efficiency of microbial strains in chemical production pipelines. In addition, multiple models of multicellular organisms have been created including plants and humans. While initially the focus here was on modeling individual cell types of the multicellular organism, this focus recently started to switch. Models of microbial communities, as well as multi-tissue models of higher organisms have been constructed. These models thereby can include different parts of a plant, like root, stem, or different tissue types in the same organ. Such models can elucidate details of the interplay between symbiotic organisms, as well as the concerted efforts of multiple tissues and can be applied to analyse the effects of drugs or mutations on a more systemic level. In this review we give an overview of the recent development of multi-tissue models using constraint based techniques and the methods employed when investigating these models. We further highlight advances in combining constraint based models with dynamic and regulatory information and give an overview of these types of hybrid or multi-level approaches. PMID:26904548

  12. A Synthetic Multicellular Memory Device.

    PubMed

    Urrios, Arturo; Macia, Javier; Manzoni, Romilde; Conde, Núria; Bonforti, Adriano; de Nadal, Eulàlia; Posas, Francesc; Solé, Ricard

    2016-08-19

    Changing environments pose a challenge to living organisms. Cells need to gather and process incoming information, adapting to changes in predictable ways. This requires in particular the presence of memory, which allows different internal states to be stored. Biological memory can be stored by switches that retain information on past and present events. Synthetic biologists have implemented a number of memory devices for biological applications, mostly in single cells. It has been shown that the use of multicellular consortia provides interesting advantages to implement biological circuits. Here we show how to build a synthetic biological memory switch using an eukaryotic consortium. We engineered yeast cells that can communicate and retain memory of changes in the extracellular environment. These cells were able to produce and secrete a pheromone and sense a different pheromone following NOT logic. When the two strains were cocultured, they behaved as a double-negative-feedback motif with memory. In addition, we showed that memory can be effectively changed by the use of external inputs. Further optimization of these modules and addition of other cells could lead to new multicellular circuits that exhibit memory over a broad range of biological inputs. PMID:27439436

  13. Constraint Based Modeling Going Multicellular.

    PubMed

    Martins Conde, Patricia do Rosario; Sauter, Thomas; Pfau, Thomas

    2016-01-01

    Constraint based modeling has seen applications in many microorganisms. For example, there are now established methods to determine potential genetic modifications and external interventions to increase the efficiency of microbial strains in chemical production pipelines. In addition, multiple models of multicellular organisms have been created including plants and humans. While initially the focus here was on modeling individual cell types of the multicellular organism, this focus recently started to switch. Models of microbial communities, as well as multi-tissue models of higher organisms have been constructed. These models thereby can include different parts of a plant, like root, stem, or different tissue types in the same organ. Such models can elucidate details of the interplay between symbiotic organisms, as well as the concerted efforts of multiple tissues and can be applied to analyse the effects of drugs or mutations on a more systemic level. In this review we give an overview of the recent development of multi-tissue models using constraint based techniques and the methods employed when investigating these models. We further highlight advances in combining constraint based models with dynamic and regulatory information and give an overview of these types of hybrid or multi-level approaches.

  14. Constraint Based Modeling Going Multicellular.

    PubMed

    Martins Conde, Patricia do Rosario; Sauter, Thomas; Pfau, Thomas

    2016-01-01

    Constraint based modeling has seen applications in many microorganisms. For example, there are now established methods to determine potential genetic modifications and external interventions to increase the efficiency of microbial strains in chemical production pipelines. In addition, multiple models of multicellular organisms have been created including plants and humans. While initially the focus here was on modeling individual cell types of the multicellular organism, this focus recently started to switch. Models of microbial communities, as well as multi-tissue models of higher organisms have been constructed. These models thereby can include different parts of a plant, like root, stem, or different tissue types in the same organ. Such models can elucidate details of the interplay between symbiotic organisms, as well as the concerted efforts of multiple tissues and can be applied to analyse the effects of drugs or mutations on a more systemic level. In this review we give an overview of the recent development of multi-tissue models using constraint based techniques and the methods employed when investigating these models. We further highlight advances in combining constraint based models with dynamic and regulatory information and give an overview of these types of hybrid or multi-level approaches. PMID:26904548

  15. Prolate spheroidal quantum cloak

    SciTech Connect

    Syue, Cheng-De; Lin, De-Hone

    2015-04-15

    To understand the propagation behavior of an oblique incident matter wave in a three-dimensional non-spherical quantum cloak, we perform the transformation design for the prolate spheroidal coordinate system and obtain a quantum cloak with an ellipsoidal shape. The mass parameters and effective potential for the creation of a perfect prolate spheroidal invisibility region are given. The analytic representations of the cloaked matter wave and probability current in the cloaking shell are presented. Special attention is paid to the discussions of the probability current in the cloaking shell for only that current can manifestly exhibit how the wave vector of the matter wave is curved, rotated, and guided in the cloaking shell to flow around the non-spherically invisible region. With the current analysis, one shows that the presented cloak can perfectly guide the matter wave in the situation of any oblique incidence. The proposed prolate spheroidal cloak for matter waves provides the first non-spherically three-dimensional setup for quantum cloaking.

  16. Prolate spheroidal quantum cloak

    NASA Astrophysics Data System (ADS)

    Syue, Cheng-De; Lin, De-Hone

    2015-04-01

    To understand the propagation behavior of an oblique incident matter wave in a three-dimensional non-spherical quantum cloak, we perform the transformation design for the prolate spheroidal coordinate system and obtain a quantum cloak with an ellipsoidal shape. The mass parameters and effective potential for the creation of a perfect prolate spheroidal invisibility region are given. The analytic representations of the cloaked matter wave and probability current in the cloaking shell are presented. Special attention is paid to the discussions of the probability current in the cloaking shell for only that current can manifestly exhibit how the wave vector of the matter wave is curved, rotated, and guided in the cloaking shell to flow around the non-spherically invisible region. With the current analysis, one shows that the presented cloak can perfectly guide the matter wave in the situation of any oblique incidence. The proposed prolate spheroidal cloak for matter waves provides the first non-spherically three-dimensional setup for quantum cloaking.

  17. Predictive modeling of multicellular structure formation by using Cellular Particle Dynamics simulations

    NASA Astrophysics Data System (ADS)

    McCune, Matthew; Shafiee, Ashkan; Forgacs, Gabor; Kosztin, Ioan

    2014-03-01

    Cellular Particle Dynamics (CPD) is an effective computational method for describing and predicting the time evolution of biomechanical relaxation processes of multicellular systems. A typical example is the fusion of spheroidal bioink particles during post bioprinting structure formation. In CPD cells are modeled as an ensemble of cellular particles (CPs) that interact via short-range contact interactions, characterized by an attractive (adhesive interaction) and a repulsive (excluded volume interaction) component. The time evolution of the spatial conformation of the multicellular system is determined by following the trajectories of all CPs through integration of their equations of motion. CPD was successfully applied to describe and predict the fusion of 3D tissue construct involving identical spherical aggregates. Here, we demonstrate that CPD can also predict tissue formation involving uneven spherical aggregates whose volumes decrease during the fusion process. Work supported by NSF [PHY-0957914]. Computer time provided by the University of Missouri Bioinformatics Consortium.

  18. The origin of multicellularity in cyanobacteria

    PubMed Central

    2011-01-01

    Background Cyanobacteria are one of the oldest and morphologically most diverse prokaryotic phyla on our planet. The early development of an oxygen-containing atmosphere approximately 2.45 - 2.22 billion years ago is attributed to the photosynthetic activity of cyanobacteria. Furthermore, they are one of the few prokaryotic phyla where multicellularity has evolved. Understanding when and how multicellularity evolved in these ancient organisms would provide fundamental information on the early history of life and further our knowledge of complex life forms. Results We conducted and compared phylogenetic analyses of 16S rDNA sequences from a large sample of taxa representing the morphological and genetic diversity of cyanobacteria. We reconstructed ancestral character states on 10,000 phylogenetic trees. The results suggest that the majority of extant cyanobacteria descend from multicellular ancestors. Reversals to unicellularity occurred at least 5 times. Multicellularity was established again at least once within a single-celled clade. Comparison to the fossil record supports an early origin of multicellularity, possibly as early as the "Great Oxygenation Event" that occurred 2.45 - 2.22 billion years ago. Conclusions The results indicate that a multicellular morphotype evolved early in the cyanobacterial lineage and was regained at least once after a previous loss. Most of the morphological diversity exhibited in cyanobacteria today —including the majority of single-celled species— arose from ancient multicellular lineages. Multicellularity could have conferred a considerable advantage for exploring new niches and hence facilitated the diversification of new lineages. PMID:21320320

  19. Antimicrobial peptides of multicellular organisms

    NASA Astrophysics Data System (ADS)

    Zasloff, Michael

    2002-01-01

    Multicellular organisms live, by and large, harmoniously with microbes. The cornea of the eye of an animal is almost always free of signs of infection. The insect flourishes without lymphocytes or antibodies. A plant seed germinates successfully in the midst of soil microbes. How is this accomplished? Both animals and plants possess potent, broad-spectrum antimicrobial peptides, which they use to fend off a wide range of microbes, including bacteria, fungi, viruses and protozoa. What sorts of molecules are they? How are they employed by animals in their defence? As our need for new antibiotics becomes more pressing, could we design anti-infective drugs based on the design principles these molecules teach us?

  20. Spatio-temporal cell dynamics in tumour spheroid irradiation

    NASA Astrophysics Data System (ADS)

    Kempf, H.; Bleicher, M.; Meyer-Hermann, M.

    2010-10-01

    Multicellular tumour spheroids are realistic in vitro systems in radiation research that integrate cell-cell interaction and cell cycle control by factors in the medium. The dynamic reaction inside a tumour spheroid triggered by radiation is not well understood. Of special interest is the amount of cell cycle synchronisation which could be triggered by irradiation, since this would allow follow-up irradiations to exploit the increased sensitivity of certain cell cycle phases. In order to investigate these questions we need to support irradiation experiments with mathematical models. In this article a new model is introduced combining the dynamics of tumour growth and irradiation treatments. The tumour spheroid growth is modelled using an agent-based Delaunay/Voronoi hybrid model in which the cells are represented by weighted dynamic vertices. Cell properties like full cell cycle dynamics are included. In order to be able to distinguish between different cell reactions in response to irradiation quality we introduce a probabilistic model for damage dynamics. The overall cell survival from this model is in agreement with predictions from the linear-quadratic model. Our model can describe the growth of avascular tumour spheroids in agreement to experimental results. Using the probabilistic model for irradiation damage dynamics the classic ‘four Rs’ of radiotherapy can be studied in silico. We found a pronounced reactivation of the tumour spheroid in response to irradiation. A majority of the surviving cells is synchronized in their cell cycle progression after irradiation. The cell synchronisation could be actively triggered and should be exploited in an advanced fractionation scheme. Thus it has been demonstrated that our model could be used to understand the dynamics of tumour growth after irradiation and to propose optimized fractionation schemes in cooperation with experimental investigations.

  1. The multicellularity genes of dictyostelid social amoebas.

    PubMed

    Glöckner, Gernot; Lawal, Hajara M; Felder, Marius; Singh, Reema; Singer, Gail; Weijer, Cornelis J; Schaap, Pauline

    2016-01-01

    The evolution of multicellularity enabled specialization of cells, but required novel signalling mechanisms for regulating cell differentiation. Early multicellular organisms are mostly extinct and the origins of these mechanisms are unknown. Here using comparative genome and transcriptome analysis across eight uni- and multicellular amoebozoan genomes, we find that 80% of proteins essential for the development of multicellular Dictyostelia are already present in their unicellular relatives. This set is enriched in cytosolic and nuclear proteins, and protein kinases. The remaining 20%, unique to Dictyostelia, mostly consists of extracellularly exposed and secreted proteins, with roles in sensing and recognition, while several genes for synthesis of signals that induce cell-type specialization were acquired by lateral gene transfer. Across Dictyostelia, changes in gene expression correspond more strongly with phenotypic innovation than changes in protein functional domains. We conclude that the transition to multicellularity required novel signals and sensors rather than novel signal processing mechanisms. PMID:27357338

  2. The multicellularity genes of dictyostelid social amoebas.

    PubMed

    Glöckner, Gernot; Lawal, Hajara M; Felder, Marius; Singh, Reema; Singer, Gail; Weijer, Cornelis J; Schaap, Pauline

    2016-06-30

    The evolution of multicellularity enabled specialization of cells, but required novel signalling mechanisms for regulating cell differentiation. Early multicellular organisms are mostly extinct and the origins of these mechanisms are unknown. Here using comparative genome and transcriptome analysis across eight uni- and multicellular amoebozoan genomes, we find that 80% of proteins essential for the development of multicellular Dictyostelia are already present in their unicellular relatives. This set is enriched in cytosolic and nuclear proteins, and protein kinases. The remaining 20%, unique to Dictyostelia, mostly consists of extracellularly exposed and secreted proteins, with roles in sensing and recognition, while several genes for synthesis of signals that induce cell-type specialization were acquired by lateral gene transfer. Across Dictyostelia, changes in gene expression correspond more strongly with phenotypic innovation than changes in protein functional domains. We conclude that the transition to multicellularity required novel signals and sensors rather than novel signal processing mechanisms.

  3. The multicellularity genes of dictyostelid social amoebas

    PubMed Central

    Glöckner, Gernot; Lawal, Hajara M.; Felder, Marius; Singh, Reema; Singer, Gail; Weijer, Cornelis J.; Schaap, Pauline

    2016-01-01

    The evolution of multicellularity enabled specialization of cells, but required novel signalling mechanisms for regulating cell differentiation. Early multicellular organisms are mostly extinct and the origins of these mechanisms are unknown. Here using comparative genome and transcriptome analysis across eight uni- and multicellular amoebozoan genomes, we find that 80% of proteins essential for the development of multicellular Dictyostelia are already present in their unicellular relatives. This set is enriched in cytosolic and nuclear proteins, and protein kinases. The remaining 20%, unique to Dictyostelia, mostly consists of extracellularly exposed and secreted proteins, with roles in sensing and recognition, while several genes for synthesis of signals that induce cell-type specialization were acquired by lateral gene transfer. Across Dictyostelia, changes in gene expression correspond more strongly with phenotypic innovation than changes in protein functional domains. We conclude that the transition to multicellularity required novel signals and sensors rather than novel signal processing mechanisms. PMID:27357338

  4. Targeting Mitochondrial Function to Treat Quiescent Tumor Cells in Solid Tumors

    PubMed Central

    Zhang, Xiaonan; de Milito, Angelo; Olofsson, Maria Hägg; Gullbo, Joachim; D’Arcy, Padraig; Linder, Stig

    2015-01-01

    The disorganized nature of tumor vasculature results in the generation of microenvironments characterized by nutrient starvation, hypoxia and accumulation of acidic metabolites. Tumor cell populations in such areas are often slowly proliferating and thus refractory to chemotherapeutical drugs that are dependent on an active cell cycle. There is an urgent need for alternative therapeutic interventions that circumvent growth dependency. The screening of drug libraries using multicellular tumor spheroids (MCTS) or glucose-starved tumor cells has led to the identification of several compounds with promising therapeutic potential and that display activity on quiescent tumor cells. Interestingly, a common theme of these drug screens is the recurrent identification of agents that affect mitochondrial function. Such data suggest that, contrary to the classical Warburg view, tumor cells in nutritionally-compromised microenvironments are dependent on mitochondrial function for energy metabolism and survival. These findings suggest that mitochondria may represent an “Achilles heel” for the survival of slowly-proliferating tumor cells and suggest strategies for the development of therapy to target these cell populations. PMID:26580606

  5. Multicellularity makes somatic differentiation evolutionarily stable

    PubMed Central

    Wahl, Mary E.; Murray, Andrew W.

    2016-01-01

    Many multicellular organisms produce two cell lineages: germ cells, whose descendants produce the next generation, and somatic cells, which support, protect, and disperse the germ cells. This germ-soma demarcation has evolved independently in dozens of multicellular taxa but is absent in unicellular species. A common explanation holds that in these organisms, inefficient intercellular nutrient exchange compels the fitness cost of producing nonreproductive somatic cells to outweigh any potential benefits. We propose instead that the absence of unicellular, soma-producing populations reflects their susceptibility to invasion by nondifferentiating mutants that ultimately eradicate the soma-producing lineage. We argue that multicellularity can prevent the victory of such mutants by giving germ cells preferential access to the benefits conferred by somatic cells. The absence of natural unicellular, soma-producing species previously prevented these hypotheses from being directly tested in vivo: to overcome this obstacle, we engineered strains of the budding yeast Saccharomyces cerevisiae that differ only in the presence or absence of multicellularity and somatic differentiation, permitting direct comparisons between organisms with different lifestyles. Our strains implement the essential features of irreversible conversion from germ line to soma, reproductive division of labor, and clonal multicellularity while maintaining sufficient generality to permit broad extension of our conclusions. Our somatic cells can provide fitness benefits that exceed the reproductive costs of their production, even in unicellular strains. We find that nondifferentiating mutants overtake unicellular populations but are outcompeted by multicellular, soma-producing strains, suggesting that multicellularity confers evolutionary stability to somatic differentiation. PMID:27402737

  6. Multicellularity makes somatic differentiation evolutionarily stable.

    PubMed

    Wahl, Mary E; Murray, Andrew W

    2016-07-26

    Many multicellular organisms produce two cell lineages: germ cells, whose descendants produce the next generation, and somatic cells, which support, protect, and disperse the germ cells. This germ-soma demarcation has evolved independently in dozens of multicellular taxa but is absent in unicellular species. A common explanation holds that in these organisms, inefficient intercellular nutrient exchange compels the fitness cost of producing nonreproductive somatic cells to outweigh any potential benefits. We propose instead that the absence of unicellular, soma-producing populations reflects their susceptibility to invasion by nondifferentiating mutants that ultimately eradicate the soma-producing lineage. We argue that multicellularity can prevent the victory of such mutants by giving germ cells preferential access to the benefits conferred by somatic cells. The absence of natural unicellular, soma-producing species previously prevented these hypotheses from being directly tested in vivo: to overcome this obstacle, we engineered strains of the budding yeast Saccharomyces cerevisiae that differ only in the presence or absence of multicellularity and somatic differentiation, permitting direct comparisons between organisms with different lifestyles. Our strains implement the essential features of irreversible conversion from germ line to soma, reproductive division of labor, and clonal multicellularity while maintaining sufficient generality to permit broad extension of our conclusions. Our somatic cells can provide fitness benefits that exceed the reproductive costs of their production, even in unicellular strains. We find that nondifferentiating mutants overtake unicellular populations but are outcompeted by multicellular, soma-producing strains, suggesting that multicellularity confers evolutionary stability to somatic differentiation. PMID:27402737

  7. Colorectal cancer derived organotypic spheroids maintain essential tissue characteristics but adapt their metabolism in culture

    PubMed Central

    2014-01-01

    Background Organotypic tumor spheroids, a 3D in vitro model derived from patient tumor material, preserve tissue heterogeneity and retain structural tissue elements, thus replicating the in vivo tumor more closely than commonly used 2D and 3D cell line models. Such structures harbour tumorigenic cells, as revealed by xenograft implantation studies in animal models and maintain the genetic makeup of the original tumor material. The aim of our work was a morphological and proteomic characterization of organotypic spheroids derived from colorectal cancer tissue in order to get insight into their composition and associated biology. Results Morphological analysis showed that spheroids were of about 250 μm in size and varied in structure, while the spheroid cells differed in shape and size and were tightly packed together by desmosomes and tight junctions. Our proteomic data revealed significant alterations in protein expression in organotypic tumor spheroids cultured as primary explants compared to primary colorectal cancer tissue. Components underlying cellular and tissue architecture were changed; nuclear DNA/ chromatin maintenance systems were up-regulated, whereas various mitochondrial components were down-regulated in spheroids. Most interestingly, the mesenchymal cells appear to be substantial component in such cellular assemblies. Thus the observed changes may partly occur in this cellular compartment. Finally, in the proteomics analysis stem cell-like characteristics were observed within the spheroid cellular assembly, reflected by accumulation of Alcam, Ctnnb1, Aldh1, Gpx2, and CD166. These findings were underlined by IHC analysis of Ctnnb1, CD24 and CD44, therefore warranting closer investigation of the tumorigenic compartment in this 3D culture model for tumor tissue. Conclusions Our analysis of organotypic CRC tumor spheroids has identified biological processes associated with a mixture of cell types and states, including protein markers for mesenchymal

  8. Spheroidal Wave Functions in Electromagnetic Theory

    NASA Astrophysics Data System (ADS)

    Li, Le-Wei; Kang, Xiao-Kang; Leong, Mook-Seng

    2001-11-01

    The flagship monograph addressing the spheroidal wave function and its pertinence to computational electromagnetics Spheroidal Wave Functions in Electromagnetic Theory presents in detail the theory of spheroidal wave functions, its applications to the analysis of electromagnetic fields in various spheroidal structures, and provides comprehensive programming codes for those computations. The topics covered in this monograph include: Spheroidal coordinates and wave functions Dyadic Green's functions in spheroidal systems EM scattering by a conducting spheroid EM scattering by a coated dielectric spheroid Spheroid antennas SAR distributions in a spheroidal head model The programming codes and their applications are provided online and are written in Mathematica 3.0 or 4.0. Readers can also develop their own codes according to the theory or routine described in the book to find subsequent solutions of complicated structures. Spheroidal Wave Functions in Electromagnetic Theory is a fundamental reference for scientists, engineers, and graduate students practicing modern computational electromagnetics or applied physics.

  9. Bacterial Ventures into Multicellularity: Collectivism through Individuality

    PubMed Central

    van Vliet, Simon; Ackermann, Martin

    2015-01-01

    Multicellular eukaryotes can perform functions that exceed the possibilities of an individual cell. These functions emerge through interactions between differentiated cells that are precisely arranged in space. Bacteria also form multicellular collectives that consist of differentiated but genetically identical cells. How does the functionality of these collectives depend on the spatial arrangement of the differentiated bacteria? In a previous issue of PLOS Biology, van Gestel and colleagues reported an elegant example of how the spatial arrangement of differentiated cells gives rise to collective behavior in Bacillus subtilus colonies, further demonstrating the similarity of bacterial collectives to higher multicellular organisms. PMID:26038821

  10. The Evolution of Multicellular Plants and Animals.

    ERIC Educational Resources Information Center

    Valentine, James W.

    1978-01-01

    Traces the evolution of unicellular organisms to the multi-cellular plants and animals in existence today. Major events are depicted in a geologic timetable. Organisms, extinct and recent, are classified by taxonomic group. (MA)

  11. Intact LKB1 activity is required for survival of dormant ovarian cancer spheroids

    PubMed Central

    Peart, Teresa; Valdes, Yudith Ramos; Correa, Rohann J. M.; Fazio, Elena; Bertrand, Monique; McGee, Jacob; Préfontaine, Michel; Sugimoto, Akira; DiMattia, Gabriel E.; Shepherd, Trevor G.

    2015-01-01

    Metastatic epithelial ovarian cancer (EOC) cells can form multicellular spheroids while in suspension and disperse directly throughout the peritoneum to seed secondary lesions. There is growing evidence that EOC spheroids are key mediators of metastasis, and they use specific intracellular signalling pathways to control cancer cell growth and metabolism for increased survival. Our laboratory discovered that AKT signalling is reduced during spheroid formation leading to cellular quiescence and autophagy, and these may be defining features of tumour cell dormancy. To further define the phenotype of EOC spheroids, we have initiated studies of the Liver kinase B1 (LKB1)-5′-AMP-activated protein kinase (AMPK) pathway as a master controller of the metabolic stress response. We demonstrate that activity of AMPK and its upstream kinase LKB1 are increased in quiescent EOC spheroids as compared with proliferating adherent EOC cells. We also show elevated AMPK activity in spheroids isolated directly from patient ascites. Functional studies reveal that treatment with the AMP mimetic AICAR or allosteric AMPK activator A-769662 led to a cytostatic response in proliferative adherent ovarian cancer cells, but they fail to elicit an effect in spheroids. Targeted knockdown of STK11 by RNAi to reduce LKB1 expression led to reduced viability and increased sensitivity to carboplatin treatment in spheroids only, a phenomenon which was AMPK-independent. Thus, our results demonstrate a direct impact of altered LKB1-AMPK signalling function in EOC. In addition, this is the first evidence in cancer cells demonstrating a pro-survival function for LKB1, a kinase traditionally thought to act as a tumour suppressor. PMID:26068970

  12. A tumor growth model with deformable ECM

    PubMed Central

    Sciumè, G; Santagiuliana, R; Ferrari, M; Decuzzi, P; Schrefler, B A

    2015-01-01

    Existing tumor growth models based on fluid analogy for the cells do not generally include the extracellular matrix (ECM), or if present, take it as rigid. The three-fluid model originally proposed by the authors and comprising tumor cells (TC), host cells (HC), interstitial fluid (IF) and an ECM, considered up to now only a rigid ECM in the applications. This limitation is here relaxed and the deformability of the ECM is investigated in detail. The ECM is modeled as a porous solid matrix with Green-elastic and elasto-visco-plastic material behavior within a large strain approach. Jauman and Truesdell objective stress measures are adopted together with the deformation rate tensor. Numerical results are first compared with those of a reference experiment of a multicellular tumor spheroid (MTS) growing in vitro, then three different tumor cases are studied: growth of an MTS in a decellularized ECM, growth of a spheroid in the presence of host cells and growth of a melanoma. The influence of the stiffness of the ECM is evidenced and comparison with the case of a rigid ECM is made. The processes in a deformable ECM are more rapid than in a rigid ECM and the obtained growth pattern differs. The reasons for this are due to the changes in porosity induced by the tumor growth. These changes are inhibited in a rigid ECM. This enhanced computational model emphasizes the importance of properly characterizing the biomechanical behavior of the malignant mass in all its components to correctly predict its temporal and spatial pattern evolution. PMID:25427284

  13. The Simplest Integrated Multicellular Organism Unveiled

    PubMed Central

    Arakaki, Yoko; Kawai-Toyooka, Hiroko; Hamamura, Yuki; Higashiyama, Tetsuya; Noga, Akira; Hirono, Masafumi; Olson, Bradley J. S. C.; Nozaki, Hisayoshi

    2013-01-01

    Volvocine green algae represent the “evolutionary time machine” model lineage for studying multicellularity, because they encompass the whole range of evolutionary transition of multicellularity from unicellular Chlamydomonas to >500-celled Volvox. Multicellular volvocalean species including Gonium pectorale and Volvox carteri generally have several common morphological features to survive as integrated multicellular organisms such as “rotational asymmetry of cells” so that the cells become components of the individual and “cytoplasmic bridges between protoplasts in developing embryos” to maintain the species-specific form of the multicellular individual before secretion of new extracellular matrix (ECM). However, these morphological features have not been studied in the four-celled colonial volvocine species Tetrabaena socialis that is positioned in the most basal lineage within the colonial or multicellular volvocine greens. Here we established synchronous cultures of T. socialis and carried out immunofluorescence microscopic and ultrastructural observations to elucidate these two morphological attributes. Based on immunofluorescence microscopy, four cells of the mature T. socialis colony were identical in morphology but had rotational asymmetry in arrangement of microtubular rootlets and separation of basal bodies like G. pectorale and V. carteri. Ultrastructural observations clearly confirmed the presence of cytoplasmic bridges between protoplasts in developing embryos of T. socialis even after the formation of new flagella in each daughter protoplast within the parental ECM. Therefore, these two morphological attributes might have evolved in the common four-celled ancestor of the colonial volvocine algae and contributed to the further increase in cell number and complexity of the multicellular individuals of this model lineage. T. socialis is one of the simplest integrated multicellular organisms in which four identical cells constitute the

  14. The simplest integrated multicellular organism unveiled.

    PubMed

    Arakaki, Yoko; Kawai-Toyooka, Hiroko; Hamamura, Yuki; Higashiyama, Tetsuya; Noga, Akira; Hirono, Masafumi; Olson, Bradley J S C; Nozaki, Hisayoshi

    2013-01-01

    Volvocine green algae represent the "evolutionary time machine" model lineage for studying multicellularity, because they encompass the whole range of evolutionary transition of multicellularity from unicellular Chlamydomonas to >500-celled Volvox. Multicellular volvocalean species including Gonium pectorale and Volvox carteri generally have several common morphological features to survive as integrated multicellular organisms such as "rotational asymmetry of cells" so that the cells become components of the individual and "cytoplasmic bridges between protoplasts in developing embryos" to maintain the species-specific form of the multicellular individual before secretion of new extracellular matrix (ECM). However, these morphological features have not been studied in the four-celled colonial volvocine species Tetrabaena socialis that is positioned in the most basal lineage within the colonial or multicellular volvocine greens. Here we established synchronous cultures of T. socialis and carried out immunofluorescence microscopic and ultrastructural observations to elucidate these two morphological attributes. Based on immunofluorescence microscopy, four cells of the mature T. socialis colony were identical in morphology but had rotational asymmetry in arrangement of microtubular rootlets and separation of basal bodies like G. pectorale and V. carteri. Ultrastructural observations clearly confirmed the presence of cytoplasmic bridges between protoplasts in developing embryos of T. socialis even after the formation of new flagella in each daughter protoplast within the parental ECM. Therefore, these two morphological attributes might have evolved in the common four-celled ancestor of the colonial volvocine algae and contributed to the further increase in cell number and complexity of the multicellular individuals of this model lineage. T. socialis is one of the simplest integrated multicellular organisms in which four identical cells constitute the individual. PMID

  15. Multicellular life cycle of magnetotactic prokaryotes.

    PubMed

    Keim, Carolina N; Martins, Juliana L; Abreu, Fernanda; Rosado, Alexandre Soares; de Barros, Henrique Lins; Borojevic, Radovan; Lins, Ulysses; Farina, Marcos

    2004-11-15

    Most multicellular organisms, prokaryotes as well as animals, plants, and algae have a unicellular stage in their life cycle. Here, we describe an uncultured prokaryotic magnetotactic multicellular organism that reproduces by binary fission. It is multicellular in all the stages of its life cycle, and during most of the life cycle the cells organize into a hollow sphere formed by a functionally coordinated and polarized single-cell layer that grows by increasing the cell size. Subsequently, all the cells divide synchronously; the organism becomes elliptical, and separates into two equal spheres with a torsional movement in the equatorial plane. Unicellular bacteria similar to the cells that compose these organisms have not been found. Molecular biology analysis showed that all the organisms studied belong to a single genetic population phylogenetically related to many-celled magnetotactic prokaryotes in the delta sub-group of the proteobacteria. This appears to be the first report of a multicellular prokaryotic organism that proliferates by dividing into two equal multicellular organisms each similar to the parent one. PMID:15522508

  16. Axisymmetric scattering of scalar waves by spheroids.

    PubMed

    Lekner, John; Boyack, Rufus

    2011-06-01

    A phase shift formulation of scattering by oblate and prolate spheroids is presented, in parallel with the partial-wave theory of scattering by spherical obstacles. The crucial step is application of a finite Legendre transform to the Helmholtz equation in spheroidal coordinates. In the long-wavelength limit the spheroidal analog of the spherical scattering length immediately gives the cross section. Analytical results are readily obtained for scattering of Schrödinger particle waves by impenetrable spheroids, and for scattering of sound waves by acoustically soft spheroidal objects. The method is restricted to scattering by spheroids whose symmetry axis is coincident with the direction of the incident plane wave. PMID:21682372

  17. Axisymmetric scattering of scalar waves by spheroids.

    PubMed

    Lekner, John; Boyack, Rufus

    2011-06-01

    A phase shift formulation of scattering by oblate and prolate spheroids is presented, in parallel with the partial-wave theory of scattering by spherical obstacles. The crucial step is application of a finite Legendre transform to the Helmholtz equation in spheroidal coordinates. In the long-wavelength limit the spheroidal analog of the spherical scattering length immediately gives the cross section. Analytical results are readily obtained for scattering of Schrödinger particle waves by impenetrable spheroids, and for scattering of sound waves by acoustically soft spheroidal objects. The method is restricted to scattering by spheroids whose symmetry axis is coincident with the direction of the incident plane wave.

  18. MUC16 mucin (CA125) regulates the formation of multicellular aggregates by altering β-catenin signaling

    PubMed Central

    Giannakouros, Panagiota; Comamala, Marina; Matte, Isabelle; Rancourt, Claudine; Piché, Alain

    2015-01-01

    After shedding from the primary tumor site, ovarian cancer cells form three-dimensional multicellular aggregates that serve as vehicle for cancer cell dissemination in the peritoneal cavity. MUC16 mucin (CA125) is aberrantly expressed by most advanced serous ovarian cancers and can promote proliferation, migration and metastasis. MUC16 associates with E-cadherin and β-catenin, two proteins involved in regulation of cell adhesion and the formation of multicellular aggregates. However, the role of MUC16 in the formation of multicellular aggregates remains to be defined. Here, we show that MUC16 alters E-cadherin cellular localization and expression. Consistent with this, MUC16 knockdown inhibited the formation of multicellular aggregates and, conversely, forced expression of MUC16 C-terminal domain (CTD) enhanced the formation of multicellular aggregates. MUC16 knockdown induces β-catenin relocation from the cell membrane to the cytoplasm, decreases its expression by increasing degradation and decreases β-catenin target gene expression. MUC16 CTD inhibits GSK-3β-mediated phosphorylation and degradation of β-catenin, leading to increased β-catenin levels. Importantly, knockdown of β-catenin inhibited multicellular aggregation. These findings indicate that MUC16 promotes the formation of multicellular aggregates by inhibiting β-catenin degradation. PMID:25628932

  19. Smart Superstructures with Ultrahigh pH-Sensitivity for Targeting Acidic Tumor Microenvironment: Instantaneous Size Switching and Improved Tumor Penetration.

    PubMed

    Li, Hong-Jun; Du, Jin-Zhi; Liu, Jing; Du, Xiao-Jiao; Shen, Song; Zhu, Yan-Hua; Wang, Xiaoyan; Ye, Xiaodong; Nie, Shuming; Wang, Jun

    2016-07-26

    The currently low delivery efficiency and limited tumor penetration of nanoparticles remain two major challenges of cancer nanomedicine. Here, we report a class of pH-responsive nanoparticle superstructures with ultrasensitive size switching in the acidic tumor microenvironment for improved tumor penetration and effective in vivo drug delivery. The superstructures were constructed from amphiphilic polymer directed assembly of platinum-prodrug conjugated polyamidoamine (PAMAM) dendrimers, in which the amphiphilic polymer contains ionizable tertiary amine groups for rapid pH-responsiveness. These superstructures had an initial size of ∼80 nm at neutral pH (e.g., in blood circulation), but once deposited in the slightly acidic tumor microenvironment (pH ∼6.5-7.0), they underwent a dramatic and sharp size transition within a very narrow range of acidity (less than 0.1-0.2 pH units) and dissociated instantaneously into the dendrimer building blocks (less than 10 nm in diameter). This rapid size-switching feature not only can facilitate nanoparticle extravasation and accumulation via the enhanced permeability and retention effect but also allows faster nanoparticle diffusion and more efficient tumor penetration. We have further carried out comparative studies of pH-sensitive and insensitive nanostructures with similar size, surface charge, and chemical composition in both multicellular spheroids and poorly permeable BxPC-3 pancreatic tumor models, whose results demonstrate that the pH-triggered size switching is a viable strategy for improving drug penetration and therapeutic efficacy.

  20. Fabrication of 3-dimensional multicellular microvascular structures

    PubMed Central

    Barreto-Ortiz, Sebastian F.; Fradkin, Jamie; Eoh, Joon; Trivero, Jacqueline; Davenport, Matthew; Ginn, Brian; Mao, Hai-Quan; Gerecht, Sharon

    2015-01-01

    Despite current advances in engineering blood vessels over 1 mm in diameter and the existing wealth of knowledge regarding capillary bed formation, studies for the development of microvasculature, the connecting bridge between them, have been extremely limited so far. Here, we evaluate the use of 3-dimensional (3D) microfibers fabricated by hydrogel electrospinning as templates for microvascular structure formation. We hypothesize that 3D microfibers improve extracellular matrix (ECM) deposition from vascular cells, enabling the formation of freestanding luminal multicellular microvasculature. Compared to 2-dimensional cultures, we demonstrate with confocal microscopy and RT-PCR that fibrin microfibers induce an increased ECM protein deposition by vascular cells, specifically endothelial colony-forming cells, pericytes, and vascular smooth muscle cells. These ECM proteins comprise different layers of the vascular wall including collagen types I, III, and IV, as well as elastin, fibronectin, and laminin. We further demonstrate the achievement of multicellular microvascular structures with an organized endothelium and a robust multicellular perivascular tunica media. This, along with the increased ECM deposition, allowed for the creation of self-supporting multilayered microvasculature with a distinct circular lumen following fibrin microfiber core removal. This approach presents an advancement toward the development of human microvasculature for basic and translational studies.—Barreto-Ortiz, S. F., Fradkin, J., Eoh, J., Trivero, J., Davenport, M., Ginn, B., Mao, H.-Q., Gerecht, S. Fabrication of 3-dimensional multicellular microvascular structures. PMID:25900808

  1. Limits to Chemically Guided Multicellular Migration

    NASA Astrophysics Data System (ADS)

    Varennes, Julien; Han, Bumsoo; Mugler, Andrew

    Collective cell migration in response to a chemical cue requires both multicellular sensing of chemical gradients and coordinated mechanical action. Examples from morphogenesis and cancer metastasis demonstrate that clusters of migratory cells are extremely sensitive, responding to gradients of less than 1% difference in chemical concentration across a cell body. While the limits to multicellular sensing are becoming known, the ensuing consequences for coherent migration remain poorly understood. We develop a model of multicellular sensing and migration based on the cellular Potts model. Multicellular sensing of noisy chemical gradients is modeled as a process of local excitation and global inhibition (LEGI) among communicating cells. The output of the sensing process is coupled to individual cells' polarization to model migratory behavior. We find that larger clusters of cells detect the gradient direction with higher precision and thus achieve stronger polarization bias. At the same time, larger clusters are also accompanied by less coherent collective motion. The trade-off between these two effects leads to an optimally efficient cluster size. We discuss how our results relate to cancer metastasis.

  2. Scaffold-Free Coculture Spheroids of Human Colonic Adenocarcinoma Cells and Normal Colonic Fibroblasts Promote Tumorigenicity in Nude Mice123

    PubMed Central

    Park, Jong-il; Lee, Jisu; Kwon, Ju-Lee; Park, Hong-Bum; Lee, Su-Yel; Kim, Ji-Yeon; Sung, Jaekye; Kim, Jin Man; Song, Kyu Sang; Kim, Kyung-Hee

    2016-01-01

    The aim of this study was to form a scaffold-free coculture spheroid model of colonic adenocarcinoma cells (CACs) and normal colonic fibroblasts (NCFs) and to use the spheroids to investigate the role of NCFs in the tumorigenicity of CACs in nude mice. We analysed three-dimensional (3D) scaffold-free coculture spheroids of CACs and NCFs. CAC Matrigel invasion assays and tumorigenicity assays in nude mice were performed to examine the effect of NCFs on CAC invasive behaviour and tumorigenicity in 3D spheroids. We investigated the expression pattern of fibroblast activation protein-α (FAP-α) by immunohistochemical staining. CAC monocultures did not form densely-packed 3D spheroids, whereas cocultured CACs and NCFs formed 3D spheroids. The 3D coculture spheroids seeded on a Matrigel extracellular matrix showed higher CAC invasiveness compared to CACs alone or CACs and NCFs in suspension. 3D spheroids injected into nude mice generated more and faster-growing tumors compared to CACs alone or mixed suspensions consisting of CACs and NCFs. FAP-α was expressed in NCFs-CACs cocultures and xenograft tumors, whereas monocultures of NCFs or CACs were negative for FAP-α expression. Our findings provide evidence that the interaction between CACs and NCFs is essential for the tumorigenicity of cancer cells as well as for tumor propagation. PMID:26947885

  3. Spheroidizing of medium carbon steels

    NASA Astrophysics Data System (ADS)

    O'Brien, James Michael

    2000-11-01

    An investigation has been made of spheroidization of medium carbon steels used in the bolt industry. Two process cycles were considered. One was the intercritical cycle, widely used in industry, in which the steel was heated above the A1 temperature for approximately 2 hours and then cooled to 688°C (1270°F) and held for various periods. The other was a subcritical cycle that involved heating to 704°C (1300°F) for various times. Wire samples were 0.4-in. diameter AISI 1541, considered high in manganese and difficult to spheroidize. Although AISI 4037 is considered easier to spheroidize, this alloy was also tested due to its extensive industrial use. It was found that the intercritical cycle produced a somewhat faster drop in hardness. However, one hour of the subcritical cycle yielded greater ductility than 32 hours of the intercritical process, as measured by tensile tests. Similar ductility results were achieved using a new flare test. The level of spheroidization was defined in this study to be the percentage of carbide particles with aspect ratios less than 3. The subcritical cycle produced the same level of spheroidization in 1/2 hour as that reached by the intercritical cycle in 32 hours. Faster spheroidization of cementite plates in the subcritical process appears to be due to the fine pearlite generated by the current practice of rapid cooling off the hot mill. This advantage is lost in the intercritical process because the original pearlite is dissolved above the A1 temperature.

  4. OVCAR-3 Spheroid-Derived Cells Display Distinct Metabolic Profiles

    PubMed Central

    Vermeersch, Kathleen A.; Wang, Lijuan; Mezencev, Roman; McDonald, John F.; Styczynski, Mark P.

    2015-01-01

    Introduction Recently, multicellular spheroids were isolated from a well-established epithelial ovarian cancer cell line, OVCAR-3, and were propagated in vitro. These spheroid-derived cells displayed numerous hallmarks of cancer stem cells, which are chemo- and radioresistant cells thought to be a significant cause of cancer recurrence and resultant mortality. Gene set enrichment analysis of expression data from the OVCAR-3 cells and the spheroid-derived putative cancer stem cells identified several metabolic pathways enriched in differentially expressed genes. Before this, there had been little previous knowledge or investigation of systems-scale metabolic differences between cancer cells and cancer stem cells, and no knowledge of such differences in ovarian cancer stem cells. Methods To determine if there were substantial metabolic changes corresponding with these transcriptional differences, we used two-dimensional gas chromatography coupled to mass spectrometry to measure the metabolite profiles of the two cell lines. Results These two cell lines exhibited significant metabolic differences in both intracellular and extracellular metabolite measurements. Principal components analysis, an unsupervised dimensional reduction technique, showed complete separation between the two cell types based on their metabolite profiles. Pathway analysis of intracellular metabolomics data revealed close overlap with metabolic pathways identified from gene expression data, with four out of six pathways found enriched in gene-level analysis also enriched in metabolite-level analysis. Some of those pathways contained multiple metabolites that were individually statistically significantly different between the two cell lines, with one of the most broadly and consistently different pathways, arginine and proline metabolism, suggesting an interesting hypothesis about cancerous and stem-like metabolic phenotypes in this pair of cell lines. Conclusions Overall, we demonstrate for the

  5. Magnetically and pH dual responsive dendrosomes for tumor accumulation enhanced folate-targeted hybrid drug delivery.

    PubMed

    Wang, Meng; Li, Jingjing; Li, Xuejuan; Mu, Hongjie; Zhang, Xuemei; Shi, Yanan; Chu, Yongchao; Wang, Aiping; Wu, Zimei; Sun, Kaoxiang

    2016-06-28

    Dendrosomes are new tumor targeted drug delivery systems to improve safety and therapeutic effects of antitumor agents. In this study we designed and synthesized magnetically and pH dual responsive dendrosomes with magnetic nanoparticles and folate-targeted dendrimers encapsulated in long-circulating pH sensitive liposomes. Cellular uptake and tissue penetration were assessed on cell lines and tumor spheroids respectively. Xenograft mice were used to study tumor accumulation. The dendrosomes were stable at pH7.4, but responsively released their content at acidic pH. In slightly acid environments, the hybrid vectors showed similar cytotoxicity and cellular uptake to the free folate-dendrimers conjugate due to rapid release. The dendrosomes showed a greater cellular uptake by HeLa cells (FA receptor positive) due to the conjugation with folic acid (FA). In multicellular tumor spheroid tests, a slightly acidic environment and the application of magnet both promoted the permeation efficiency of the hybrid vectors. In the xenograft mice model both in vivo images and tissue distribution assessment indicated that the dendrosomes had higher peak intensity and a longer residence time. Through the synergistic effects of magnetic responsiveness and both passive and active targeting properties, the multi-functional dendrosomes were demonstrated to have great potential as a promising anticancer drug delivery platform. PMID:27090165

  6. Dynamic Change of Polarity in Primary Cultured Spheroids of Human Colorectal Adenocarcinoma and Its Role in Metastasis.

    PubMed

    Okuyama, Hiroaki; Kondo, Jumpei; Sato, Yumi; Endo, Hiroko; Nakajima, Aya; Piulats, Jose M; Tomita, Yasuhiko; Fujiwara, Takeshi; Itoh, Yu; Mizoguchi, Akira; Ohue, Masayuki; Inoue, Masahiro

    2016-04-01

    Intestinal epithelial cells possess apical-basal polarity, which governs the exchange of nutrients and waste. Perturbation of cell polarity appears to be a general feature of cancers, although most colorectal cancers are differentiated adenocarcinomas, in which polarity is maintained to some extent. Little is known about the role of dysregulated polarity in cancer. The cancer tissue-originated spheroid method was applied to the preparation and culture of spheroids. Spheroids were cultured in suspension or in type I collagen gel. Polarity was assessed by IHC of apical markers and electron microscopy. Two types of polarity status in spheroids were observed: apical-in, with apical membrane located at cavities inside the spheroids in type I collagen gel; and apical-out, with apical membrane located at the outermost layer of spheroids in suspension. These polarities were highly interchangeable. Inhibitors of Src and dynamin attenuated the polarity switch. In patients, clusters of cancer cells that invaded vessels had both apical-in and apical-out morphologic features, whereas primary and metastatic tumors had apical-in features. In a mouse liver metastasis model, apical-out spheroids injected into the portal vein became apical-in spheroids in the liver within a few days. Inhibitors of Src and dynamin significantly decreased liver metastasis. Polarity switching was observed in spheroids and human cancer. The polarity switch was critical in an experimental liver metastasis model.

  7. High-content analysis of tumour cell invasion in three-dimensional spheroid assays

    PubMed Central

    Cheng, Vinton; Esteves, Filomena; Chakrabarty, Aruna; Cockle, Julia; Short, Susan; Brüning-Richardson, Anke

    2015-01-01

    Targeting infiltrating tumour cells is an attractive way of combating cancer invasion and metastasis. Here we describe a novel and reproducible method for high content analysis of invading cells using multicellular tumour spheroid assays in a high grade glioma model. Live cell imaging of spheroids generated from glioma cell lines, U87 and U251, gave insight into migration dynamics and cell morphology in response to anti-migratory drugs. Immunofluorescence imaging confirmed cytoskeletal rearrangements in the treated cells indicating a direct effect on cell morphology. Effect on migration was determined by a Migration Index (MI) from brightfield images which confirmed anti-migratory activity of the drugs. A marked effect on the core with treatment suggestive of disordered proliferation was also observed. A newly developed technique to prepare the spheroids and migratory cells for immunohistochemistry allowed an assessment of response to drug treatment with a selection of markers. A difference in protein expression was noted between cells maintained within the core and migratory cells indicative of the presence of cell subpopulations within the spheroid core. We conclude that this high content analysis allows researchers to perform screening of anti-tumour invasion compounds and study their effects on cellular dynamics, particularly in relation to protein expression, for the first time. PMID:26244167

  8. High-content analysis of tumour cell invasion in three-dimensional spheroid assays.

    PubMed

    Cheng, Vinton; Esteves, Filomena; Chakrabarty, Aruna; Cockle, Julia; Short, Susan; Brüning-Richardson, Anke

    2015-01-01

    Targeting infiltrating tumour cells is an attractive way of combating cancer invasion and metastasis. Here we describe a novel and reproducible method for high content analysis of invading cells using multicellular tumour spheroid assays in a high grade glioma model. Live cell imaging of spheroids generated from glioma cell lines, U87 and U251, gave insight into migration dynamics and cell morphology in response to anti-migratory drugs. Immunofluorescence imaging confirmed cytoskeletal rearrangements in the treated cells indicating a direct effect on cell morphology. Effect on migration was determined by a Migration Index (MI) from brightfield images which confirmed anti-migratory activity of the drugs. A marked effect on the core with treatment suggestive of disordered proliferation was also observed. A newly developed technique to prepare the spheroids and migratory cells for immunohistochemistry allowed an assessment of response to drug treatment with a selection of markers. A difference in protein expression was noted between cells maintained within the core and migratory cells indicative of the presence of cell subpopulations within the spheroid core. We conclude that this high content analysis allows researchers to perform screening of anti-tumour invasion compounds and study their effects on cellular dynamics, particularly in relation to protein expression, for the first time. PMID:26244167

  9. A multiphase model for three-dimensional tumor growth

    PubMed Central

    Sciumè, G; Shelton, S; Gray, WG; Miller, CT; Hussain, F; Ferrari, M; Decuzzi, P; Schrefler, BA

    2014-01-01

    Several mathematical formulations have analyzed the time-dependent behaviour of a tumor mass. However, most of these propose simplifications that compromise the physical soundness of the model. Here, multiphase porous media mechanics is extended to model tumor evolution, using governing equations obtained via the Thermodynamically Constrained Averaging Theory (TCAT). A tumor mass is treated as a multiphase medium composed of an extracellular matrix (ECM); tumor cells (TC), which may become necrotic depending on the nutrient concentration and tumor phase pressure; healthy cells (HC); and an interstitial fluid (IF) for the transport of nutrients. The equations are solved by a Finite Element method to predict the growth rate of the tumor mass as a function of the initial tumor-to-healthy cell density ratio, nutrient concentration, mechanical strain, cell adhesion and geometry. Results are shown for three cases of practical biological interest such as multicellular tumor spheroids (MTS) and tumor cords. First, the model is validated by experimental data for time-dependent growth of an MTS in a culture medium. The tumor growth pattern follows a biphasic behaviour: initially, the rapidly growing tumor cells tend to saturate the volume available without any significant increase in overall tumor size; then, a classical Gompertzian pattern is observed for the MTS radius variation with time. A core with necrotic cells appears for tumor sizes larger than 150 μm, surrounded by a shell of viable tumor cells whose thickness stays almost constant with time. A formula to estimate the size of the necrotic core is proposed. In the second case, the MTS is confined within a healthy tissue. The growth rate is reduced, as compared to the first case – mostly due to the relative adhesion of the tumor and healthy cells to the ECM, and the less favourable transport of nutrients. In particular, for tumor cells adhering less avidly to the ECM, the healthy tissue is progressively displaced

  10. Hydrodynamic simulation of multicellular embryo invagination

    NASA Astrophysics Data System (ADS)

    Pouille, Philippe-Alexandre; Farge, Emmanuel

    2008-03-01

    The mechanical aspects of embryonic morphogenesis have been widely analysed by numerical simulations of invagination in sea urchins and Drosophila gastrulation. Finite element models, which describe the tissue as a continuous medium, lead to the global invagination morphogenesis observed in vivo. Here we develop a simulation of multicellular embryo invagination that allows access to both cellular and multicellular mechanical behaviours of the embryo. In this model, the tissue is composed of adhesive individual cells, in which shape change dynamics is governed by internal acto-myosin forces and the hydrodynamic flow associated with membrane movements. We investigated the minimal structural and force elements sufficient to phenocopy mesoderm invagination. The minimal structures are cell membranes characterized by an acto-myosin cortical tension and connected by apical and basal junctions and an acto-myosin contractile ring connected to the apical junctions. An increase in the apical-cortical surface tension is the only control parameter change required to phenocopy most known multicellular and cellular shape changes of Drosophila gastrulation. Specifically, behaviours observed in vivo, including apical junction movements at the onset of gastrulation, cell elongation and subsequent shortening during invagination, and the development of a dorso-ventral gradient of thickness of the embryo, are predicted by this model as passive mechanical consequences of the genetically controlled increase in the apical surface tension in invaginating mesoderm cells, thus demonstrating the accurate description of structures at both global and single cell scales.

  11. Cooperation, clumping and the evolution of multicellularity

    PubMed Central

    Biernaskie, Jay M.; West, Stuart A.

    2015-01-01

    The evolution of multicellular organisms represents one of the major evolutionary transitions in the history of life. A potential advantage of forming multicellular clumps is that it provides an efficiency benefit to pre-existing cooperation, such as the production of extracellular ‘public goods’. However, this is complicated by the fact that cooperation could jointly evolve with clumping, and clumping could have multiple consequences for the evolution of cooperation. We model the evolution of clumping and a cooperative public good, showing that (i) when considered separately, both clumping and public goods production gradually increase with increasing genetic relatedness; (ii) in contrast, when the traits evolve jointly, a small increase in relatedness can lead to a major shift in evolutionary outcome—from a non-clumping state with low public goods production to a cooperative clumping state with high values of both traits; (iii) high relatedness makes it easier to get to the cooperative clumping state and (iv) clumping can be inhibited when it increases the number of cells that the benefits of cooperation must be shared with, but promoted when it increases relatedness between those cells. Overall, our results suggest that public goods sharing can facilitate the formation of well-integrated cooperative clumps as a first step in the evolution of multicellularity. PMID:26246549

  12. Collective Chemotaxis through Noisy Multicellular Gradient Sensing

    NASA Astrophysics Data System (ADS)

    Varennes, Julien; Han, Bumsoo; Mugler, Andrew

    2016-08-01

    Collective cell migration in response to a chemical cue occurs in many biological processes such as morphogenesis and cancer metastasis. Clusters of migratory cells in these systems are capable of responding to gradients of less than 1% difference in chemical concentration across a cell length. Multicellular systems are extremely sensitive to their environment and while the limits to multicellular sensing are becoming known, how this information leads to coherent migration remains poorly understood. We develop a computational model of multicellular sensing and migration in which groups of cells collectively measure noisy chemical gradients. The output of the sensing process is coupled to individual cells polarization to model migratory behavior. Through the use of numerical simulations, we find that larger clusters of cells detect the gradient direction with higher precision and thus achieve stronger polarization bias, but larger clusters also induce more drag on collective motion. The trade-off between these two effects leads to an optimal cluster size for most efficient migration. We discuss how our model could be validated using simple, phenomenological experiments.

  13. Cooperation, clumping and the evolution of multicellularity.

    PubMed

    Biernaskie, Jay M; West, Stuart A

    2015-08-22

    The evolution of multicellular organisms represents one of the major evolutionary transitions in the history of life. A potential advantage of forming multicellular clumps is that it provides an efficiency benefit to pre-existing cooperation, such as the production of extracellular 'public goods'. However, this is complicated by the fact that cooperation could jointly evolve with clumping, and clumping could have multiple consequences for the evolution of cooperation. We model the evolution of clumping and a cooperative public good, showing that (i) when considered separately, both clumping and public goods production gradually increase with increasing genetic relatedness; (ii) in contrast, when the traits evolve jointly, a small increase in relatedness can lead to a major shift in evolutionary outcome—from a non-clumping state with low public goods production to a cooperative clumping state with high values of both traits; (iii) high relatedness makes it easier to get to the cooperative clumping state and (iv) clumping can be inhibited when it increases the number of cells that the benefits of cooperation must be shared with, but promoted when it increases relatedness between those cells. Overall, our results suggest that public goods sharing can facilitate the formation of well-integrated cooperative clumps as a first step in the evolution of multicellularity.

  14. Collective Chemotaxis through Noisy Multicellular Gradient Sensing.

    PubMed

    Varennes, Julien; Han, Bumsoo; Mugler, Andrew

    2016-08-01

    Collective cell migration in response to a chemical cue occurs in many biological processes such as morphogenesis and cancer metastasis. Clusters of migratory cells in these systems are capable of responding to gradients of <1% difference in chemical concentration across a cell length. Multicellular systems are extremely sensitive to their environment, and although the limits to multicellular sensing are becoming known, how this information leads to coherent migration remains poorly understood. We develop a computational model of multicellular sensing and migration in which groups of cells collectively measure noisy chemical gradients. The output of the sensing process is coupled to the polarization of individual cells to model migratory behavior. Through the use of numerical simulations, we find that larger clusters of cells detect the gradient direction with higher precision and thus achieve stronger polarization bias, but larger clusters also induce more drag on collective motion. The trade-off between these two effects leads to an optimal cluster size for most efficient migration. We discuss how our model could be validated using simple, phenomenological experiments.

  15. Collective Chemotaxis through Noisy Multicellular Gradient Sensing.

    PubMed

    Varennes, Julien; Han, Bumsoo; Mugler, Andrew

    2016-08-01

    Collective cell migration in response to a chemical cue occurs in many biological processes such as morphogenesis and cancer metastasis. Clusters of migratory cells in these systems are capable of responding to gradients of <1% difference in chemical concentration across a cell length. Multicellular systems are extremely sensitive to their environment, and although the limits to multicellular sensing are becoming known, how this information leads to coherent migration remains poorly understood. We develop a computational model of multicellular sensing and migration in which groups of cells collectively measure noisy chemical gradients. The output of the sensing process is coupled to the polarization of individual cells to model migratory behavior. Through the use of numerical simulations, we find that larger clusters of cells detect the gradient direction with higher precision and thus achieve stronger polarization bias, but larger clusters also induce more drag on collective motion. The trade-off between these two effects leads to an optimal cluster size for most efficient migration. We discuss how our model could be validated using simple, phenomenological experiments. PMID:27508447

  16. 3D cell culture systems modeling tumor growth determinants in cancer target discovery.

    PubMed

    Thoma, Claudio R; Zimmermann, Miriam; Agarkova, Irina; Kelm, Jens M; Krek, Wilhelm

    2014-04-01

    Phenotypic heterogeneity of cancer cells, cell biological context, heterotypic crosstalk and the microenvironment are key determinants of the multistep process of tumor development. They sign responsible, to a significant extent, for the limited response and resistance of cancer cells to molecular-targeted therapies. Better functional knowledge of the complex intra- and intercellular signaling circuits underlying communication between the different cell types populating a tumor tissue and of the systemic and local factors that shape the tumor microenvironment is therefore imperative. Sophisticated 3D multicellular tumor spheroid (MCTS) systems provide an emerging tool to model the phenotypic and cellular heterogeneity as well as microenvironmental aspects of in vivo tumor growth. In this review we discuss the cellular, chemical and physical factors contributing to zonation and cellular crosstalk within tumor masses. On this basis, we further describe 3D cell culture technologies for growth of MCTS as advanced tools for exploring molecular tumor growth determinants and facilitating drug discovery efforts. We conclude with a synopsis on technological aspects for on-line analysis and post-processing of 3D MCTS models.

  17. Predicting the growth of glioblastoma multiforme spheroids using a multiphase porous media model.

    PubMed

    Mascheroni, Pietro; Stigliano, Cinzia; Carfagna, Melania; Boso, Daniela P; Preziosi, Luigi; Decuzzi, Paolo; Schrefler, Bernhard A

    2016-10-01

    Tumor spheroids constitute an effective in vitro tool to investigate the avascular stage of tumor growth. These three-dimensional cell aggregates reproduce the nutrient and proliferation gradients found in the early stages of cancer and can be grown with a strict control of their environmental conditions. In the last years, new experimental techniques have been developed to determine the effect of mechanical stress on the growth of tumor spheroids. These studies report a reduction in cell proliferation as a function of increasingly applied stress on the surface of the spheroids. This work presents a specialization for tumor spheroid growth of a previous more general multiphase model. The equations of the model are derived in the framework of porous media theory, and constitutive relations for the mass transfer terms and the stress are formulated on the basis of experimental observations. A set of experiments is performed, investigating the growth of U-87MG spheroids both freely growing in the culture medium and subjected to an external mechanical pressure induced by a Dextran solution. The growth curves of the model are compared to the experimental data, with good agreement for both the experimental settings. A new mathematical law regulating the inhibitory effect of mechanical compression on cancer cell proliferation is presented at the end of the paper. This new law is validated against experimental data and provides better results compared to other expressions in the literature.

  18. Cancer cell spheroids for screening of chemotherapeutics and drug-delivery systems.

    PubMed

    Patel, Niravkumar R; Aryasomayajula, Bhawani; Abouzeid, Abraham H; Torchilin, Vladimir P

    2015-01-01

    Over the last few decades, the most popular platform to perform high-throughput screening for viable anti-neoplastic compounds has been monolayer cell culture. However, cells in monolayer culture lose many of their in vivo characteristics. As a result, this platform provides a limited predictive value in determining the clinical outcome of the compounds of interest. Using a technique known as 3D spheroid culture, may be the answer to this conundrum. Spheroids have been shown to mimic the tissue-like properties of tumors necessary for the proper evaluation of compounds. In this review, production of cancer cell spheroids, utilization of these spheroids in understanding various therapeutic mechanisms and the potential for their use in high-throughput screening of drugs and drug-delivery systems are discussed in detail. PMID:25996047

  19. TGFβ signaling regulates epithelial-mesenchymal plasticity in ovarian cancer ascites-derived spheroids.

    PubMed

    Rafehi, Samah; Ramos Valdes, Yudith; Bertrand, Monique; McGee, Jacob; Préfontaine, Michel; Sugimoto, Akira; DiMattia, Gabriel E; Shepherd, Trevor G

    2016-03-01

    Epithelial-mesenchymal transition (EMT) serves as a key mechanism driving tumor cell migration, invasion, and metastasis in many carcinomas. Transforming growth factor-beta (TGFβ) signaling is implicated in several steps during cancer pathogenesis and acts as a classical inducer of EMT. Since epithelial ovarian cancer (EOC) cells have the potential to switch between epithelial and mesenchymal states during metastasis, we predicted that modulation of TGFβ signaling would significantly impact EMT and the malignant potential of EOC spheroid cells. Ovarian cancer patient ascites-derived cells naturally underwent an EMT response when aggregating into spheroids, and this was reversed upon spheroid re-attachment to a substratum. CDH1/E-cadherin expression was markedly reduced in spheroids compared with adherent cells, in concert with an up-regulation of several transcriptional repressors, i.e., SNAI1/Snail, TWIST1/2, and ZEB2. Treatment of EOC spheroids with the TGFβ type I receptor inhibitor, SB-431542, potently blocked the endogenous activation of EMT in spheroids. Furthermore, treatment of spheroids with SB-431542 upon re-attachment enhanced the epithelial phenotype of dispersing cells and significantly decreased cell motility and Transwell migration. Spheroid formation was significantly compromised by exposure to SB-431542 that correlated with a reduction in cell viability particularly in combination with carboplatin treatment. Thus, our findings are the first to demonstrate that intact TGFβ signaling is required to control EMT in EOC ascites-derived cell spheroids, and it promotes the malignant characteristics of these structures. As such, we show the therapeutic potential for targeted inhibition of this pathway in ovarian cancer patients with late-stage disease. PMID:26647384

  20. [Spheroid body myopathy: case report].

    PubMed

    Scola, Rosana Hermínia; Trentin, Alcides Júnior; Vaez, Rodrigo; Gignon, Vinicius de Faria; Costa, Thaís Gurgel; Werneck, Lineu Cesar

    2005-06-01

    Spheroid body myopathy is a rare illness classified in the group of the congenital myopathies as a desmin-related neuromuscular disorder, presenting dominant autosomical origin with the beginning of the symptoms in the adult phase. We report on a seven years old girl with facial paresia, generalized muscular hypotrophy and hypotony, generalized deep areflexia, proximal upper and lower limbs muscular strengh and distal upper limbs grade 3 and distal lower limbs grade 1. Needle electromyography evidenced increased conscription and potentials of motor unit of short duration and low amplitude, characterizing a myopathic standard. The muscle biopsy disclosed mixed standard to myopathy, denervation and inclusion bodies that are consistent to spheroid body myopathy. In this case, the patient presented, in advance, early beginning of the symptoms and there are no similar cases in the family.

  1. Silica bioreplication preserves three-dimensional spheroid structures of human pluripotent stem cells and HepG2 cells

    PubMed Central

    Lou, Yan-Ru; Kanninen, Liisa; Kaehr, Bryan; Townson, Jason L.; Niklander, Johanna; Harjumäki, Riina; Jeffrey Brinker, C.; Yliperttula, Marjo

    2015-01-01

    Three-dimensional (3D) cell cultures produce more in vivo-like multicellular structures such as spheroids that cannot be obtained in two-dimensional (2D) cell cultures. Thus, they are increasingly employed as models for cancer and drug research, as well as tissue engineering. It has proven challenging to stabilize spheroid architectures for detailed morphological examination. Here we overcome this issue using a silica bioreplication (SBR) process employed on spheroids formed from human pluripotent stem cells (hPSCs) and hepatocellular carcinoma HepG2 cells cultured in the nanofibrillar cellulose (NFC) hydrogel. The cells in the spheroids are more round and tightly interacting with each other than those in 2D cultures, and they develop microvilli-like structures on the cell membranes as seen in 2D cultures. Furthermore, SBR preserves extracellular matrix-like materials and cellular proteins. These findings provide the first evidence of intact hPSC spheroid architectures and similar fine structures to 2D-cultured cells, providing a pathway to enable our understanding of morphogenesis in 3D cultures. PMID:26323570

  2. Silica bioreplication preserves three-dimensional spheroid structures of human pluripotent stem cells and HepG2 cells

    SciTech Connect

    Lou, Yan-Ru; Kanninen, Liisa; Kaehr, Bryan; Townson, Jason L.; Niklander, Johanna; Harjumäki, Riina; Jeffrey Brinker, C.; Yliperttula, Marjo

    2015-09-01

    Three-dimensional (3D) cell cultures produce more in vivo-like multicellular structures such as spheroids that cannot be obtained in two-dimensional (2D) cell cultures. Thus, they are increasingly employed as models for cancer and drug research, as well as tissue engineering. It has proven challenging to stabilize spheroid architectures for detailed morphological examination. Here we overcome this issue using a silica bioreplication (SBR) process employed on spheroids formed from human pluripotent stem cells (hPSCs) and hepatocellular carcinoma HepG2 cells cultured in the nanofibrillar cellulose (NFC) hydrogel. The cells in the spheroids are more round and tightly interacting with each other than those in 2D cultures, and they develop microvilli-like structures on the cell membranes as seen in 2D cultures. Furthermore, SBR preserves extracellular matrix-like materials and cellular proteins. In conclusion, these findings provide the first evidence of intact hPSC spheroid architectures and similar fine structures to 2D-cultured cells, providing a pathway to enable our understanding of morphogenesis in 3D cultures.

  3. Silica bioreplication preserves three-dimensional spheroid structures of human pluripotent stem cells and HepG2 cells

    DOE PAGES

    Lou, Yan-Ru; Kanninen, Liisa; Kaehr, Bryan; Townson, Jason L.; Niklander, Johanna; Harjumäki, Riina; Jeffrey Brinker, C.; Yliperttula, Marjo

    2015-09-01

    Three-dimensional (3D) cell cultures produce more in vivo-like multicellular structures such as spheroids that cannot be obtained in two-dimensional (2D) cell cultures. Thus, they are increasingly employed as models for cancer and drug research, as well as tissue engineering. It has proven challenging to stabilize spheroid architectures for detailed morphological examination. Here we overcome this issue using a silica bioreplication (SBR) process employed on spheroids formed from human pluripotent stem cells (hPSCs) and hepatocellular carcinoma HepG2 cells cultured in the nanofibrillar cellulose (NFC) hydrogel. The cells in the spheroids are more round and tightly interacting with each other than thosemore » in 2D cultures, and they develop microvilli-like structures on the cell membranes as seen in 2D cultures. Furthermore, SBR preserves extracellular matrix-like materials and cellular proteins. In conclusion, these findings provide the first evidence of intact hPSC spheroid architectures and similar fine structures to 2D-cultured cells, providing a pathway to enable our understanding of morphogenesis in 3D cultures.« less

  4. Cell spheroids: the new frontiers in in vitro models for cancer drug validation.

    PubMed

    Chatzinikolaidou, Maria

    2016-09-01

    During the past decades, evaluation of anticancer drugs utilizing 2D cell cultures has been in common usage. In contrast to 2D cell cultures however, which lack many characteristics of the complex in vivo situation, 3D cell or tissue culture systems, such as cellular spheroids, better mimic the crucial tumor tissue properties and the microenvironment, and are thus more appropriate for the evaluation of pharmaceutical candidates. Taking the characteristics of the tumor microenvironment into consideration, crucial aspects and recent advances related to cell spheroids in the validation of anticancer drugs are discussed here. PMID:27371506

  5. Spheroid Culture of Mesenchymal Stem Cells

    PubMed Central

    Cesarz, Zoe; Tamama, Kenichi

    2016-01-01

    Compared with traditional 2D adherent cell culture, 3D spheroidal cell aggregates, or spheroids, are regarded as more physiological, and this technique has been exploited in the field of oncology, stem cell biology, and tissue engineering. Mesenchymal stem cells (MSCs) cultured in spheroids have enhanced anti-inflammatory, angiogenic, and tissue reparative/regenerative effects with improved cell survival after transplantation. Cytoskeletal reorganization and drastic changes in cell morphology in MSC spheroids indicate a major difference in mechanophysical properties compared with 2D culture. Enhanced multidifferentiation potential, upregulated expression of pluripotency marker genes, and delayed replicative senescence indicate enhanced stemness in MSC spheroids. Furthermore, spheroid formation causes drastic changes in the gene expression profile of MSC in microarray analyses. In spite of these significant changes, underlying molecular mechanisms and signaling pathways triggering and sustaining these changes are largely unknown. PMID:26649054

  6. Characterizing the Role of Nanoparticle Design on Tumor Transport and Stability in the Extracellular Environment

    NASA Astrophysics Data System (ADS)

    Albanese, Alexandre

    Nanotechnology has emerged as an exciting strategy for the delivery of diagnostic and therapeutic agents into established tumors. Advancements in nanomaterial synthesis have generated an extensive number of nanoparticle designs made from different materials. Unfortunately, it remains impossible to predict a design's effectiveness for in vivo tumor accumulation. Little is known about how a nanoparticle's morphology and surface chemistry affect its interactions with cells and proteins inside the tumor tissue. This thesis focuses on the development of in vitro experimental tools to evaluate how nanoparticle design affects transport in a three-dimensional tumor tissue and stability in the tumor microenvironment. Nanoparticle transport was evaluated using a novel 'tumor-on-a-chip' system where multicellular tumor spheroids were immobilized in a microfluidic channel. This setup created a three-dimensional tumor environment displaying physiological cell density, extracellular matrix organization, and interstitial flow rates. The tumor-on-a-chip demonstrated that accumulation of nanoparticles was limited to diameters below 110 nm and was improved by receptor targeting. Nanoparticle stability in the tumor microenvironment was evaluated using media isolated from different tumor cell lines. Nanoparticle diameter and surface chemistry were important determinants of stability in cancer cell-conditioned media. Small nanoparticles with unstable surface chemistries adsorbed cellular proteins on their surface and were prone to aggregation. Nanoparticle aggregation altered cellular interactions leading to changes in cell uptake. Using a novel technique to generate different aggregate sizes possessing a uniform surface composition, it was determined that aggregation can change receptor affinity, cell internalization mechanisms and sub-cellular sequestration patterns. Data from this thesis characterize the behavior of nanoparticles within modeled tumor environments and provide some

  7. Molecular imaging of temporal dynamics and spatial heterogeneity of hypoxia-inducible factor-1 signal transduction activity in tumors in living mice.

    PubMed

    Serganova, Inna; Doubrovin, Michael; Vider, Jelena; Ponomarev, Vladimir; Soghomonyan, Suren; Beresten, Tatiana; Ageyeva, Ludmila; Serganov, Alexander; Cai, Shangde; Balatoni, Julius; Blasberg, Ronald; Gelovani, Juri

    2004-09-01

    Tumor hypoxia is a spatially and temporally heterogeneous phenomenon, which results from several tumor and host tissue-specific processes. To study the dynamics and spatial heterogeneity of hypoxia-inducible factor-1 (HIF-1)-specific transcriptional activity in tumors, we used repetitive noninvasive positron emission tomography (PET) imaging of hypoxia-induced HIF-1 transcriptional activity in tumors in living mice. This approach uses a novel retroviral vector bearing a HIF-1-inducible "sensor" reporter gene (HSV1-tk/GFP fusion) and a constitutively expressed "beacon" reporter gene (DsRed2/XPRT). C6 glioma cells transduced with this multireporter system revealed dose-dependent patterns in temporal dynamics of HIF-1 transcriptional activity induced by either CoCl2 or decreased atmospheric oxygen concentration. Multicellular spheroids of C6 reporter cells developed a hypoxic core when >350 microm in diameter. 18F-2'-fluoro-2'deoxy-1beta-D-arabionofuranosyl-5-ethyl-uracil (FEAU) PET revealed spatial heterogeneity of HIF-1 transcriptional activity in reporter xenografts in mice as a function of size or ischemia-reperfusion injury. With increasing tumor diameter (>3 mm), a marked increase in HIF-1 transcriptional activity was observed in the core regions of tumors. Even a moderate ischemia-reperfusion injury in small C6 tumors caused a rapid induction of HIF-1 transcriptional activity, which persisted for a long time because of the inability of C6 tumors to rapidly compensate acute changes in tumor microcirculation.

  8. Spheroid Culture of Head and Neck Cancer Cells Reveals an Important Role of EGFR Signalling in Anchorage Independent Survival

    PubMed Central

    Braunholz, Diana; Saki, Mohammad; Niehr, Franziska; Öztürk, Merve; Borràs Puértolas, Berta; Konschak, Robert; Budach, Volker; Tinhofer, Ingeborg

    2016-01-01

    In solid tumours millions of cells are shed into the blood circulation each day. Only a subset of these circulating tumour cells (CTCs) survive, many of them presumable because of their potential to form multi-cellular clusters also named spheroids. Tumour cells within these spheroids are protected from anoikis, which allows them to metastasize to distant organs or re-seed at the primary site. We used spheroid cultures of head and neck squamous cell carcinoma (HNSCC) cell lines as a model for such CTC clusters for determining the role of the epidermal growth factor receptor (EGFR) in cluster formation ability and cell survival after detachment from the extra-cellular matrix. The HNSCC cell lines FaDu, SCC-9 and UT-SCC-9 (UT-SCC-9P) as well as its cetuximab (CTX)-resistant sub-clone (UT-SCC-9R) were forced to grow in an anchorage-independent manner by coating culture dishes with the anti-adhesive polymer poly-2-hydroxyethylmethacrylate (poly-HEMA). The extent of apoptosis, clonogenic survival and EGFR signalling under such culture conditions was evaluated. The potential of spheroid formation in suspension culture was found to be positively correlated with the proliferation rate of HNSCC cell lines as well as their basal EGFR expression levels. CTX and gefitinib blocked, whereas the addition of EGFR ligands promoted anchorage-independent cell survival and spheroid formation. Increased spheroid formation and growth were associated with persistent activation of EGFR and its downstream signalling component (MAPK/ERK). Importantly, HNSCC cells derived from spheroid cultures retained their clonogenic potential in the absence of cell-matrix contact. Addition of CTX under these conditions strongly inhibited colony formation in CTX-sensitive cell lines but not their resistant subclones. Altogether, EGFR activation was identified as crucial factor for anchorage-independent survival of HNSCC cells. Targeting EGFR in CTC cluster formation might represent an attractive anti

  9. Spheroid Culture of Head and Neck Cancer Cells Reveals an Important Role of EGFR Signalling in Anchorage Independent Survival.

    PubMed

    Braunholz, Diana; Saki, Mohammad; Niehr, Franziska; Öztürk, Merve; Borràs Puértolas, Berta; Konschak, Robert; Budach, Volker; Tinhofer, Ingeborg

    2016-01-01

    In solid tumours millions of cells are shed into the blood circulation each day. Only a subset of these circulating tumour cells (CTCs) survive, many of them presumable because of their potential to form multi-cellular clusters also named spheroids. Tumour cells within these spheroids are protected from anoikis, which allows them to metastasize to distant organs or re-seed at the primary site. We used spheroid cultures of head and neck squamous cell carcinoma (HNSCC) cell lines as a model for such CTC clusters for determining the role of the epidermal growth factor receptor (EGFR) in cluster formation ability and cell survival after detachment from the extra-cellular matrix. The HNSCC cell lines FaDu, SCC-9 and UT-SCC-9 (UT-SCC-9P) as well as its cetuximab (CTX)-resistant sub-clone (UT-SCC-9R) were forced to grow in an anchorage-independent manner by coating culture dishes with the anti-adhesive polymer poly-2-hydroxyethylmethacrylate (poly-HEMA). The extent of apoptosis, clonogenic survival and EGFR signalling under such culture conditions was evaluated. The potential of spheroid formation in suspension culture was found to be positively correlated with the proliferation rate of HNSCC cell lines as well as their basal EGFR expression levels. CTX and gefitinib blocked, whereas the addition of EGFR ligands promoted anchorage-independent cell survival and spheroid formation. Increased spheroid formation and growth were associated with persistent activation of EGFR and its downstream signalling component (MAPK/ERK). Importantly, HNSCC cells derived from spheroid cultures retained their clonogenic potential in the absence of cell-matrix contact. Addition of CTX under these conditions strongly inhibited colony formation in CTX-sensitive cell lines but not their resistant subclones. Altogether, EGFR activation was identified as crucial factor for anchorage-independent survival of HNSCC cells. Targeting EGFR in CTC cluster formation might represent an attractive anti

  10. Collective Calcium Signaling of Defective Multicellular Networks

    NASA Astrophysics Data System (ADS)

    Potter, Garrett; Sun, Bo

    2015-03-01

    A communicating multicellular network processes environmental cues into collective cellular dynamics. We have previously demonstrated that, when excited by extracellular ATP, fibroblast monolayers generate correlated calcium dynamics modulated by both the stimuli and gap junction communication between the cells. However, just as a well-connected neural network may be compromised by abnormal neurons, a tissue monolayer can also be defective with cancer cells, which typically have down regulated gap junctions. To understand the collective cellular dynamics in a defective multicellular network we have studied the calcium signaling of co-cultured breast cancer cells and fibroblast cells in various concentrations of ATP delivered through microfluidic devices. Our results demonstrate that cancer cells respond faster, generate singular spikes, and are more synchronous across all stimuli concentrations. Additionally, fibroblast cells exhibit persistent calcium oscillations that increase in regularity with greater stimuli. To interpret these results we quantitatively analyzed the immunostaining of purigenic receptors and gap junction channels. The results confirm our hypothesis that collective dynamics are mainly determined by the availability of gap junction communications.

  11. Magnetic Optimization in a Multicellular Magnetotactic Organism

    PubMed Central

    Winklhofer, Michael; Abraçado, Leida G.; Davila, Alfonso F.; Keim, Carolina N.; Lins de Barros, Henrique G. P.

    2007-01-01

    Unicellular magnetotactic prokaryotes, which typically carry a natural remanent magnetic moment equal to the saturation magnetic moment, are the prime example of magnetically optimized organisms. We here report magnetic measurements on a multicellular magnetotactic prokaryote (MMP) consisting of 17 undifferentiated cells (mean from 148 MMPs) with chains of ferrimagnetic particles in each cell. To test if the chain polarities of each cell contribute coherently to the total magnetic moment of the MMP, we used a highly sensitive magnetization measurement technique (1 fAm2) that enabled us to determine the degree of magnetic optimization (DMO) of individual MMPs in vivo. We obtained DMO values consistently above 80%. Numerical modeling shows that the probability of reaching a DMO > 80% would be as low as 0.017 for 17 randomly oriented magnetic dipoles. We simulated different scenarios to test whether high DMOs are attainable by aggregation or self-organization of individual magnetic cells. None of the scenarios investigated is likely to yield consistently high DMOs in each generation of MMPs. The observed high DMO values require strong Darwinian selection and a sophisticated reproduction mechanism. We suggest a multicellular life cycle as the most plausible scenario for transmitting the high DMO from one generation to the next. PMID:17071652

  12. Integration of single and multicellular wound responses

    PubMed Central

    Clark, Andrew G.; Miller, Ann L.; Vaughan, Emily; Yu, Hoi-Ying E.; Penkert, Rhiannon; Bement, William M.

    2013-01-01

    Summary Single cells and multicellular tissues rapidly heal wounds. These processes are considered distinct, but one mode of healing—Rho GTPase-dependent formation and closure of a purse string of actin filaments (F-actin) and myosin-2 around wounds—occurs in single cells (1,2) and in epithelia (3-10). Here we show that wounding of one cell in Xenopus embryos elicits Rho GTPase activation around the wound and at the nearest cell-cell junctions in the neighbor cells. F-actin and myosin-2 accumulate at the junctions as well as around the wound itself, and as the resultant actomyosin array closes over the wound site, junctional F-actin and myosin-2 become mechanically integrated with the actin and myosin-2 around the wound, forming a hybrid purse string. When cells are ablated rather than wounded, Rho GTPase activation and F-actin accumulation occur at cell-cell junctions surrounding the ablated cell, and the purse string closes the hole in the epithelium. Elevation of intracellular free calcium, an essential upstream signal for the single cell wound response (2,11), also occurs at the cell-cell contacts and in neighbor cells. Thus, the single and multicellular purse string wound responses represent points on a signaling and mechanical continuum that are integrated by cell-cell junctions. PMID:19631537

  13. Spheroid culture of LuCaP 136 patient-derived xenograft enables versatile preclinical models of prostate cancer.

    PubMed

    Valta, Maija P; Zhao, Hongjuan; Saar, Matthias; Tuomela, Johanna; Nolley, Rosalie; Linxweiler, Johannes; Sandholm, Jouko; Lehtimäki, Jaakko; Härkönen, Pirkko; Coleman, Ilsa; Nelson, Peter S; Corey, Eva; Peehl, Donna M

    2016-04-01

    LuCaP serially transplantable patient-derived xenografts (PDXs) are valuable preclinical models of locally advanced or metastatic prostate cancer. Using spheroid culture methodology, we recently established cell lines from several LuCaP PDXs. Here, we characterized in depth the features of xenografts derived from LuCaP 136 spheroid cultures and found faithful retention of the phenotype of the original PDX. In vitro culture enabled luciferase transfection into LuCaP 136 spheroids, facilitating in vivo imaging. We showed that LuCaP 136 spheroids formed intratibial, orthotopic, and subcutaneous tumors when re-introduced into mice. Intratibial tumors responded to castration and were highly osteosclerotic. LuCaP 136 is a realistic in vitro-in vivo preclinical model of a subtype of bone metastatic prostate cancer.

  14. Deciphering unusual uncultured magnetotactic multicellular prokaryotes through genomics

    PubMed Central

    Abreu, Fernanda; Morillo, Viviana; Nascimento, Fabrícia F; Werneck, Clarissa; Cantão, Mauricio Egidio; Ciapina, Luciane Prioli; de Almeida, Luiz Gonzaga Paula; Lefèvre, Christopher T; Bazylinski, Dennis A; de Vasconcelos, Ana Tereza Ribeiro; Lins, Ulysses

    2014-01-01

    Candidatus Magnetoglobus multicellularis (Ca. M. multicellularis) is a member of a group of uncultured magnetotactic prokaryotes that possesses a unique multicellular morphology. To better understand this organism's physiology, we used a genomic approach through pyrosequencing. Genomic data analysis corroborates previous structural studies and reveals the proteins that are likely involved in multicellular morphogenesis of this microorganism. Interestingly, some detected protein sequences that might be involved in cell adhesion are homologues to phylogenetically unrelated filamentous multicellular bacteria proteins, suggesting their contribution in the early development of multicellular organization in Bacteria. Genes related to the behavior of Ca. M. multicellularis (chemo-, photo- and magnetotaxis) and its metabolic capabilities were analyzed. On the basis of the genomic–physiologic information, enrichment media were tested. One medium supported chemoorganoheterotrophic growth of Ca. M. multicellularis and allowed the microorganisms to maintain their multicellular morphology and cell cycle, confirming for the first time that the entire life cycle of the MMP occurs in a multicellular form. Because Ca. M. multicellularis has a unique multicellular life style, its cultivation is an important achievement for further studies regarding the multicellular evolution in prokaryotes. PMID:24196322

  15. Elucidation of spheroid formation with and without the extrusion step.

    PubMed

    Liew, Celine V; Chua, Siang Meng; Heng, Paul W S

    2007-01-01

    Spheroid formation mechanisms were investigated using extrusion-spheronization (ES) and rotary processing (RP). Using ES (cross-hatch), ES (teardrop), and RP (teardrop), spheroids with similar mass median diameter (MMD) and span were produced using equivalent formulation and spheronization conditions. During spheronization, the teardrop-studded rotating frictional surface, with increased peripheral tip speed and duration, produced spheroids of equivalent MMD and span to those produced by the cross-hatch rotating frictional plate surface. The roundness of these spheroids was also similar. RP required less water to produce spheroids of MMD similar to that of spheroids produced by ES. However, these RP spheroids were less spherical. Image analysis of 625 spheroids per batch indicated that the size distribution of RP spheroids had significantly greater SD, positive skewness, and kurtosis. Morphological examination of time-sampled spheroids produced by ES indicated that spheroid formation occurred predominantly by attrition and layering, while RP spheroids were formed by nucleation, agglomeration, layering, and coalescence. RP produced spheroids with higher crushing strength than that of ES-produced spheroids. The amount of moisture lost during spheronization for spheroids produced by ES had minimal influence on their eventual size. Differences in process and formulation parameters, in addition to size distribution and observed morphological changes, enabled a greater understanding of spheroid formation and methods to optimize spheroid production. PMID:17408210

  16. Multicellular density fluctuations in epithelial monolayers

    NASA Astrophysics Data System (ADS)

    Zehnder, Steven M.; Wiatt, Marina K.; Uruena, Juan M.; Dunn, Alison C.; Sawyer, W. Gregory; Angelini, Thomas E.

    2015-09-01

    Changes in cell size often accompany multicellular motion in tissue, and cell number density is known to strongly influence collective migration in monolayers. Density fluctuations in other forms of active matter have been explored extensively, but not the potential role of density fluctuations in collective cell migration. Here we investigate collective motion in cell monolayers, focusing on the divergent component of the migration velocity field to probe density fluctuations. We find spatial patterns of diverging and converging cell groups throughout the monolayers, which oscillate in time with a period of approximately 3-4 h. Simultaneous fluorescence measurements of a cytosol dye within the cells show that fluid passes between groups of cells, facilitating these oscillations in cell density. Our findings reveal that cell-cell interactions in monolayers may be mediated by intercellular fluid flow.

  17. The multicellular complexity of peripheral nerve regeneration.

    PubMed

    Cattin, Anne-Laure; Lloyd, Alison C

    2016-08-01

    Peripheral nerves show a remarkable ability to regenerate following a transection injury. Downstream of the cut, the axons degenerate and so to regenerate the nerve, the severed axons need to regrow back to their targets and regain function. This requires the axons to navigate through two different environments. (1) The bridge of new tissue that forms between the two nerve stumps and (2) the distal stump of the nerve that remains associated with the target tissues. This involves distinct, complex multicellular responses that guide and sustain axonal regrowth. These processes have important implications for our understanding of the regeneration of an adult tissue and have parallels to aspects of tumour formation and spread. PMID:27128880

  18. Role of Multicellular Aggregates in Biofilm Formation

    PubMed Central

    Kragh, Kasper N.; Hutchison, Jaime B.; Melaugh, Gavin; Rodesney, Chris; Roberts, Aled E. L.; Irie, Yasuhiko; Jensen, Peter Ø.; Diggle, Stephen P.; Allen, Rosalind J.

    2016-01-01

    ABSTRACT In traditional models of in vitro biofilm development, individual bacterial cells seed a surface, multiply, and mature into multicellular, three-dimensional structures. Much research has been devoted to elucidating the mechanisms governing the initial attachment of single cells to surfaces. However, in natural environments and during infection, bacterial cells tend to clump as multicellular aggregates, and biofilms can also slough off aggregates as a part of the dispersal process. This makes it likely that biofilms are often seeded by aggregates and single cells, yet how these aggregates impact biofilm initiation and development is not known. Here we use a combination of experimental and computational approaches to determine the relative fitness of single cells and preformed aggregates during early development of Pseudomonas aeruginosa biofilms. We find that the relative fitness of aggregates depends markedly on the density of surrounding single cells, i.e., the level of competition for growth resources. When competition between aggregates and single cells is low, an aggregate has a growth disadvantage because the aggregate interior has poor access to growth resources. However, if competition is high, aggregates exhibit higher fitness, because extending vertically above the surface gives cells at the top of aggregates better access to growth resources. Other advantages of seeding by aggregates, such as earlier switching to a biofilm-like phenotype and enhanced resilience toward antibiotics and immune response, may add to this ecological benefit. Our findings suggest that current models of biofilm formation should be reconsidered to incorporate the role of aggregates in biofilm initiation. PMID:27006463

  19. Multicellular computing using conjugation for wiring.

    PubMed

    Goñi-Moreno, Angel; Amos, Martyn; de la Cruz, Fernando

    2013-01-01

    Recent efforts in synthetic biology have focussed on the implementation of logical functions within living cells. One aim is to facilitate both internal "re-programming" and external control of cells, with potential applications in a wide range of domains. However, fundamental limitations on the degree to which single cells may be re-engineered have led to a growth of interest in multicellular systems, in which a "computation" is distributed over a number of different cell types, in a manner analogous to modern computer networks. Within this model, individual cell type perform specific sub-tasks, the results of which are then communicated to other cell types for further processing. The manner in which outputs are communicated is therefore of great significance to the overall success of such a scheme. Previous experiments in distributed cellular computation have used global communication schemes, such as quorum sensing (QS), to implement the "wiring" between cell types. While useful, this method lacks specificity, and limits the amount of information that may be transferred at any one time. We propose an alternative scheme, based on specific cell-cell conjugation. This mechanism allows for the direct transfer of genetic information between bacteria, via circular DNA strands known as plasmids. We design a multi-cellular population that is able to compute, in a distributed fashion, a Boolean XOR function. Through this, we describe a general scheme for distributed logic that works by mixing different strains in a single population; this constitutes an important advantage of our novel approach. Importantly, the amount of genetic information exchanged through conjugation is significantly higher than the amount possible through QS-based communication. We provide full computational modelling and simulation results, using deterministic, stochastic and spatially-explicit methods. These simulations explore the behaviour of one possible conjugation-wired cellular computing

  20. Geometry shapes evolution of early multicellularity.

    PubMed

    Libby, Eric; Ratcliff, William; Travisano, Michael; Kerr, Ben

    2014-09-01

    Organisms have increased in complexity through a series of major evolutionary transitions, in which formerly autonomous entities become parts of a novel higher-level entity. One intriguing feature of the higher-level entity after some major transitions is a division of reproductive labor among its lower-level units in which reproduction is the sole responsibility of a subset of units. Although it can have clear benefits once established, it is unknown how such reproductive division of labor originates. We consider a recent evolution experiment on the yeast Saccharomyces cerevisiae as a unique platform to address the issue of reproductive differentiation during an evolutionary transition in individuality. In the experiment, independent yeast lineages evolved a multicellular "snowflake-like" cluster formed in response to gravity selection. Shortly after the evolution of clusters, the yeast evolved higher rates of cell death. While cell death enables clusters to split apart and form new groups, it also reduces their performance in the face of gravity selection. To understand the selective value of increased cell death, we create a mathematical model of the cellular arrangement within snowflake yeast clusters. The model reveals that the mechanism of cell death and the geometry of the snowflake interact in complex, evolutionarily important ways. We find that the organization of snowflake yeast imposes powerful limitations on the available space for new cell growth. By dying more frequently, cells in clusters avoid encountering space limitations, and, paradoxically, reach higher numbers. In addition, selection for particular group sizes can explain the increased rate of apoptosis both in terms of total cell number and total numbers of collectives. Thus, by considering the geometry of a primitive multicellular organism we can gain insight into the initial emergence of reproductive division of labor during an evolutionary transition in individuality.

  1. Light scattering by a spheroidal particle.

    PubMed

    Asano, S; Yamamoto, G

    1975-01-01

    The solution of electromagnetic scattering by a homogeneous prolate (or oblate) spheroidal particle with an arbitrary size and refractive index is obtained for any angle of incidence by solving Maxwell's equations under given boundary conditions. The method used is that of separating the vector wave equations in the spheroidal coordinates and expanding them in terms of the spheroidal wavefunctions. The unknown coefficients for the expansion are determined by a system of equations derived from the boundary conditions regarding the continuity of tangential components of the electric and magnetic vectors across the surface of the spheroid. The solutions both in the prolate and oblate spheroidal coordinate systems result in a same form, and the equations for the oblate spheroidal system can be obtained from those for the prolate one by replacing the prolate spheroidal wavefunctions with the oblate ones and vice versa. For an oblique incidence, the polarized incident wave is resolved into two components, the TM mode for which the magnetic vector vibrates perpendicularly to the incident plane and the TE mode for which the electric vector vibrates perpendicularly to this plane. For the incidence along the rotation axis the resultant equations are given in the form similar to the one for a sphere given by the Mie theory. The physical parameters involved are the following five quantities: the size parameter defined by the product of the semifocal distance of the spheroid and the propagation constant of the incident wave, the eccentricity, the refractive index of the spheroid relative to the surrounding medium, the incident angle between the direction of the incident wave and the rotation axis, and the angles that specify the direction of the scattered wave.

  2. A multiphase model for three-dimensional tumor growth

    NASA Astrophysics Data System (ADS)

    Sciumè, G.; Shelton, S.; Gray, W. G.; Miller, C. T.; Hussain, F.; Ferrari, M.; Decuzzi, P.; Schrefler, B. A.

    2013-01-01

    Several mathematical formulations have analyzed the time-dependent behavior of a tumor mass. However, most of these propose simplifications that compromise the physical soundness of the model. Here, multiphase porous media mechanics is extended to model tumor evolution, using governing equations obtained via the thermodynamically constrained averaging theory. A tumor mass is treated as a multiphase medium composed of an extracellular matrix (ECM); tumor cells (TCs), which may become necrotic depending on the nutrient concentration and tumor phase pressure; healthy cells (HCs); and an interstitial fluid for the transport of nutrients. The equations are solved by a finite element method to predict the growth rate of the tumor mass as a function of the initial tumor-to-healthy cell density ratio, nutrient concentration, mechanical strain, cell adhesion and geometry. Results are shown for three cases of practical biological interest such as multicellular tumor spheroids (MTSs) and tumor cords. First, the model is validated by experimental data for time-dependent growth of an MTS in a culture medium. The tumor growth pattern follows a biphasic behavior: initially, the rapidly growing TCs tend to saturate the volume available without any significant increase in overall tumor size; then, a classical Gompertzian pattern is observed for the MTS radius variation with time. A core with necrotic cells appears for tumor sizes larger than 150 μm, surrounded by a shell of viable TCs whose thickness stays almost constant with time. A formula to estimate the size of the necrotic core is proposed. In the second case, the MTS is confined within a healthy tissue. The growth rate is reduced, as compared to the first case—mostly due to the relative adhesion of the TCs and HCs to the ECM, and the less favorable transport of nutrients. In particular, for HCs adhering less avidly to the ECM, the healthy tissue is progressively displaced as the malignant mass grows, whereas TC

  3. Spheroidization of medium-carbon steels

    SciTech Connect

    O`Brien, J.M.; Hosford, W.F.

    1997-02-01

    Spheroidization experiments were made on a medium-carbon AISI 4037 steel, using both intercritical and subcritical annealing cycles. The results indicate that in the subcritical cycle the spheroidization occurred much more quickly than expected, so that shorter times were sufficient to achieve high formability. On the other hand, the hardness dropped faster in the intercritical cycle. Although more work needs to be done, these results suggest that using a subcritical spheroidization process instead of an intercritical process could achieve considerable savings in time, energy, and cost.

  4. Rapid Formation of Cell Aggregates and Spheroids Induced by a "Smart" Boronic Acid Copolymer.

    PubMed

    Amaral, Adérito J R; Pasparakis, George

    2016-09-01

    Cell surface engineering has emerged as a powerful approach to forming cell aggregates/spheroids and cell-biomaterial ensembles with significant uses in tissue engineering and cell therapeutics. Herein, we demonstrate that cell membrane remodeling with a thermoresponsive boronic acid copolymer induces the rapid formation of spheroids using either cancer or cardiac cell lines under conventional cell culture conditions at minute concentrations. It is shown that the formation of well-defined spheroids is accelerated by at least 24 h compared to non-polymer-treated controls, and, more importantly, the polymer allows for fine control of the aggregation kinetics owing to its stimulus response to temperature and glucose content. On the basis of its simplicity and effectiveness to promote cellular aggregation, this platform holds promise in three-dimensional tissue/tumor modeling and tissue engineering applications. PMID:27571512

  5. Light scattering by a multilayered spheroidal particle

    NASA Astrophysics Data System (ADS)

    Farafonov, Victor G.; Voshchinnikov, Nikolai V.

    2012-04-01

    The light scattering problem for a confocal multilayered spheroid has been solved by the extended boundary condition method (EBCM) with a corresponding spheroidal basis. The solution preserves the advantages of the approach applied previously to homogeneous and core-mantle spheroids, i.e. the separation of the radiation fields into two parts and a special choice of scalar potentials for each of the parts. The method is known to be useful in a wide range of the particle parameters. It is particularly efficient for strongly prolate and oblate spheroids. Numerical tests are described. Illustrative calculations have shown that the extinction factors to converge to average values with a growing number of layers and how the extinction vary with a growth of particle porosity.

  6. Bar-spheroid interaction in galaxies

    NASA Technical Reports Server (NTRS)

    Hernquist, Lars; Weinberg, Martin D.

    1992-01-01

    N-body simulation and linear analysis is employed to investigate the secular evolution of barred galaxies, with emphasis on the interaction between bars and spheroidal components of galaxies. This interaction is argued to drive secular transfer of angular momentum from bars to spheroids, primarily through resonant coupling. A moderately strong bar, having mass within corotation about 0.3 times the enclosed spheroid mass, is predicted to shed all its angular momentum typically in less than about 10 exp 9 yr. Even shorter depletion time scales are found for relatively more massive bars. It is suggested either that spheroids around barred galaxies are structured so as to inhibit strong coupling with bars, or that bars can form by unknown processes long after disks are established. The present models reinforce the notion that bars can drive secular evolution in galaxies.

  7. Organ printing: tissue spheroids as building blocks.

    PubMed

    Mironov, Vladimir; Visconti, Richard P; Kasyanov, Vladimir; Forgacs, Gabor; Drake, Christopher J; Markwald, Roger R

    2009-04-01

    Organ printing can be defined as layer-by-layer additive robotic biofabrication of three-dimensional functional living macrotissues and organ constructs using tissue spheroids as building blocks. The microtissues and tissue spheroids are living materials with certain measurable, evolving and potentially controllable composition, material and biological properties. Closely placed tissue spheroids undergo tissue fusion - a process that represents a fundamental biological and biophysical principle of developmental biology-inspired directed tissue self-assembly. It is possible to engineer small segments of an intraorgan branched vascular tree by using solid and lumenized vascular tissue spheroids. Organ printing could dramatically enhance and transform the field of tissue engineering by enabling large-scale industrial robotic biofabrication of living human organ constructs with "built-in" perfusable intraorgan branched vascular tree. Thus, organ printing is a new emerging enabling technology paradigm which represents a developmental biology-inspired alternative to classic biodegradable solid scaffold-based approaches in tissue engineering.

  8. Self-electrophoresis of spheroidal electrocatalytic swimmers

    NASA Astrophysics Data System (ADS)

    Nourhani, Amir; Crespi, Vincent H.; Lammert, Paul E.; Borhan, Ali

    2015-09-01

    Using the method of matched asymptotic expansions, we derive a general expression for the speed of a prolate spheroidal electrocatalytic nanomotor in terms of interfacial potential and physical properties of the motor environment in the limit of small Debye length and Péclet number. This greatly increases the range of geometries that can be handled without resorting to numerical simulations, since a wide range of shapes from spherical to needle-like, and in particular the common cylindrical shape, can be well-approximated by prolate spheroids. For piecewise-uniform distribution of surface cation flux with fixed average absolute value, the mobility of a prolate spheroidal motor with a symmetric cation source/sink configuration is a monotonically decreasing function of eccentricity. A prolate spheroidal motor with an asymmetric sink/source configuration moves faster than its symmetric counterpart and can exhibit a non-monotonic dependence of motor speed on eccentricity for a highly asymmetric design.

  9. Infection and cancer in multicellular organisms

    PubMed Central

    Ewald, Paul W.; Swain Ewald, Holly A.

    2015-01-01

    Evolutionary considerations suggest that oncogenic infections should be pervasive among animal species. Infection-associated cancers are well documented in humans and domestic animals, less commonly reported in undomesticated captive animals, and rarely documented in nature. In this paper, we review the literature associating infectious agents with cancer to evaluate the reasons for this pattern. Non-malignant infectious neoplasms occur pervasively in multicellular life, but oncogenic progression to malignancy is often uncertain. Evidence from humans and domestic animals shows that non-malignant infectious neoplasms can develop into cancer, although generally with low frequency. Malignant neoplasms could be difficult to find in nature because of a low frequency of oncogenic transformation, short survival after malignancy and reduced survival prior to malignancy. Moreover, the evaluation of malignancy can be ambiguous in nature, because criteria for malignancy may be difficult to apply consistently across species. The information available in the literature therefore does not allow for a definitive assessment of the pervasiveness of infectious cancers in nature, but the presence of infectious neoplasias and knowledge about the progression of benign neoplasias to cancer is consistent with a widespread but largely undetected occurrence. PMID:26056368

  10. Apprehending multicellularity: regulatory networks, genomics and evolution

    PubMed Central

    Aravind, L.; Anantharaman, Vivek; Venancio, Thiago M.

    2009-01-01

    The genomic revolution has provided the first glimpses of the architecture of regulatory networks. Combined with evolutionary information, the “network view” of life processes leads to remarkable insights into how biological systems have been shaped by various forces. This understanding is critical because biological systems, including regulatory networks, are not products of engineering but of historical contingencies. In this light, we attempt a synthetic overview of the natural history of regulatory networks operating in the development and differentiation of multicellular organisms. We first introduce regulatory networks and their organizational principles as can be deduced using ideas from the graph theory. We then discuss findings from comparative genomics to illustrate the effects of lineage-specific expansions, gene-loss, and non-protein-coding DNA on the architecture of networks. We consider the interaction between expansions of transcription factors, and cis regulatory and more general chromatin state stabilizing elements in the emergence of morphological complexity. Finally, we consider a case study of the Notch sub-network, which is present throughout Metazoa, to examine how such a regulatory system has been pieced together in evolution from new innovations and pre-existing components that were originally functionally distinct. PMID:19530132

  11. Multicellular oxidant defense in unicellular organisms.

    PubMed Central

    Ma, M; Eaton, J W

    1992-01-01

    Although catalase is thought to be a major defense against hydrogen peroxide (H2O2), the catalase activity within individual Escherichia coli fails to protect against exogenous H2O2. Contrary to earlier reports, we find that dilute suspensions of wild-type and catalase-deficient E. coli are identical in their sensitivity to H2O2, perhaps because even wild-type, catalase-positive E. coli cannot maintain an internal/external concentration gradient of this highly diffusible oxidant. However, concentrated suspensions or colonies of catalase-positive E. coli do preferentially survive H2O2 challenge and can even cross-protect adjacent catalase-deficient organisms. Furthermore, high-density catalase-positive--but not catalase-negative--E. coli can survive and multiply in the presence of competitive, peroxide-generating streptococci. These observations support the concept that bacterial catalase may defend colonial, but not individual, E. coli against environmental H2O2. Group protection by the activity of enzymes that mitigate oxidative stress may have been a driving force in the evolution of multicellular organisms. Images PMID:1518815

  12. Infection and cancer in multicellular organisms.

    PubMed

    Ewald, Paul W; Swain Ewald, Holly A

    2015-07-19

    Evolutionary considerations suggest that oncogenic infections should be pervasive among animal species. Infection-associated cancers are well documented in humans and domestic animals, less commonly reported in undomesticated captive animals, and rarely documented in nature. In this paper, we review the literature associating infectious agents with cancer to evaluate the reasons for this pattern. Non-malignant infectious neoplasms occur pervasively in multicellular life, but oncogenic progression to malignancy is often uncertain. Evidence from humans and domestic animals shows that non-malignant infectious neoplasms can develop into cancer, although generally with low frequency. Malignant neoplasms could be difficult to find in nature because of a low frequency of oncogenic transformation, short survival after malignancy and reduced survival prior to malignancy. Moreover, the evaluation of malignancy can be ambiguous in nature, because criteria for malignancy may be difficult to apply consistently across species. The information available in the literature therefore does not allow for a definitive assessment of the pervasiveness of infectious cancers in nature, but the presence of infectious neoplasias and knowledge about the progression of benign neoplasias to cancer is consistent with a widespread but largely undetected occurrence. PMID:26056368

  13. Communication shapes sensory response in multicellular networks.

    PubMed

    Potter, Garrett D; Byrd, Tommy A; Mugler, Andrew; Sun, Bo

    2016-09-13

    Collective sensing by interacting cells is observed in a variety of biological systems, and yet, a quantitative understanding of how sensory information is collectively encoded is lacking. Here, we investigate the ATP-induced calcium dynamics of monolayers of fibroblast cells that communicate via gap junctions. Combining experiments and stochastic modeling, we find that increasing the ATP stimulus increases the propensity for calcium oscillations, despite large cell-to-cell variability. The model further predicts that the oscillation propensity increases with not only the stimulus, but also the cell density due to increased communication. Experiments confirm this prediction, showing that cell density modulates the collective sensory response. We further implicate cell-cell communication by coculturing the fibroblasts with cancer cells, which we show act as "defects" in the communication network, thereby reducing the oscillation propensity. These results suggest that multicellular networks sit at a point in parameter space where cell-cell communication has a significant effect on the sensory response, allowing cells to simultaneously respond to a sensory input and the presence of neighbors.

  14. Global gene expression analysis of early response to chemotherapy treatment in ovarian cancer spheroids

    PubMed Central

    L'Espérance, Sylvain; Bachvarova, Magdalena; Tetu, Bernard; Mes-Masson, Anne-Marie; Bachvarov, Dimcho

    2008-01-01

    Background Chemotherapy (CT) resistance in ovarian cancer (OC) is broad and encompasses diverse unrelated drugs, suggesting more than one mechanism of resistance. To better understand the molecular mechanisms controlling the immediate response of OC cells to CT exposure, we have performed gene expression profiling in spheroid cultures derived from six OC cell lines (OVCAR3, SKOV3, TOV-112, TOV-21, OV-90 and TOV-155), following treatment with 10,0 μM cisplatin, 2,5 μM paclitaxel or 5,0 μM topotecan for 72 hours. Results Exposure of OC spheroids to these CT drugs resulted in differential expression of genes associated with cell growth and proliferation, cellular assembly and organization, cell death, cell cycle control and cell signaling. Genes, functionally involved in DNA repair, DNA replication and cell cycle arrest were mostly overexpressed, while genes implicated in metabolism (especially lipid metabolism), signal transduction, immune and inflammatory response, transport, transcription regulation and protein biosynthesis, were commonly suppressed following all treatments. Cisplatin and topotecan treatments triggered similar alterations in gene and pathway expression patterns, while paclitaxel action was mainly associated with induction of genes and pathways linked to cellular assembly and organization (including numerous tubulin genes), cell death and protein synthesis. The microarray data were further confirmed by pathway and network analyses. Conclusion Most alterations in gene expression were directly related to mechanisms of the cytotoxics actions in OC spheroids. However, the induction of genes linked to mechanisms of DNA replication and repair in cisplatin- and topotecan-treated OC spheroids could be associated with immediate adaptive response to treatment. Similarly, overexpression of different tubulin genes upon exposure to paclitaxel could represent an early compensatory effect to this drug action. Finally, multicellular growth conditions that are

  15. Collective behavior of brain tumor cells: The role of hypoxia

    NASA Astrophysics Data System (ADS)

    Khain, Evgeniy; Katakowski, Mark; Hopkins, Scott; Szalad, Alexandra; Zheng, Xuguang; Jiang, Feng; Chopp, Michael

    2011-03-01

    We consider emergent collective behavior of a multicellular biological system. Specifically, we investigate the role of hypoxia (lack of oxygen) in migration of brain tumor cells. We performed two series of cell migration experiments. In the first set of experiments, cell migration away from a tumor spheroid was investigated. The second set of experiments was performed in a typical wound-healing geometry: Cells were placed on a substrate, a scratch was made, and cell migration into the gap was investigated. Experiments show a surprising result: Cells under normal and hypoxic conditions have migrated the same distance in the “spheroid” experiment, while in the “scratch” experiment cells under normal conditions migrated much faster than under hypoxic conditions. To explain this paradox, we formulate a discrete stochastic model for cell dynamics. The theoretical model explains our experimental observations and suggests that hypoxia decreases both the motility of cells and the strength of cell-cell adhesion. The theoretical predictions were further verified in independent experiments.

  16. Dwarf spheroidal galaxies and resonant orbital coupling

    NASA Technical Reports Server (NTRS)

    Kuhn, J. R.; Miller, R. H.

    1989-01-01

    The structural properties of the dwarf spheroidal satellite galaxies of the Milky Way may be strongly affected by their time-dependent interactions with the 'tidal' field of the Milky Way. A low Q resonance of the tidal driving force with collective oscillation modes of the dwarf system can produce many of the observed properties of the Local Group dwarf spheroidal galaxies, including large velocity dispersions that would normally be interpreted as indicating large dynamical masses.

  17. Approximating spheroid inductive responses using spheres

    SciTech Connect

    Smith, J. Torquil; Morrison, H. Frank

    2003-12-12

    The response of high permeability ({mu}{sub r} {ge} 50) conductive spheroids of moderate aspect ratios (0.25 to 4) to excitation by uniform magnetic fields in the axial or transverse directions is approximated by the response of spheres of appropriate diameters, of the same conductivity and permeability, with magnitude rescaled based on the differing volumes, D.C. magnetizations, and high frequency limit responses of the spheres and modeled spheroids.

  18. Evaluation of drug penetration with cationic micelles and their penetration mechanism using an in vitro tumor model.

    PubMed

    Suzuki, Hidenori; Bae, You Han

    2016-08-01

    Elevated interstitial fluid pressure (IFP) and abnormal extracellular matrix (ECM) are major factors causing significant barriers to penetration of nanomedicines in solid tumors. To better understand the barriers, various in vitro tumor models including multicellular spheroids and multilayered cell cultures (MCCs) have been developing. Recently, we have established a unique in vitro tumor model composed of a MCC and an Ussing chamber system which is modified to add a hydraulic pressure gradient through the MCC. In this study, we evaluated the drug penetration ability of cationic micelles using the unique in vitro tumor model. The doxorubicin (DOX)-loaded cationic micelles, which are formed from a triblock copolymer of poly(d,l-lactide-co-glycolide)-block-branched polyethyleneimine-block-poly(d,l-lactide-co-glycolide), deeply penetrated and released the DOX throughout the MCC against convectional flow caused by a hydraulic pressure gradient. The studies using endocytosis markers and inhibitors showed that the micelles utilized mainly macropinocytosis as an internalization pathway. Furthermore, the penetration was apparently inhibited by treatment with exocytosis inhibitor Exo1. These results suggest that the active penetration of the cationic micelles is induced by iterative transcytosis via macropinocytosis and exocytosis. Our findings could be beneficial information to improve intratumoral penetration in new nanomedicines for solid tumors. PMID:27182814

  19. Tumor

    MedlinePlus

    ... plants (aflatoxins) Excessive sunlight exposure Genetic problems Obesity Radiation exposure Viruses Types of tumors known to be caused by viruses are: Cervical cancer (human papillomavirus) Hepatocellular carcinoma (hepatitis B and hepatitis C ...

  20. Mechanical Trade-offs in Experimentally Evolved Multicellular Yeast

    NASA Astrophysics Data System (ADS)

    Jacobeen, Shane; Pentz, Jennifer; Ratcliff, William; Yunker, Peter

    The evolution of multicellularity as much about physics as it is about biology, as selection acts on the physical properties of multicellular bodies. Nascent multicellular organisms are confronted by internal and external forces that act on large length scales and are capable of fracturing intercellular bonds. We study the evolution of the mechanical properties of multicellular `snowflake' yeast that were selected for increased size over ~1,500 generations. While these snowflakes evolve to be larger by mitigating internal forces, they also become more susceptible to fracturing when faced with external compressive forces. Using confocal microscopy and direct mechanical measurements, we investigate the physical underpinnings and consequences of this strength-toughness trade-off.

  1. Aggregative multicellularity evolved independently in the eukaryotic supergroup Rhizaria.

    PubMed

    Brown, Matthew W; Kolisko, Martin; Silberman, Jeffrey D; Roger, Andrew J

    2012-06-19

    Multicellular forms of life have evolved many times, independently giving rise to a diversity of organisms such as animals, plants, and fungi that together comprise the visible biosphere. Yet multicellular life is far more widespread among eukaryotes than just these three lineages. A particularly common form of multicellularity is a social aggregative fruiting lifestyle whereby individual cells associate to form a "fungus-like" sorocarp. This complex developmental process that requires the interaction of thousands of cells working in concert was made famous by the "cellular slime mold"Dictyostelium discoideum, which became an important model organism. Although sorocarpic protistan lineages have been identified in five of the major eukaryote groups, the ubiquitous and globally distributed species Guttulinopsis vulgaris has eluded proper classification. Here we demonstrate, by phylogenomic analyses of a 159-protein data set, that G. vulgaris is a member of Rhizaria and is thus the first member of this eukaryote supergroup known to be capable of aggregative multicellularity. PMID:22608512

  2. α2β1 integrin affects metastatic potential of ovarian carcinoma spheroids by supporting disaggregation and proteolysis

    PubMed Central

    Shield, Kristy; Riley, Clyde; Quinn, Michael A; Rice, Gregory E; Ackland, Margaret L; Ahmed, Nuzhat

    2007-01-01

    Background Ovarian cancer is characterized by a wide-spread intra-abdominal metastases which represents a major clinical hurdle in the prognosis and management of the disease. A significant proportion of ovarian cancer cells in peritoneal ascites exist as multicellular aggregates or spheroids. We hypothesize that these cellular aggregates or spheroids are invasive with the capacity to survive and implant on the peritoneal surface. This study was designed to elucidate early inherent mechanism(s) of spheroid survival, growth and disaggregation required for peritoneal metastases Methods In this study, we determined the growth pattern and adhesive capacity of ovarian cancer cell lines (HEY and OVHS1) grown as spheroids, using the well established liquid overlay technique, and compared them to a normal ovarian cell line (IOSE29) and cancer cells grown as a monolayer. The proteolytic capacity of these spheroids was compared with cells grown as a monolayer using a gelatin zymography assay to analyze secreted MMP-2/9 in conditioned serum-free medium. The disaggregation of cancer cell line spheroids was determined on extracellular matrices (ECM) such as laminin (LM), fibronectin (FN) and collagen (CI) and the expression of α2, α3, αv, α6 and β1 interin was determined by flow cytometric analysis. Neutralizing antibodies against α2, β1 subunits and α2β1 integrin was used to inhibit disaggregation as well as activation of MMPs in spheroids. Results We demonstrate that ovarian cancer cell lines grown as spheroids can sustain growth for 10 days while the normal ovarian cell line failed to grow beyond 2 days. Compared to cells grown as a monolayer, cancer cells grown as spheroids demonstrated no change in adhesion for up to 4 days, while IOSE29 cells had a 2–4-fold loss of adhesion within 2 days. Cancer cell spheroids disaggregated on extracellular matrices (ECM) and demonstrated enhanced expression of secreted pro-MMP2 as well as activated MMP2/MMP9 with no such

  3. Versatile Functions of p53 Protein in Multicellular Organisms

    PubMed Central

    Chumakov, P. M.

    2009-01-01

    The p53 tumor suppressor plays a pivotal role by controlling virtually all processes in the cell. The functions of p53 determine modes of behavior of cells in multicellular organisms and ensure priorities of interests of the organism as a whole above the interests of an individual cell. Multiple signaling pathways of the cell report signals modifying the activities of p53 through numerous connections, ensuring highly selective and gradual regulation of functions that depend on the ongoing events in the cell. The task of p53 is to control the integrity and correctness of all processes in each individual cell and in the organism as a whole. The changes in the activity of p53 depend on the degree of errors or faults, and the effect is directed either toward correction of an imbalance or damage, or, in case of severe damages, leads to the prevention of multiplication of abnormal cells or their death. The strategy of p53 ensures genetic identity of cells and prevents the selection of cells having growth or other advantages. By accomplishing these strategic tasks, p53 may use a wide spectrum of activities. The majority of the activities are due to the ability of p53 to function as a transcription factor, by inducing or repressing different genes. However, p53 can also function as an enzyme, acting as an exonuclease during DNA reparation, or as an adaptor or a regulatory protein, intervening into functions of numerous signaling pathways. It can also act as direct inducer of apoptosis by translocation into mitochondria. Loss of function of the p53 gene occurs in virtually every case of cancer, and deficiency in p53 is an unavoidable prerequisite to the development of malignancies. The functions of p53 play substantial roles in many other pathologies as well as in the aging process. This review is focused on strategies of the p53 gene, demonstrating individual mechanisms underlying its functions. The p53 tumor suppressor plays a pivotal role in multicellular organism by

  4. Boswellia sacra essential oil induces tumor cell-specific apoptosis and suppresses tumor aggressiveness in cultured human breast cancer cells

    PubMed Central

    2011-01-01

    Background Gum resins obtained from trees of the Burseraceae family (Boswellia sp.) are important ingredients in incense and perfumes. Extracts prepared from Boswellia sp. gum resins have been shown to possess anti-inflammatory and anti-neoplastic effects. Essential oil prepared by distillation of the gum resin traditionally used for aromatic therapy has also been shown to have tumor cell-specific anti-proliferative and pro-apoptotic activities. The objective of this study was to optimize conditions for preparing Boswellea sacra essential oil with the highest biological activity in inducing tumor cell-specific cytotoxicity and suppressing aggressive tumor phenotypes in human breast cancer cells. Methods Boswellia sacra essential oil was prepared from Omani Hougari grade resins through hydrodistillation at 78 or 100 oC for 12 hours. Chemical compositions were identified by gas chromatography-mass spectrometry; and total boswellic acids contents were quantified by high-performance liquid chromatography. Boswellia sacra essential oil-mediated cell viability and death were studied in established human breast cancer cell lines (T47D, MCF7, MDA-MB-231) and an immortalized normal human breast cell line (MCF10-2A). Apoptosis was assayed by genomic DNA fragmentation. Anti-invasive and anti-multicellular tumor properties were evaluated by cellular network and spheroid formation models, respectively. Western blot analysis was performed to study Boswellia sacra essential oil-regulated proteins involved in apoptosis, signaling pathways, and cell cycle regulation. Results More abundant high molecular weight compounds, including boswellic acids, were present in Boswellia sacra essential oil prepared at 100 oC hydrodistillation. All three human breast cancer cell lines were sensitive to essential oil treatment with reduced cell viability and elevated cell death, whereas the immortalized normal human breast cell line was more resistant to essential oil treatment. Boswellia sacra

  5. Significantly increased expression of OCT4 and ABCG2 in spheroid body-forming cells of the human gastric cancer MKN-45 cell line.

    PubMed

    Liu, Jianming; Wang, Lei; Ma, Lilin; Xu, Junfei; Liu, Chun; Zhang, Jianguo; Liu, Jie; Chen, Ruixin

    2013-10-01

    The cancer stem cell (CSC) theory hypothesizes that CSCs are the cause of tumor formation, recurrence and metastasis. Key to the study of CSCs is their isolation and identification. The present study investigated whether spheroid body-forming cells in the human gastric cancer (GC) MKN-45 cell line are enriched for CSC properties, and also assessed the expression of the candidate CSC markers, octamer-binding transcription factor-4 (OCT4) and adenosine triphosphate-binding cassette transporter G2 (ABCG2) in the MKN-45 spheroid body cells. The MKN-45 cells were plated in a stem cell-conditioned culture system to allow for spheroid body formation. The expression levels of OCT4 and ABCG2 in the spheroid body cells were assessed by qPCR, western blot analysis and immunofluorescence staining, while the tumorigenicity of the spheroid body-forming cells was assessed by in vivo xenograft studies in nude mice. The MKN-45 cells were able to form spheroid bodies when cultured in stem cell-conditioned medium. The spheroid body-forming cells showed a significantly higher (P<0.01) expression of OCT4 and ABCG2 compared with the parental cells. These data suggest that the spheroid body cells from the MKN-45 GC cell line cultured in stem cell-conditioned medium possessed gastric CSC properties. The co-expression of OCT4 and ABCG2 by these cells may represent the presence of a subpopulation of gastric CSCs.

  6. Duplicate gene enrichment and expression pattern diversification in multicellularity

    PubMed Central

    Padawer, Timothy; Leighty, Ralph E.; Wang, Degeng

    2012-01-01

    The enrichment of duplicate genes, and therefore paralogs (proteins coded by duplicate genes), in multicellular versus unicellular organisms enhances genomic functional innovation. This study quantitatively examined relationships among paralog enrichment, expression pattern diversification and multicellularity, aiming to better understand genomic basis of multicellularity. Paralog abundance in specific cells was compared with those in unicellular proteomes and the whole proteomes of multicellular organisms. The budding yeast, Saccharomyces cerevisiae and the nematode, Caenorhabditis elegans, for which the gene sets expressed in specific cells are available, were used as uni and multicellular models, respectively. Paralog count (K) distributions [P(k)] follow a power-law relationship [P(k) ∝ k−α] in the whole proteomes of both species and in specific C. elegans cells. The value of the constant α can be used as a gauge of paralog abundance; the higher the value, the lower the paralog abundance. The α-value is indeed lower in the whole proteome of C. elegans (1.74) than in S. cerevisiae (2.34), quantifying the enrichment of paralogs in multicellular species. We also found that the power-law relationship applies to the proteomes of specific C. elegans cells. Strikingly, values of α in specific cells are higher and comparable to that in S. cerevisiae. Thus, paralog abundance in specific cells is lower and comparable to that in unicellular species. Furthermore, how much the expression level of a gene fluctuates across different C. elegans cells correlates positively with its paralog count, which is further confirmed by human gene-expression patterns across different tissues. Taken together, these results quantitatively and mechanistically establish enrichment of paralogs with diversifying expression patterns as genomic and evolutionary basis of multicellularity. PMID:22645319

  7. Effects of cobalt on haem proteins of erythropoietin-producing HepG2 cells in multicellular spheroid culture.

    PubMed

    Görlach, A; Fandrey, J; Holtermann, G; Acker, H

    1994-07-11

    The hypoxia-induced increase of spectrophotometrically measured light absorption at 560 nm, considered as reduced cytochrome b, in HepG2 cells is diminished after exposure to cobalt chloride (50 or 100 microM) for 18-36 h. The redox state of cytochrome c and cytochrome aa3, however, remains stable, indicating a particular affinity of cytochrome b for cobalt. Erythropoietin production of HepG2 cells increases after application of cobalt chloride, whereas H2O2 production, as measured by the dihydrorhodamine technique, decreases. It is concluded that cobalt stimulates a signal cascade with cytochrome b as receptor and H2O2 as second messenger for regulating erythropoietin production.

  8. Accumulation and toxicity of antibody-targeted doxorubicin-loaded PEG-PE micelles in ovarian cancer cell spheroid model.

    PubMed

    Perche, Federico; Patel, Niravkumar R; Torchilin, Vladimir P

    2012-11-28

    We describe the evaluation of doxorubicin-loaded PEG-PE micelles targeting using an ovarian cancer cell spheroid model. Most ovarian cancer patients present at an advanced clinical stage and develop resistance to standard of care platinum/taxane therapy. Doxorubicin is also approved for ovarian cancer but had limited benefits in refractory patients. In this study, we used drug-resistant spheroid cultures of ovarian carcinoma to evaluate the uptake and cytotoxicity of an antibody-targeted doxorubicin formulation. Doxorubicin was encapsulated in polyethylene glycol-phosphatidyl ethanolamine (PEG-PE) conjugated micelles. The doxorubicin-loaded PEG-PE micelles (MDOX) were further decorated with a cancer cell-specific monoclonal 2C5 antibody to obtain doxorubicin-loaded immunomicelles (2C5-MDOX). Targeting and resulting toxicity of doxorubicin-loaded PEG-PE micelles were evaluated in three dimensional cancer cell spheroids. Superior accumulation of 2C5-MDOX compared to free doxorubicin or untargeted MDOX in spheroids was evidenced both by flow cytometry, fluorescence and confocal microscopy. Interestingly, even higher toxicity was measured by lactate dehydrogenase release and terminal deoxynucleotidyl transferase dUTP nick end labeling of targeted doxorubicin micelles in Bcl-2 overexpressing adriamycin-resistant spheroids. Overall, these results support use of spheroids to evaluate tumor targeted drug delivery. PMID:22974689

  9. Accumulation and toxicity of antibody-targeted doxorubicin-loaded PEG-PE micelles in ovarian cancer cell spheroid model

    PubMed Central

    Perche, Federico; Torchilin, Vladimir P.

    2012-01-01

    We describe the evaluation of doxorubicin-loaded PEG-PE micelles targeting using an ovarian cancer cell spheroid model. Most ovarian cancer patients present at an advanced clinical stage and develop resistance to standard of care platinum/taxane therapy. Doxorubicin is also approved for ovarian cancer but had limited benefits in refractory patients. In this study, we used drug-resistant spheroid cultures of ovarian carcinoma to evaluate the uptake and cytotoxicity of an antibody-targeted doxorubicin formulation. Doxorubicin was encapsulated in polyethylene glycol-phosphatidyl ethanolamine (PEG-PE) conjugated micelles. The doxorubicin-loaded PEG-PE micelles (MDOX) were further decorated with a cancer cell-specific monoclonal 2C5 antibody to obtain doxorubicin-loaded immunomicelles (2C5-MDOX). Targeting and resulting toxicity of doxorubicin-loaded PEG-PE micelles were evaluated in three dimensional cancer cell spheroids. Superior accumulation of 2C5-MDOX compared to free doxorubicin or untargeted MDOX in spheroids was evidenced both by flow cytometry, fluorescence and confocal microscopy. Interestingly, even higher toxicity was measured by lactate dehydrogenase release and terminal deoxynucleotidyl transferase dUTP nick end labeling of targeted doxorubicin micelles in Bcl-2 overexpressing adriamycin-resistant spheroids. Overall, these results support use of spheroids to evaluate tumor targeted drug delivery. PMID:22974689

  10. Development of size-customized hepatocarcinoma spheroids as a potential drug testing platform using a sacrificial gelatin microsphere system.

    PubMed

    Leong, Wenyan; Kremer, Antje; Wang, Dong-An

    2016-06-01

    Sacrificial gelatin microspheres can be developed as a cell delivery vehicle for non-anchorage dependent cells - its incorporation into a macroscopic scaffold system not only allows the cells to be cultured in suspension within cavities left behind by the sacrificial material, it also allows scaffold-free tissue development to be confined within the cavities. In this study, dense and highly viable hepatocarcinoma spheroids were developed by means of encapsulation in sacrificial gelatin microspheres produced via a simple water-in-oil emulsion technique. By initial selection of microsphere size and distribution, spheroid size can be controlled for various applications such as uniform tumor spheroids as a reproducible three-dimensional drug screening and testing platform that better mimics the in vivo nature of tumors (instead of conventional monolayer culture), as this study has suggested as a proof-of-concept with chemotherapy drug Doxorubicin. PMID:27040260

  11. Development of size-customized hepatocarcinoma spheroids as a potential drug testing platform using a sacrificial gelatin microsphere system.

    PubMed

    Leong, Wenyan; Kremer, Antje; Wang, Dong-An

    2016-06-01

    Sacrificial gelatin microspheres can be developed as a cell delivery vehicle for non-anchorage dependent cells - its incorporation into a macroscopic scaffold system not only allows the cells to be cultured in suspension within cavities left behind by the sacrificial material, it also allows scaffold-free tissue development to be confined within the cavities. In this study, dense and highly viable hepatocarcinoma spheroids were developed by means of encapsulation in sacrificial gelatin microspheres produced via a simple water-in-oil emulsion technique. By initial selection of microsphere size and distribution, spheroid size can be controlled for various applications such as uniform tumor spheroids as a reproducible three-dimensional drug screening and testing platform that better mimics the in vivo nature of tumors (instead of conventional monolayer culture), as this study has suggested as a proof-of-concept with chemotherapy drug Doxorubicin.

  12. Spreading and spontaneous motility of multicellular aggregates on soft substrates

    NASA Astrophysics Data System (ADS)

    Brochard-Wyart, Françoise

    2013-03-01

    We first describe the biomechanics of multicellular aggregates, a model system for tissues and tumors. We first characterize the tissue mechanical properties (surface tension, elasticity, viscosity) by a new pipette aspiration technique. The aggregate exhibits a viscoelastic response but, unlike an inert fluid, we observe aggregate reinforcement with pressure, which for a narrow range of pressures results in pulsed contractions or shivering. We interpret this reinforcement as a mechanosensitive active response of the acto-myosin cortex. Such an active behavior has previously been found to cause tissue pulsation during dorsal closure of Drosophila embryo. We then describe the spreading of aggregates on rigid glass substrates, varying both intercellular and substrate adhesion. We find both partial and complete wetting regimes. For the dynamics, we find a universal spreading law at short time, analogous to that of a viscoelastic drop. At long time, we observe, for strong substrate adhesion, a precursor film spreading around the aggregate. Depending on aggregate cohesion, this precursor film can be a dense cellular monolayer (liquid state) or consist of individual cells escaping from the aggregate body (gas state). The transition from liquid to gas state appears also to be present in the progression of a tumor from noninvasive to metastatic, known as the epithelial-mesenchymal transition. Finally, we describe the effect of the substrate rigidity on the phase diagram of wetting. On soft gels decorated with fibronectin and strongly cohesive aggregates, we have observed a wetting transition induced by the substrate rigidity: on ultra soft gels, below an elastic modulus Ec the aggregates do not spread, whereas above Ec we observe a precursor film expending with a diffusive law. The diffusion coefficient D(E) present a maximum for E =Em. A maximum of mobility versus the substrate rigidity had also been observed for single cells. Near Em, we observe a new phenomenon: a cell

  13. Sulphur isotope ratios in the Canyon Diablo metallic spheroids

    NASA Astrophysics Data System (ADS)

    McEwing, C. E.; Rees, C. E.; Thode, H. G.

    1983-09-01

    Nininger (1956) has discovered metallic spheroids in the soil surrounding Meteor Crater in Arizona. Nininger suggested that the spheroids condensed from the center of a homogeneous explosion-produced metallic vapor cloud. The present investigation is concerned with measurements of sulfur contents and delta S-34 values of metallic spheroids from the vicinity of Meteor Crater. It is found that the small metallic spheroids have lower sulfur contents and higher delta S-34 values than do the large spheroids. It is concluded that the observed isotopic patterns are unlikely to have arisen during desulfurization of the metallic liquid from which the spheroids were formed or during high temperature oxidation or the spheroids. The most likely process for the production of the observed delta S-34 values and sulfur contents is low temperature oxidation reactions experienced by the spheroids during their surface exposure following formation.

  14. Screening of charged spheroidal colloidal particles

    NASA Astrophysics Data System (ADS)

    Álvarez, Carlos; Téllez, Gabriel

    2010-10-01

    We study the effective screened electrostatic potential created by a spheroidal colloidal particle immersed in an electrolyte, within the mean field approximation, using Poisson-Boltzmann equation in its linear and nonlinear forms, and also beyond the mean field by means of Monte Carlo computer simulation. The anisotropic shape of the particle has a strong effect on the screened potential, even at large distances (compared to the Debye length) from it. To quantify this anisotropy effect, we focus our study on the dependence of the potential on the position of the observation point with respect with the orientation of the spheroidal particle. For several different boundary conditions (constant potential, or constant surface charge) we find that, at large distance, the potential is higher in the direction of the large axis of the spheroidal particle.

  15. Prolate spheroidal harmonic expansion of gravitational field

    SciTech Connect

    Fukushima, Toshio

    2014-06-01

    As a modification of the oblate spheroidal case, a recursive method is developed to compute the point value and a few low-order derivatives of the prolate spheroidal harmonics of the second kind, Q{sub nm} (y), namely the unnormalized associated Legendre function (ALF) of the second kind with its argument in the domain, 1 < y < ∞. They are required in evaluating the prolate spheroidal harmonic expansion of the gravitational field in addition to the point value and the low-order derivatives of P-bar {sub nm}(t), the 4π fully normalized ALF of the first kind with its argument in the domain, |t| ≤ 1. The new method will be useful in the gravitational field computation of elongated celestial objects.

  16. Multicellularity in green algae: upsizing in a walled complex.

    PubMed

    Domozych, David S; Domozych, Catherine E

    2014-01-01

    Modern green algae constitute a large and diverse taxonomic assemblage that encompasses many multicellular phenotypes including colonial, filamentous, and parenchymatous forms. In all multicellular green algae, each cell is surrounded by an extracellular matrix (ECM), most often in the form of a cell wall. Volvocalean taxa like Volvox have an elaborate, gel-like, hydroxyproline rich glycoprotein covering that contains the cells of the colony. In "ulvophytes," uronic acid-rich and sulfated polysaccharides are the likely adhesion agents that maintain the multicellular habit. Charophytes also produce polysaccharide-rich cell walls and in late divergent taxa, pectin plays a critical role in cell adhesion in the multicellular complex. Cell walls are products of coordinated interaction of membrane trafficking, cytoskeletal dynamics and the cell's signal transduction machinery responding both to precise internal clocks and external environmental cues. Most often, these activities must be synchronized with the secretion, deposition and remodeling of the polymers of the ECM. Rapid advances in molecular genetics, cell biology and cell wall biochemistry of green algae will soon provide new insights into the evolution and subcellular processes leading to multicellularity. PMID:25477895

  17. Multicellularity in green algae: upsizing in a walled complex

    PubMed Central

    Domozych, David S.; Domozych, Catherine E.

    2014-01-01

    Modern green algae constitute a large and diverse taxonomic assemblage that encompasses many multicellular phenotypes including colonial, filamentous, and parenchymatous forms. In all multicellular green algae, each cell is surrounded by an extracellular matrix (ECM), most often in the form of a cell wall. Volvocalean taxa like Volvox have an elaborate, gel-like, hydroxyproline rich glycoprotein covering that contains the cells of the colony. In “ulvophytes,” uronic acid-rich and sulfated polysaccharides are the likely adhesion agents that maintain the multicellular habit. Charophytes also produce polysaccharide-rich cell walls and in late divergent taxa, pectin plays a critical role in cell adhesion in the multicellular complex. Cell walls are products of coordinated interaction of membrane trafficking, cytoskeletal dynamics and the cell’s signal transduction machinery responding both to precise internal clocks and external environmental cues. Most often, these activities must be synchronized with the secretion, deposition and remodeling of the polymers of the ECM. Rapid advances in molecular genetics, cell biology and cell wall biochemistry of green algae will soon provide new insights into the evolution and subcellular processes leading to multicellularity. PMID:25477895

  18. Triassic origin and early radiation of multicellular volvocine algae

    PubMed Central

    Herron, Matthew D.; Hackett, Jeremiah D.; Aylward, Frank O.; Michod, Richard E.

    2009-01-01

    Evolutionary transitions in individuality (ETIs) underlie the watershed events in the history of life on Earth, including the origins of cells, eukaryotes, plants, animals, and fungi. Each of these events constitutes an increase in the level of complexity, as groups of individuals become individuals in their own right. Among the best-studied ETIs is the origin of multicellularity in the green alga Volvox, a model system for the evolution of multicellularity and cellular differentiation. Since its divergence from unicellular ancestors, Volvox has evolved into a highly integrated multicellular organism with cellular specialization, a complex developmental program, and a high degree of coordination among cells. Remarkably, all of these changes were previously thought to have occurred in the last 50–75 million years. Here we estimate divergence times using a multigene data set with multiple fossil calibrations and use these estimates to infer the times of developmental changes relevant to the evolution of multicellularity. Our results show that Volvox diverged from unicellular ancestors at least 200 million years ago. Two key innovations resulting from an early cycle of cooperation, conflict and conflict mediation led to a rapid integration and radiation of multicellular forms in this group. This is the only ETI for which a detailed timeline has been established, but multilevel selection theory predicts that similar changes must have occurred during other ETIs. PMID:19223580

  19. Cold storage of rat hepatocyte spheroids.

    PubMed

    Liu, Hongling; Yu, Yue; Glorioso, Jaime; Mao, Shennen; Rodysil, Brian; Amiot, Bruce P; Rinaldo, Piero; Nyberg, Scott L

    2014-01-01

    Cell-based therapies for liver disease rely on a high-quality supply of hepatocytes and a means for storage during transportation from site of isolation to site of usage. Unfortunately, frozen cryopreservation is associated with unacceptable loss of hepatocyte viability after thawing. The purpose of this study was to optimize conditions for cold storage of rat hepatocyte spheroids without freezing. Rat hepatocytes were isolated by a two-step perfusion method; hepatocyte spheroids were formed during 48 h of rocked culture in serum-free medium (SFM). Spheroids were then maintained in rocked culture at 37 °C (control condition) or cold stored at 4 °C for 24 or 48 h in six different cold storage solutions: SFM alone; SFM + 1 mM deferoxamine (Def); SFM + 1 μM cyclosporin A (CsA); SFM + 1 mM Def + 1 μM CsA, University of Wisconsin (UW) solution alone, UW + 1 mM Def. Performance metrics after cold storage included viability, gene expression, albumin production, and functional activity of cytochrome P450 enzymes and urea cycle proteins. We observed that cold-induced injury was reduced significantly by the addition of the iron chelator (Def) to both SFM and UW solution. Performance metrics (ammonia detoxification, albumin production) of rat hepatocyte spheroids stored in SFM + Def for 24 h were significantly increased from SFM alone and approached those in control conditions, while performance metrics after cold storage in SFM alone or cold storage for 48 h were both significantly reduced. A serum-free medium supplemented with Def allowed hepatocyte spheroids to tolerate 24 h of cold storage with less than 10% loss in viability and functionality. Further research is warranted to optimize a solution for extended cold storage of hepatocyte spheroids.

  20. Dwarf spheroidal galaxies: Keystones of galaxy evolution

    NASA Technical Reports Server (NTRS)

    Gallagher, John S., III; Wyse, Rosemary F. G.

    1994-01-01

    Dwarf spheroidal galaxies are the most insignificant extragalactic stellar systems in terms of their visibility, but potentially very significant in terms of their role in the formation and evolution of much more luminous galaxies. We discuss the present observational data and their implications for theories of the formation and evolution of both dwarf and giant galaxies. The putative dark-matter content of these low-surface-brightness systems is of particular interest, as is their chemical evolution. Surveys for new dwarf spheroidals hidden behind the stars of our Galaxy and those which are not bound to giant galaxies may give new clues as to the origins of this unique class of galaxy.

  1. The dwarf spheroidal galaxy Andromeda I

    SciTech Connect

    Mould, J.; Kristian, J. Mount Wilson and Las Campanas Observatories, Pasadena, CA )

    1990-05-01

    Images of Andromeda I in the visual and near-infrared show a giant branch characteristic of galactic globular clusters of intermediate metallicity. The distance of the galaxy is estimated from the tip of the giant branch to be 790 + or - 60 kpc. The physical dimensions and luminosity are similar to those of the dwarf spheroidal in Sculptor. There is no evidence for an intermediate age population in Andromeda I, and appropriate upper limits are specified. There is marginal evidence for a color gradient in the galaxy, a phenomenon not previously noted in a dwarf spheroidal. 21 refs.

  2. Bacterial swarming: an example of prokaryotic differentiation and multicellular behaviour.

    PubMed

    Allison, C; Hughes, C

    1991-01-01

    Bacterial swarming involves the differentiation of vegetative cells into hyperflagellated swarm cells which undergo cycles of rapid and coordinated population migration across solid surfaces. Species capable of this simple form of developmental behaviour lie on the boundary between unicellular and multicellular organisms and provide processes for study which are not only of intrinsic interest but which are analogous to components of more complex eukaryotic systems. This review attempts to place current knowledge of bacterial swarming within the framework provided by more extensively studied forms of prokaryotic multicellular behaviour. It discusses the potential of swarming as a readily accessible model of differentiation and multicellular behaviour and describes evidence indicating that swarming differentiation plays an important role in bacterial virulence.

  3. A conceptual framework for the evolutionary origins of multicellularity

    NASA Astrophysics Data System (ADS)

    Libby, Eric; Rainey, Paul B.

    2013-06-01

    The evolution of multicellular organisms from unicellular counterparts involved a transition in Darwinian individuality from single cells to groups. A particular challenge is to understand the nature of the earliest groups, the causes of their evolution, and the opportunities for emergence of Darwinian properties. Here we outline a conceptual framework based on a logical set of possible pathways for evolution of the simplest self-replicating groups. Central to these pathways is the recognition of a finite number of routes by which genetic information can be transmitted between individual cells and groups. We describe the form and organization of each primordial group state and consider factors affecting persistence and evolution of the nascent multicellular forms. Implications arising from our conceptual framework become apparent when attempting to partition fitness effects at individual and group levels. These are discussed with reference to the evolutionary emergence of individuality and its manifestation in extant multicellular life—including those of marginal Darwinian status.

  4. Tyrosine kinase signaling and the emergence of multicellularity.

    PubMed

    Miller, W Todd

    2012-06-01

    Tyrosine phosphorylation is an essential element of signal transduction in multicellular animals. Although tyrosine kinases were originally regarded as specific to the metazoan lineage, it is now clear that they evolved prior to the split between unicellular and multicellular eukaryotes (≈600million years ago). Genome analyses of choanoflagellates and other protists show an abundance of tyrosine kinases that rivals the most complex animals. Some of these kinases are orthologs of metazoan enzymes (e.g., Src), but others display unique domain compositions not seen in any metazoan. Biochemical experiments have highlighted similarities and differences between the unicellular and multicellular tyrosine kinases. In particular, it appears that the complex systems of kinase autoregulation may have evolved later in the metazoan lineage. PMID:22480439

  5. Tyrosine kinase signaling and the emergence of multicellularity.

    PubMed

    Miller, W Todd

    2012-06-01

    Tyrosine phosphorylation is an essential element of signal transduction in multicellular animals. Although tyrosine kinases were originally regarded as specific to the metazoan lineage, it is now clear that they evolved prior to the split between unicellular and multicellular eukaryotes (≈600million years ago). Genome analyses of choanoflagellates and other protists show an abundance of tyrosine kinases that rivals the most complex animals. Some of these kinases are orthologs of metazoan enzymes (e.g., Src), but others display unique domain compositions not seen in any metazoan. Biochemical experiments have highlighted similarities and differences between the unicellular and multicellular tyrosine kinases. In particular, it appears that the complex systems of kinase autoregulation may have evolved later in the metazoan lineage.

  6. Plasmon Resonances of Nanoshells of Spheroidal Shape

    PubMed Central

    Norton, Stephen J.; Vo-Dinh, Tuan

    2009-01-01

    Plasmon resonances are computed for nanoshells of prolate and oblate spheroidal shape. Both longitudinal and transverse resonances are investigated as a function of aspect ratio and shell thickness. Formulas for the surface charge density on the outside and inside shell surfaces are derived. PMID:23976876

  7. Convection in Slab and Spheroidal Geometries

    NASA Technical Reports Server (NTRS)

    Porter, David H.; Woodward, Paul R.; Jacobs, Michael L.

    2000-01-01

    Three-dimensional numerical simulations of compressible turbulent thermally driven convection, in both slab and spheroidal geometries, are reviewed and analyzed in terms of velocity spectra and mixing-length theory. The same ideal gas model is used in both geometries, and resulting flows are compared. The piecewise-parabolic method (PPM), with either thermal conductivity or photospheric boundary conditions, is used to solve the fluid equations of motion. Fluid motions in both geometries exhibit a Kolmogorov-like k(sup -5/3) range in their velocity spectra. The longest wavelength modes are energetically dominant in both geometries, typically leading to one convection cell dominating the flow. In spheroidal geometry, a dipolar flow dominates the largest scale convective motions. Downflows are intensely turbulent and up drafts are relatively laminar in both geometries. In slab geometry, correlations between temperature and velocity fluctuations, which lead to the enthalpy flux, are fairly independent of depth. In spheroidal geometry this same correlation increases linearly with radius over the inner 70 percent by radius, in which the local pressure scale heights are a sizable fraction of the radius. The effects from the impenetrable boundary conditions in the slab geometry models are confused with the effects from non-local convection. In spheroidal geometry nonlocal effects, due to coherent plumes, are seen as far as several pressure scale heights from the lower boundary and are clearly distinguishable from boundary effects.

  8. Engineered three-dimensional microfluidic device for interrogating cell-cell interactions in the tumor microenvironment

    PubMed Central

    Hockemeyer, K.; Janetopoulos, C.; Terekhov, A.; Hofmeister, W.; Vilgelm, A.; Costa, Lino; Wikswo, J. P.; Richmond, A.

    2014-01-01

    Stromal cells in the tumor microenvironment play a key role in the metastatic properties of a tumor. It is recognized that cancer-associated fibroblasts (CAFs) and endothelial cells secrete factors capable of influencing tumor cell migration into the blood or lymphatic vessels. We developed a microfluidic device that can be used to image the interactions between stromal cells and tumor cell spheroids in a three dimensional (3D) microenvironment while enabling external control of interstitial flow at an interface, which supports endothelial cells. The apparatus couples a 200-μm channel with a semicircular well to mimic the interface of a blood vessel with the stroma, and the design allows for visualization of the interactions of interstitial flow, endothelial cells, leukocytes, and fibroblasts with the tumor cells. We observed that normal tissue-associated fibroblasts (NAFs) contribute to the “single file” pattern of migration of tumor cells from the spheroid in the 3D microenvironment. In contrast, CAFs induce a rapid dispersion of tumor cells out of the spheroid with migration into the 3D matrix. Moreover, treatment of tumor spheroid cultures with the chemokine CXCL12 mimics the effect of the CAFs, resulting in similar patterns of dispersal of the tumor cells from the spheroid. Conversely, addition of CXCL12 to co-cultures of NAFs with tumor spheroids did not mimic the effects observed with CAF co-cultures, suggesting that NAFs produce factors that stabilize the tumor spheroids to reduce their migration in response to CXCL12. PMID:25379090

  9. Transformation properties of spheroidal multipole moments and potentials

    NASA Astrophysics Data System (ADS)

    Jansen, Georg

    2000-02-01

    Introducing definitions of solid spheroidal harmonics which contain those of solid spherical harmonics as special cases for vanishing ellipticity it is shown that the formalism of the multipole expansion of a 1/R -potential can be consistently extended to incorporate prolate and oblate spheroidal multipole moments. For finite ellipticity one can transform between regular solid spheroidal and spherical harmonics and multipole moments through simple relations given before and independently proven here. Corresponding relations between irregular solid spheroidal and spherical harmonics are presented for the first time, together with an investigation of the convergence properties of the resulting series expansions. Explicit formulae are derived for the transformations between spheroidal multipoles calculated in coordinate systems of different ellipticity, origin and orientation. These fromulae can be utilized to calculate the energy of interaction between two arbitrarily oriented spheroidal charge or mass distributions of different ellipticity. The performance of spheroidal multipole expansions is illustrated with some numerical examples.

  10. Regulated aggregative multicellularity in a close unicellular relative of metazoa.

    PubMed

    Sebé-Pedrós, Arnau; Irimia, Manuel; Del Campo, Javier; Parra-Acero, Helena; Russ, Carsten; Nusbaum, Chad; Blencowe, Benjamin J; Ruiz-Trillo, Iñaki

    2013-12-24

    The evolution of metazoans from their unicellular ancestors was one of the most important events in the history of life. However, the cellular and genetic changes that ultimately led to the evolution of multicellularity are not known. In this study, we describe an aggregative multicellular stage in the protist Capsaspora owczarzaki, a close unicellular relative of metazoans. Remarkably, transition to the aggregative stage is associated with significant upregulation of orthologs of genes known to establish multicellularity and tissue architecture in metazoans. We further observe transitions in regulated alternative splicing during the C. owczarzaki life cycle, including the deployment of an exon network associated with signaling, a feature of splicing regulation so far only observed in metazoans. Our results reveal the existence of a highly regulated aggregative stage in C. owczarzaki and further suggest that features of aggregative behavior in an ancestral protist may had been co-opted to develop some multicellular properties currently seen in metazoans. DOI: http://dx.doi.org/10.7554/eLife.01287.001.

  11. Regulated aggregative multicellularity in a close unicellular relative of metazoa

    PubMed Central

    Sebé-Pedrós, Arnau; Irimia, Manuel; del Campo, Javier; Parra-Acero, Helena; Russ, Carsten; Nusbaum, Chad; Blencowe, Benjamin J; Ruiz-Trillo, Iñaki

    2013-01-01

    The evolution of metazoans from their unicellular ancestors was one of the most important events in the history of life. However, the cellular and genetic changes that ultimately led to the evolution of multicellularity are not known. In this study, we describe an aggregative multicellular stage in the protist Capsaspora owczarzaki, a close unicellular relative of metazoans. Remarkably, transition to the aggregative stage is associated with significant upregulation of orthologs of genes known to establish multicellularity and tissue architecture in metazoans. We further observe transitions in regulated alternative splicing during the C. owczarzaki life cycle, including the deployment of an exon network associated with signaling, a feature of splicing regulation so far only observed in metazoans. Our results reveal the existence of a highly regulated aggregative stage in C. owczarzaki and further suggest that features of aggregative behavior in an ancestral protist may had been co-opted to develop some multicellular properties currently seen in metazoans. DOI: http://dx.doi.org/10.7554/eLife.01287.001 PMID:24368732

  12. Kinetic Monte Carlo and cellular particle dynamics simulations of multicellular systems

    NASA Astrophysics Data System (ADS)

    Flenner, Elijah; Janosi, Lorant; Barz, Bogdan; Neagu, Adrian; Forgacs, Gabor; Kosztin, Ioan

    2012-03-01

    Computer modeling of multicellular systems has been a valuable tool for interpreting and guiding in vitro experiments relevant to embryonic morphogenesis, tumor growth, angiogenesis and, lately, structure formation following the printing of cell aggregates as bioink particles. Here we formulate two computer simulation methods: (1) a kinetic Monte Carlo (KMC) and (2) a cellular particle dynamics (CPD) method, which are capable of describing and predicting the shape evolution in time of three-dimensional multicellular systems during their biomechanical relaxation. Our work is motivated by the need of developing quantitative methods for optimizing postprinting structure formation in bioprinting-assisted tissue engineering. The KMC and CPD model parameters are determined and calibrated by using an original computational-theoretical-experimental framework applied to the fusion of two spherical cell aggregates. The two methods are used to predict the (1) formation of a toroidal structure through fusion of spherical aggregates and (2) cell sorting within an aggregate formed by two types of cells with different adhesivities.

  13. Empirical chemosensitivity testing in a spheroid model of ovarian cancer using a microfluidics-based multiplex platform

    PubMed Central

    Das, Tamal; Meunier, Liliane; Barbe, Laurent; Provencher, Diane; Guenat, Olivier; Gervais, Thomas; Mes-Masson, Anne-Marie

    2013-01-01

    The use of biomarkers to infer drug response in patients is being actively pursued, yet significant challenges with this approach, including the complicated interconnection of pathways, have limited its application. Direct empirical testing of tumor sensitivity would arguably provide a more reliable predictive value, although it has garnered little attention largely due to the technical difficulties associated with this approach. We hypothesize that the application of recently developed microtechnologies, coupled to more complex 3-dimensional cell cultures, could provide a model to address some of these issues. As a proof of concept, we developed a microfluidic device where spheroids of the serous epithelial ovarian cancer cell line TOV112D are entrapped and assayed for their chemoresponse to carboplatin and paclitaxel, two therapeutic agents routinely used for the treatment of ovarian cancer. In order to index the chemoresponse, we analyzed the spatiotemporal evolution of the mortality fraction, as judged by vital dyes and confocal microscopy, within spheroids subjected to different drug concentrations and treatment durations inside the microfluidic device. To reflect microenvironment effects, we tested the effect of exogenous extracellular matrix and serum supplementation during spheroid formation on their chemotherapeutic response. Spheroids displayed augmented chemoresistance in comparison to monolayer culturing. This resistance was further increased by the simultaneous presence of both extracellular matrix and high serum concentration during spheroid formation. Following exposure to chemotherapeutics, cell death profiles were not uniform throughout the spheroid. The highest cell death fraction was found at the center of the spheroid and the lowest at the periphery. Collectively, the results demonstrate the validity of the approach, and provide the basis for further investigation of chemotherapeutic responses in ovarian cancer using microfluidics technology. In

  14. Oxygen Partial Pressure Is a Rate-Limiting Parameter for Cell Proliferation in 3D Spheroids Grown in Physioxic Culture Condition

    PubMed Central

    Gomes, Aurélie; Guillaume, Ludivine; Grimes, David Robert; Fehrenbach, Jérôme; Lobjois, Valérie; Ducommun, Bernard

    2016-01-01

    The in situ oxygen partial pressure in normal and tumor tissues is in the range of a few percent. Therefore, when studying cell growth in 3D culture systems, it is essential to consider how the physiological oxygen concentration, rather than the one in the ambient air, influences the proliferation parameters. Here, we investigated the effect of reducing oxygen partial pressure from 21% to 5% on cell proliferation rate and regionalization in a 3D tumor spheroid model. We found that 5% oxygen concentration strongly inhibited spheroid growth, changed the proliferation gradient and reduced the 50% In Depth Proliferation index (IDP50), compared with culture at 21% oxygen. We then modeled the oxygen partial pressure profiles using the experimental data generated by culturing spheroids in physioxic and normoxic conditions. Although hypoxia occurred at similar depth in spheroids grown in the two conditions, oxygen partial pressure was a major rate-limiting factor with a critical effect on cell proliferation rate and regionalization only in spheroids grown in physioxic condition and not in spheroids grown at atmospheric normoxia. Our findings strengthen the need to consider conducting experiment in physioxic conditions (i.e., tissue normoxia) for proper understanding of cancer cell biology and the evaluation of anticancer drugs in 3D culture systems. PMID:27575790

  15. Oxygen Partial Pressure Is a Rate-Limiting Parameter for Cell Proliferation in 3D Spheroids Grown in Physioxic Culture Condition.

    PubMed

    Gomes, Aurélie; Guillaume, Ludivine; Grimes, David Robert; Fehrenbach, Jérôme; Lobjois, Valérie; Ducommun, Bernard

    2016-01-01

    The in situ oxygen partial pressure in normal and tumor tissues is in the range of a few percent. Therefore, when studying cell growth in 3D culture systems, it is essential to consider how the physiological oxygen concentration, rather than the one in the ambient air, influences the proliferation parameters. Here, we investigated the effect of reducing oxygen partial pressure from 21% to 5% on cell proliferation rate and regionalization in a 3D tumor spheroid model. We found that 5% oxygen concentration strongly inhibited spheroid growth, changed the proliferation gradient and reduced the 50% In Depth Proliferation index (IDP50), compared with culture at 21% oxygen. We then modeled the oxygen partial pressure profiles using the experimental data generated by culturing spheroids in physioxic and normoxic conditions. Although hypoxia occurred at similar depth in spheroids grown in the two conditions, oxygen partial pressure was a major rate-limiting factor with a critical effect on cell proliferation rate and regionalization only in spheroids grown in physioxic condition and not in spheroids grown at atmospheric normoxia. Our findings strengthen the need to consider conducting experiment in physioxic conditions (i.e., tissue normoxia) for proper understanding of cancer cell biology and the evaluation of anticancer drugs in 3D culture systems. PMID:27575790

  16. Phase behavior of shape-changing spheroids

    NASA Astrophysics Data System (ADS)

    Teixeira, P. I. C.; Masters, A. J.

    2015-12-01

    We introduce a simple model for a biaxial nematic liquid crystal. This consists of hard spheroids that can switch shape between prolate (rodlike) and oblate (platelike) subject to an energy penalty Δ ɛ . The spheroids are approximated as hard Gaussian overlap particles and are treated at the level of Onsager's second-virial description. We use both bifurcation analysis and a numerical minimization of the free energy to show that, for additive particle shapes, (i) there is no stable biaxial phase even for Δ ɛ =0 (although there is a metastable biaxial phase in the same density range as the stable uniaxial phase) and (ii) the isotropic-to-nematic transition is into either one of two degenerate uniaxial phases, rod rich or plate rich. We confirm that even a small amount of shape nonadditivity may stabilize the biaxial nematic phase.

  17. Dynamics of spheroid particles in channel flow

    NASA Astrophysics Data System (ADS)

    Mao, Wenbin; Alexeev, Alexander

    2012-11-01

    The effect of inertia on the dynamics of rigid spheroid microparticles in a pressure-driven channel flow is studied using a hybrid lattice Boltzmann and lattice spring method. We find distinctive behaviors of particles depending on the particle shape, initial orientation, and ratio of particle size to the channel size. Two possible stable modes of motion are found for prolate spheroids. Particles either tumble in a shear plane or spin with the axis parallel to the vortex direction. We present a phase diagram showing the transition between these two modes. Cross-stream migration and equilibrium trajectories of particles are also investigated and found to depend on the particle shape and mode of motion. The simulations results are compared with experimental data showing favorable agreement. Our results will be useful for separating biological and synthetic particles by size and shape.

  18. Dipolophoresis of dielectric spheroids under asymmetric fields

    NASA Astrophysics Data System (ADS)

    Frankel, Itzchak; Yossifon, Gilad; Miloh, Touvia

    2012-01-01

    Non-spherical particles are common in colloidal science. Spheroidal shapes are particularly convenient for the analysis of the pertinent electrostatic and hydrodynamic problems and are thus widely used to model the manipulation of biological cells as well as deformed drops and bubbles. We study the rotary motion of a dielectric spheroidal micro-particle which is freely suspended in an unbounded electrolyte solution in the presence of a uniform applied electric field, assuming a thin Debye layer. For the common case of a uniform distribution of the native surface-charge density, the rotary motion of the particle is generated by the contributions of the induced-charge electro-osmotic (ICEO) slip and the dielectrophoresis associated with the distribution of the Maxwell stress, respectively. Series solutions are obtained by using spheroidal (prolate or oblate) coordinates. Explicit results are presented for the angular velocity of particles spanning the entire spectrum from rod-like to disk-like shapes. These results demonstrate the non-monotonic variation of the angular speed with the eccentricity of particle shape and the singularity of the multiple limits corresponding to conducting (ideally polarizable) particles of extreme eccentricity (e ≈ 1). The non-monotonic variation of the angular speed with the particle dielectric permittivity is related to the induced-charge contribution. We apply these results to describe the motion of particles subject to a uniform field rotating in the plane. For a sufficiently slow rotation rate, prolate particles eventually become "locked" to the external field with their stationary relative orientation in the plane of rotation being determined by the particle eccentricity and dielectric constant. This effect may be of potential use in the manipulation of poly-disperse suspensions of dielectric non-spherical particles. Oblate spheroids invariably approach a uniform orientation with their symmetry axes directed normal to the external

  19. Cancer cell spheroids as a model to evaluate chemotherapy protocols.

    PubMed

    Perche, Federico; Torchilin, Vladimir P

    2012-10-01

    To determine whether the spheroid culture can be used to evaluate drug efficacy, we have evaluated the toxicity of free or carrier-associated doxorubicin as a single drug or in combination with other antineoplastic agents using the spheroid cultures of drug-resistant cancer cells. Paclitaxel, cisplatin, dexamethasone, mitoxantrone, sclareol or methotrexate were used in combination with doxorubicin. The effect of the treatment protocols on free, micellar and liposomal doxorubicin accumulation in spheroids and on resulting toxicity was evaluated by fluorescence and lactate dehydrogenase release, respectively. Enhanced doxorubicin accumulation and toxicity were observed after spheroid pretreatment with mitoxantrone or paclitaxel. Effects of the drug combination with doxorubicin were sequence dependent, use of doxorubicin as the first drug being the least inducer of toxicity. Finally, spheroids were recognized by a cancer cell-specific antibody. Our results suggest the usefulness of spheroids to evaluate chemotherapy combinations. PMID:22892843

  20. Cancer cell spheroids as a model to evaluate chemotherapy protocols

    PubMed Central

    Perche, Federico; Torchilin, Vladimir P.

    2012-01-01

    To determine whether the spheroid culture can be used to evaluate drug efficacy, we have evaluated the toxicity of free or carrier-associated doxorubicin as a single drug or in combination with other antineoplastic agents using the spheroid cultures of drug-resistant cancer cells. Paclitaxel, cisplatin, dexamethasone, mitoxantrone, sclareol or methotrexate were used in combination with doxorubicin. The effect of the treatment protocols on free, micellar and liposomal doxorubicin accumulation in spheroids and on resulting toxicity was evaluated by fluorescence and lactate dehydrogenase release, respectively. Enhanced doxorubicin accumulation and toxicity were observed after spheroid pretreatment with mitoxantrone or paclitaxel. Effects of the drug combination with doxorubicin were sequence dependent, use of doxorubicin as the first drug being the least inducer of toxicity. Finally, spheroids were recognized by a cancer cell-specific antibody. Our results suggest the usefulness of spheroids to evaluate chemotherapy combinations. PMID:22892843

  1. An evolutionary scenario for the transition to undifferentiated multicellularity

    PubMed Central

    Pfeiffer, Thomas; Bonhoeffer, Sebastian

    2003-01-01

    The evolutionary transition from single cells toward multicellular forms of life represents one of the major transitions in the evolution of complex organisms. In this transition, single autonomously reproducing cells became parts of larger reproducing entities that eventually constituted a new unit of selection. The first step in the evolutionary transition to multicellularity likely was the evolution of simple, undifferentiated cell clusters. However, what the selective advantage of such cell clusters may have been remains unclear. Here, we argue that in populations of unicellular organisms with cooperative behavior, clustering may be beneficial by reducing interactions with noncooperative individuals. In support of this hypothesis, we present a set of computer simulations showing that clustering can evolve as a biological, heritable trait for cells that cooperate in the use of external energy resources. Following the evolution of simple cell clusters, further benefits could have arisen from the exchange of resources between cells of a cluster. PMID:12547910

  2. Life cycles, fitness decoupling and the evolution of multicellularity.

    PubMed

    Hammerschmidt, Katrin; Rose, Caroline J; Kerr, Benjamin; Rainey, Paul B

    2014-11-01

    Cooperation is central to the emergence of multicellular life; however, the means by which the earliest collectives (groups of cells) maintained integrity in the face of destructive cheating types is unclear. One idea posits cheats as a primitive germ line in a life cycle that facilitates collective reproduction. Here we describe an experiment in which simple cooperating lineages of bacteria were propagated under a selective regime that rewarded collective-level persistence. Collectives reproduced via life cycles that either embraced, or purged, cheating types. When embraced, the life cycle alternated between phenotypic states. Selection fostered inception of a developmental switch that underpinned the emergence of collectives whose fitness, during the course of evolution, became decoupled from the fitness of constituent cells. Such development and decoupling did not occur when groups reproduced via a cheat-purging regime. Our findings capture key events in the evolution of Darwinian individuality during the transition from single cells to multicellularity.

  3. Detecting tree-like multicellular life on extrasolar planets.

    PubMed

    Doughty, Christopher E; Wolf, Adam

    2010-11-01

    Over the next two decades, NASA and ESA are planning a series of space-based observatories to find Earth-like planets and determine whether life exists on these planets. Previous studies have assessed the likelihood of detecting life through signs of biogenic gases in the atmosphere or a red edge. Biogenic gases and the red edge could be signs of either single-celled or multicellular life. In this study, we propose a technique with which to determine whether tree-like multicellular life exists on extrasolar planets. For multicellular photosynthetic organisms on Earth, competition for light and the need to transport water and nutrients has led to a tree-like body plan characterized by hierarchical branching networks. This design results in a distinct bidirectional reflectance distribution function (BRDF) that causes differing reflectance at different sun/view geometries. BRDF arises from the changing visibility of the shadows cast by objects, and the presence of tree-like structures is clearly distinguishable from flat ground with the same reflectance spectrum. We examined whether the BRDF could detect the existence of tree-like structures on an extrasolar planet by using changes in planetary albedo as a planet orbits its star. We used a semi-empirical BRDF model to simulate vegetation reflectance at different planetary phase angles and both simulated and real cloud cover to calculate disk and rotation-averaged planetary albedo for a vegetated and non-vegetated planet with abundant liquid water. We found that even if the entire planetary albedo were rendered to a single pixel, the rate of increase of albedo as a planet approaches full illumination would be comparatively greater on a vegetated planet than on a non-vegetated planet. Depending on how accurately planetary cloud cover can be resolved and the capabilities of the coronagraph to resolve exoplanets, this technique could theoretically detect tree-like multicellular life on exoplanets in 50 stellar systems

  4. Multi-cellular, three-dimensional living mammalian tissue

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J. (Inventor); Wolf, David A. (Inventor)

    1994-01-01

    The present invention relates to a multicellular, three-dimensional, living mammalian tissue. The tissue is produced by a co-culture process wherein two distinct types of mammalian cells are co-cultured in a rotating bioreactor which is completely filled with culture media and cell attachment substrates. As the size of the tissue assemblies formed on the attachment substrates changes, the rotation of the bioreactor is adjusted accordingly.

  5. Detecting tree-like multicellular life on extrasolar planets.

    PubMed

    Doughty, Christopher E; Wolf, Adam

    2010-11-01

    Over the next two decades, NASA and ESA are planning a series of space-based observatories to find Earth-like planets and determine whether life exists on these planets. Previous studies have assessed the likelihood of detecting life through signs of biogenic gases in the atmosphere or a red edge. Biogenic gases and the red edge could be signs of either single-celled or multicellular life. In this study, we propose a technique with which to determine whether tree-like multicellular life exists on extrasolar planets. For multicellular photosynthetic organisms on Earth, competition for light and the need to transport water and nutrients has led to a tree-like body plan characterized by hierarchical branching networks. This design results in a distinct bidirectional reflectance distribution function (BRDF) that causes differing reflectance at different sun/view geometries. BRDF arises from the changing visibility of the shadows cast by objects, and the presence of tree-like structures is clearly distinguishable from flat ground with the same reflectance spectrum. We examined whether the BRDF could detect the existence of tree-like structures on an extrasolar planet by using changes in planetary albedo as a planet orbits its star. We used a semi-empirical BRDF model to simulate vegetation reflectance at different planetary phase angles and both simulated and real cloud cover to calculate disk and rotation-averaged planetary albedo for a vegetated and non-vegetated planet with abundant liquid water. We found that even if the entire planetary albedo were rendered to a single pixel, the rate of increase of albedo as a planet approaches full illumination would be comparatively greater on a vegetated planet than on a non-vegetated planet. Depending on how accurately planetary cloud cover can be resolved and the capabilities of the coronagraph to resolve exoplanets, this technique could theoretically detect tree-like multicellular life on exoplanets in 50 stellar systems.

  6. Division of labour and the evolution of multicellularity.

    PubMed

    Ispolatov, Iaroslav; Ackermann, Martin; Doebeli, Michael

    2012-05-01

    Understanding the emergence and evolution of multicellularity and cellular differentiation is a core problem in biology. We develop a quantitative model that shows that a multicellular form emerges from genetically identical unicellular ancestors when the compartmentalization of poorly compatible physiological processes into component cells of an aggregate produces a fitness advantage. This division of labour between the cells in the aggregate occurs spontaneously at the regulatory level owing to mechanisms present in unicellular ancestors and does not require any genetic predisposition for a particular role in the aggregate or any orchestrated cooperative behaviour of aggregate cells. Mathematically, aggregation implies an increase in the dimensionality of phenotype space that generates a fitness landscape with new fitness maxima, in which the unicellular states of optimized metabolism become fitness saddle points. Evolution of multicellularity is modelled as evolution of a hereditary parameter: the propensity of cells to stick together, which determines the fraction of time a cell spends in the aggregate form. Stickiness can increase evolutionarily owing to the fitness advantage generated by the division of labour between cells in an aggregate. PMID:22158952

  7. Heritable Remodeling of Yeast Multicellularity by an Environmentally Responsive Prion

    PubMed Central

    Holmes, Daniel L.; Lancaster, Alex K.; Lindquist, Susan; Halfmann, Randal

    2013-01-01

    SUMMARY Prion proteins undergo self-sustaining conforma-tional conversions that heritably alter their activities. Many of these proteins operate at pivotal positions in determining how genotype is translated into pheno-type. But the breadth of prion influences on biology and their evolutionary significance are just beginning to be explored. We report that a prion formed by the Mot3 transcription factor, [MOT3+], governs the acquisition of facultative multicellularity in the budding yeast Saccharomyces cerevisiae. The traits governed by [MOT3+] involved both gains and losses of Mot3 regulatory activity. [MOT3+]-dependent expression of FLO11, a major determinant of cell-cell adhesion, produced diverse lineage-specific multicellular phenotypes in response to nutrient deprivation. The prions themselves were induced by ethanol and eliminated by hypoxia—conditions that occur sequentially in the natural respiro-fermen-tative cycles of yeast populations. These data demonstrate that prions can act as environmentally responsive molecular determinants of multicellularity and contribute to the natural morphological diversity of budding yeast. PMID:23540696

  8. Ruthenium(II) Complexes with 2-Phenylimidazo[4,5-f][1,10]phenanthroline Derivatives that Strongly Combat Cisplatin-Resistant Tumor Cells

    PubMed Central

    Zeng, Leli; Chen, Yu; Liu, Jiangping; Huang, Huaiyi; Guan, Ruilin; Ji, Liangnian; Chao, Hui

    2016-01-01

    Cisplatin was the first metal-based therapeutic agent approved for the treatment of human cancers, but its clinical activity is greatly limited by tumor drug resistance. This work utilized the parent complex [Ru(phen)2(PIP)]2+ (1) to develop three Ru(II) complexes (2–4) with different positional modifications. These compounds exhibited similar or superior cytotoxicities compared to cisplatin in HeLa, A549 and multidrug-resistant (A549R) tumor cell lines. Complex 4, the most potent member of the series, was highly active against A549R cancer cells (IC50 = 0.8 μM). This complex exhibited 178-fold better activity than cisplatin (IC50 = 142.5 μM) in A549R cells. 3D multicellular A549R tumor spheroids were also used to confirm the high proliferative and cytotoxic activity of complex 4. Complex 4 had the greatest cellular uptake and had a tendency to accumulate in the mitochondria of A549R cells. Further mechanistic studies showed that complex 4 induced A549R cell apoptosis via inhibition of thioredoxin reductase (TrxR), elevated intracellular ROS levels, mitochondrial dysfunction and cell cycle arrest, making it an outstanding candidate for overcoming cisplatin resistance. PMID:26763798

  9. Janus magnetic cellular spheroids for vascular tissue engineering

    PubMed Central

    Mattix, Brandon M.; Olsen, Timothy R.; Casco, Megan; Reese, Laura; Poole, John T.; Zhang, Jing; Visconti, Richard P.; Simionescu, Agneta; Simionescu, Dan T.; Alexis, Frank

    2016-01-01

    Cell aggregates, or spheroids, have been used as building blocks to fabricate scaffold-free tissues that can closely mimic the native three-dimensional in vivo environment for broad applications including regenerative medicine and high throughput testing of drugs. The incorporation of magnetic nanoparticles (MNPs) into spheroids permits the manipulation of spheroids into desired shapes, patterns, and tissues using magnetic forces. Current strategies incorporating MNPs often involve cellular uptake, and should therefore be avoided because it induces adverse effects on cell activity, viability, and phenotype. Here, we report a Janus structure of magnetic cellular spheroids (JMCS) with spatial control of MNPs to form two distinct domains: cells and extracellular MNPs. This separation of cells and MNPs within magnetic cellular spheroids was successfully incorporated into cellular spheroids with various cellular and extracellular compositions and contents. The amount of cells that internalized MNPs was quantified and showed that JMCSs resulted in significantly lower internalization (35%) compared to uptake spheroids (83%, p < 0.05). Furthermore, the addition of MNPs to cellular spheroids using the Janus method has no adverse effects on cellular viability up to seven weeks, with spheroids maintaining at least 82% viability over 7 weeks when compared to control spheroids without MNPs. By safely incorporating MNPs into cellular spheroids, results demonstrated that JMCSs were capable of magnetic manipulation, and that magnetic forces used during magnetic force assembly mediate fusion into controlled patterns and complex tissues. Finally, JMCSs were assembled and fused into a vascular tissue construct 5 mm in diameter using magnetic force assembly. PMID:24183699

  10. Global Landslides on Rapidly Spinning Spheroids

    NASA Astrophysics Data System (ADS)

    Scheeres, Daniel J.; Sanchez, P.

    2013-10-01

    The angle of repose and conditions for global landslides on the surfaces of small, rapidly spinning, spheroidal asteroids are studied. Applying techniques of soil mechanics, we develop a theory for, and examples of, how regolith will fail and flow in this microgravity environment. Our motivation is to develop an understanding of the "top-shaped" class of asteroids based on analytical soil mechanics. Our analysis transforms the entire asteroid surface into a local frame where we can model it as a conventional granular pile with a surface slope, acceleration and height variations as a function of the body's spin rate, shape and density. A general finding is that the lowest point on a rapidly spinning spheroid is at the equator with the effective height of surface material monotonically increasing towards the polar regions, where the height can be larger than the physical radius of the body. We study the failure conditions of both cohesionless and cohesive regolith, and develop specific predictions of the surface profile as a function of the regolith angle of friction and the maximum spin rate experienced by the body. The theory also provides simple guidelines on what the shape may look like, although we do not analyze gravitationally self-consistent evolution of the body shape. The theory is tested with soft-sphere discrete element method granular mechanics simulations to better understand the dynamical aspects of global asteroid landslides. We find significant differences between failure conditions for cohesive and cohesionless regolith. In the case of cohesive regolith, we show that extremely small values of strength (much less than that found in lunar regolith) can stabilize a surface even at very rapid spin rates. Cohesionless surfaces, as expected, fail whenever their surface slopes exceed the angle of friction. Based on our analysis we propose that global landslides and the flow of material towards the equator on spheroidal bodies are precipitated by exogenous

  11. Spheroid body-forming cells in the human gastric cancer cell line MKN-45 possess cancer stem cell properties.

    PubMed

    Liu, Jianming; Ma, Lilin; Xu, Junfei; Liu, Chun; Zhang, Jianguo; Liu, Jie; Chen, Ruixin; Zhou, Youlang

    2013-02-01

    The cancer stem cell theory hypothesizes that cancer stem cells (CSCs), which possess self-renewal and other stem cell properties, are regarded as the cause of tumor formation, recurrence and metastasis. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. In this study, we enriched gastric cancer stem cells through spheroid body formation by cultivating the human gastric cancer cell line MKN-45 in defined serum-free medium. The stemness characteristics of spheroid body-forming cells, including self-renewal, proliferation, chemoresistance, tumorigenicity of the MKN-45 spheroid body-forming cells were evaluated, and the expression levels of stemness genes and related proteins in the MKN-45 spheroid body-forming cells were assessed. Furthermore, immunofluorescence staining for the stem cell markers on spheroid body-forming cells was examined to evaluate the association between stemness factors (Oct4, Sox2, Nanog) and the proposed CSC marker CD44. Our data demonstrated that non-adherent spheroid body-forming cells from the gastric cancer cell line MKN-45 cultured in stem cell-conditioned medium possessed gastric CSC properties, such as persistent self-renewal, extensive proliferation, drug resistance, high tumorigenic capacity and overexpression of CSC-related genes and proteins (Oct4, Sox2, Nanog and CD44), compared with the parental cells. More importantly, CD44-positive cells co-expressing the pluripotency genes Oct4, Sox2 and Nanog may represent gastric CSCs. Further experiments using more refined selection criteria such as a combination of two or multiple markers would be useful to specifically identify and purify CSCs.

  12. Transition from one- to two-dimensional development facilitates maintenance of multicellularity

    PubMed Central

    Manjarrez-Casas, Alejandra M.; Bagheri, Homayoun C.

    2016-01-01

    Filamentous organisms represent an example where incomplete separation after cell division underlies the development of multicellular formations. With a view to understanding the evolution of more complex multicellular structures, we explore the transition of multicellular growth from one to two dimensions. We develop a computational model to simulate multicellular development in populations where cells exhibit density-dependent division and death rates. In both the one- and two-dimensional contexts, multicellular formations go through a developmental cycle of growth and subsequent decay. However, the model shows that a transition to a higher dimension increases the size of multicellular formations and facilitates the maintenance of large cell clusters for significantly longer periods of time. We further show that the turnover rate for cell division and death scales with the number of iterations required to reach the stationary multicellular size at equilibrium. Although size and life cycles of multicellular organisms are affected by other environmental and genetic factors, the model presented here evaluates the extent to which the transition of multicellular growth from one to two dimensions contributes to the maintenance of multicellular structures during development. PMID:27703714

  13. Action of 50 Hz magnetic fields on cyclic AMP and intercellular communication in monolayers and spheroids of mammalian cells

    SciTech Connect

    Schimmelpfeng, J.; Stein, J.C.; Dertinger, H.

    1995-12-31

    To investigate the influence of physiological parameters such as cell density and three-dimensional cell contact on the biological action of a 2mT/50 Hz magnetic field, mouse fibroblasts were exposed as monolayers and as multicellular spheroids. Changes in cyclic AMP content of cells and alterations in gap junction-mediated intercellular communication were measured immediately after 5 min of exposure to the field. In monolayers of intermediate cell density (1 {times} 10{sup 5} cells/cm{sup 2}), the field treatment caused an increase in cAMP to 121% of the control level, whereas, at 3 {times} 10{sup 5} cells/cm{sup 2} (near confluence), a decrease to 88% of the unexposed cells was observed. Furthermore, field exposure stimulated gap-junction communication to 160% of the control level as determined by Lucifer yellow dye exchange. In spheroids, alterations in the radial profile of cellular cAMP were observed that were due both to field-induced local cAMP changes and to increased gap-junction permeability for this second messenger, the latter causing radial cAMP gradients to be flattened. The results indicate a strong dependence of field action on physiological parameters of the system exposed.

  14. Attenuation of microwaves by poly-disperse small spheroid particles

    NASA Astrophysics Data System (ADS)

    Zhang, Peichang; Wang, Zhenhui

    1998-08-01

    Expressions for calculating the attenuation cross sections of poly-disperse, small spheroids, whose rotatory axes are in specific status, have been derived from a universal formula for calculating the attenuation cross section of a particle of arbitrary shape. Attenuation cross sections of liquid, ice, and spongy spheroidal droplets in different size and eccentricity at different wave lengths have been computed and analyzed.

  15. Generation of an in vitro 3D PDAC stroma rich spheroid model.

    PubMed

    Ware, Matthew J; Keshishian, Vazrik; Law, Justin J; Ho, Jason C; Favela, Carlos A; Rees, Paul; Smith, Billie; Mohammad, Sayeeduddin; Hwang, Rosa F; Rajapakshe, Kimal; Coarfa, Cristian; Huang, Shixia; Edwards, Dean P; Corr, Stuart J; Godin, Biana; Curley, Steven A

    2016-11-01

    Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent desmoplastic/stromal reaction, which contributes to the poor clinical outcome of this disease. Therefore, greater understanding of the stroma development and tumor-stroma interactions is highly required. Pancreatic stellate cells (PSC) are myofibroblast-like cells located in exocrine areas of the pancreas, which as a result of inflammation produced by PDAC migrate and accumulate in the tumor mass, secreting extracellular matrix components and producing the dense PDAC stroma. Currently, only a few orthotopic or ectopic animal tumor models, where PDAC cells are injected into the pancreas or subcutaneous tissue layer, or genetically engineered animals offer tumors that encompass some stromal component. Herein, we report generation of a simple 3D PDAC in vitro micro-tumor model without an addition of external extracellular matrix, which encompasses a rich, dense and active stromal compartment. We have achieved this in vitro model by incorporating PSCs into 3D PDAC cell culture using a modified hanging drop method. It is now known that PSCs are the principal source of fibrosis in the stroma and interact closely with cancer cells to create a tumor facilitatory environment that stimulates local and distant tumor growth. The 3D micro-stroma models are highly reproducible with excellent uniformity, which can be used for PDAC-stroma interaction analysis and high throughput automated drug-screening assays. Additionally, the increased expression of collagenous regions means that molecular based perfusion and cytostaticity of gemcitabine is decreased in our Pancreatic adenocarcinoma stroma spheroids (PDAC-SS) model when compared to spheroids grown without PSCs. We believe this model will allow an improved knowledge of PDAC biology and has the potential to provide an insight into pathways that may be therapeutically targeted to inhibit PSC activation, thereby inhibiting the development of fibrosis in PDAC

  16. What Do We Learn from Spheroid Culture Systems? Insights from Tumorspheres Derived from Primary Colon Cancer Tissue

    PubMed Central

    Qureshi-Baig, Komal; Ullmann, Pit; Rodriguez, Fabien; Frasquilho, Sónia; Nazarov, Petr V.; Haan, Serge; Letellier, Elisabeth

    2016-01-01

    Due to their self-renewal and tumorigenic properties, tumor-initiating cells (TICs) have been hypothesized to be important targets for colorectal cancer (CRC). However the study of TICs is hampered by the fact that the identification and culturing of TICs is still a subject of extensive debate. Floating three-dimensional spheroid cultures (SC) that grow in serum-free medium supplemented with growth factors are supposed to be enriched in TICs. We generated SC from fresh clinical tumor specimens and compared them to SC isolated from CRC cell-lines as well as to adherent differentiated counterparts. Patient-derived SC display self-renewal capacity and can induce serial transplantable tumors in immuno-deficient mice, which phenotypically resemble the tumor of origin. In addition, the original tumor tissue and established SC retain several similar CRC-relevant mutations. Primary SC express key stemness proteins such as SOX2, OCT4, NANOG and LGR5 and importantly show increased chemoresistance ability compared to their adherent differentiated counterparts and to cell line-derived SC. Strikingly, cells derived from spheroid or adherent differentiating culture conditions displayed similar self-renewal capacity and equally formed tumors in immune-deficient mice, suggesting that self-renewal and tumor-initiation capacity of TICs is not restricted to phenotypically immature spheroid cells, which we describe to be highly plastic and able to reacquire stem-cell traits even after long differentiation processes. Finally, we identified two genes among a sphere gene expression signature that predict disease relapse in CRC patients. Here we propose that SC derived from fresh patient tumor tissue present interesting phenotypic features that may have clinical relevance for chemoresistance and disease relapse and therefore represent a valuable tool to test for new CRC-therapies that overcome drug resistance. PMID:26745821

  17. What Do We Learn from Spheroid Culture Systems? Insights from Tumorspheres Derived from Primary Colon Cancer Tissue.

    PubMed

    Qureshi-Baig, Komal; Ullmann, Pit; Rodriguez, Fabien; Frasquilho, Sónia; Nazarov, Petr V; Haan, Serge; Letellier, Elisabeth

    2016-01-01

    Due to their self-renewal and tumorigenic properties, tumor-initiating cells (TICs) have been hypothesized to be important targets for colorectal cancer (CRC). However the study of TICs is hampered by the fact that the identification and culturing of TICs is still a subject of extensive debate. Floating three-dimensional spheroid cultures (SC) that grow in serum-free medium supplemented with growth factors are supposed to be enriched in TICs. We generated SC from fresh clinical tumor specimens and compared them to SC isolated from CRC cell-lines as well as to adherent differentiated counterparts. Patient-derived SC display self-renewal capacity and can induce serial transplantable tumors in immuno-deficient mice, which phenotypically resemble the tumor of origin. In addition, the original tumor tissue and established SC retain several similar CRC-relevant mutations. Primary SC express key stemness proteins such as SOX2, OCT4, NANOG and LGR5 and importantly show increased chemoresistance ability compared to their adherent differentiated counterparts and to cell line-derived SC. Strikingly, cells derived from spheroid or adherent differentiating culture conditions displayed similar self-renewal capacity and equally formed tumors in immune-deficient mice, suggesting that self-renewal and tumor-initiation capacity of TICs is not restricted to phenotypically immature spheroid cells, which we describe to be highly plastic and able to reacquire stem-cell traits even after long differentiation processes. Finally, we identified two genes among a sphere gene expression signature that predict disease relapse in CRC patients. Here we propose that SC derived from fresh patient tumor tissue present interesting phenotypic features that may have clinical relevance for chemoresistance and disease relapse and therefore represent a valuable tool to test for new CRC-therapies that overcome drug resistance. PMID:26745821

  18. Scattering of polarized Gaussian light by a spheroidal particle

    NASA Astrophysics Data System (ADS)

    Sun, Xianming; Xiao, Sai; Ma, Lixiu; Su, Baochen

    2015-02-01

    Light scattering by a small particle can produce light with polarization characteristics different from those of the incident beam. In this article, we studied the polarized Gaussian beam scattered by a spheroidal particle within the generalized Lorenz-Mie theory framework. A theoretical procedure is given to expand an incident Gaussian beam in terms of spheroidal vector wave functions. We studied the single scattering properties of a single spheroidal particle with varying aspect ratios and size parameters. Exact analytic solutions are obtained for computing the amplitude matrix and single scattering Muller matrix for a single spheroid with normal illumination. The Muller scattering matrix elements of a single spheroid are compared between plane wave and Gaussian light beam as incident light source.

  19. On Convergence Aspects of Spheroidal Monogenics

    NASA Astrophysics Data System (ADS)

    Georgiev, S.; Morais, J.

    2011-09-01

    Orthogonal polynomials have found wide applications in mathematical physics, numerical analysis, and other fields. Accordingly there is an enormous amount of variety of such polynomials and relations that describe their properties. The paper's main results are the discussion of approximation properties for monogenic functions over prolate spheroids in R3 in terms of orthogonal monogenic polynomials and their interdependences. Certain results are stated without proof for now. The motivation for the present study stems from the fact that these polynomials play an important role in the calculation of the Bergman kernel and Green's monogenic functions in a spheroid. Once these functions are known, it is possible to solve both basic boundary value and conformal mapping problems. Interestingly, most of the used methods have a n-dimensional counterpart and can be extended to arbitrary ellipsoids. But such a procedure would make the further study of the underlying ellipsoidal monogenics somewhat laborious, and for this reason we shall not discuss these general cases here. To the best of our knowledge, this does not appear to have been done in literature before.

  20. Design of a Uranium Dioxide Spheroidization System

    NASA Technical Reports Server (NTRS)

    Cavender, Daniel P.; Mireles, Omar R.; Frendi, Abdelkader

    2013-01-01

    The plasma spheroidization system (PSS) is the first process in the development of tungsten-uranium dioxide (W-UO2) fuel cermets. The PSS process improves particle spherocity and surface morphology for coating by chemical vapor deposition (CVD) process. Angular fully dense particles melt in an argon-hydrogen plasma jet at between 32-36 kW, and become spherical due to surface tension. Surrogate CeO2 powder was used in place of UO2 for system and process parameter development. Particles range in size from 100 - 50 microns in diameter. Student s t-test and hypothesis testing of two proportions statistical methods were applied to characterize and compare the spherocity of pre and post process powders. Particle spherocity was determined by irregularity parameter. Processed powders show great than 800% increase in the number of spherical particles over the stock powder with the mean spherocity only mildly improved. It is recommended that powders be processed two-three times in order to reach the desired spherocity, and that process parameters be optimized for a more narrow particles size range. Keywords: spherocity, spheroidization, plasma, uranium-dioxide, cermet, nuclear, propulsion

  1. Corelation between Complexity and Stability in Multicellular Organisms

    NASA Astrophysics Data System (ADS)

    Werman, Steven D.; Qaddour, Jihad; Deyoung, Gary; Misra, Prasanta K.

    1997-03-01

    We present a model to study the corelation between the complexity and stability of unicellular and multicellular organisms. We state the postulates made by us and define the thermodynamic functions and other parameters used to formulate the problem. We have also used several theorems based on reasonable assumptions to analyze the functional interactions which are non-symmetric, non-local and non-instantaneous. The mathematical model developed by using these hypotheses is explained in detail and a set of non-linear equations for each type of organism is presented. We present the results obtained by us by solving these equations.

  2. Waltzing Volvox/: Orbiting Bound States of Flagellated Multicellular Algae

    NASA Astrophysics Data System (ADS)

    Drescher, K.; Leptos, K.; Pedley, T. J.; Goldstein, R. E.; Ishikawa, T.

    2008-11-01

    The spherical colonial alga Volvox swims by means of flagella on thousands of surface somatic cells. This geometry and its large size makes it a model organism for the fluid dynamics of multicellularity. Remarkably, when two nearby colonies swim close to a solid surface, they are attracted together and can form a stable bound state in which they continuously waltz around each other. A surface-mediated hydrodynamic attraction between colonies combined with the rotational motion of bottom-heavy Volvox are shown to explain the stability and dynamics of the bound state. This phenomenon is suggested to underlie observed clustering of colonies at surfaces.

  3. Experimental evolution of an alternating uni- and multicellular life cycle in Chlamydomonas reinhardtii

    PubMed Central

    Ratcliff, William C.; Herron, Matthew D.; Howell, Kathryn; Pentz, Jennifer T.; Rosenzweig, Frank; Travisano, Michael

    2013-01-01

    The transition to multicellularity enabled the evolution of large, complex organisms, but early steps in this transition remain poorly understood. Here we show that multicellular complexity, including development from a single cell, can evolve rapidly in a unicellular organism that has never had a multicellular ancestor. We subject the alga Chlamydomonas reinhardtii to conditions that favour multicellularity, resulting in the evolution of a multicellular life cycle in which clusters reproduce via motile unicellular propagules. While a single-cell genetic bottleneck during ontogeny is widely regarded as an adaptation to limit among-cell conflict, its appearance very early in this transition suggests that it did not evolve for this purpose. Instead, we find that unicellular propagules are adaptive even in the absence of intercellular conflict, maximizing cluster-level fecundity. These results demonstrate that the unicellular bottleneck, a trait essential for evolving multicellular complexity, can arise rapidly via co-option of the ancestral unicellular form. PMID:24193369

  4. Cancer across the tree of life: cooperation and cheating in multicellularity

    PubMed Central

    Aktipis, C. Athena; Boddy, Amy M.; Jansen, Gunther; Hibner, Urszula; Hochberg, Michael E.; Maley, Carlo C.; Wilkinson, Gerald S.

    2015-01-01

    Multicellularity is characterized by cooperation among cells for the development, maintenance and reproduction of the multicellular organism. Cancer can be viewed as cheating within this cooperative multicellular system. Complex multicellularity, and the cooperation underlying it, has evolved independently multiple times. We review the existing literature on cancer and cancer-like phenomena across life, not only focusing on complex multicellularity but also reviewing cancer-like phenomena across the tree of life more broadly. We find that cancer is characterized by a breakdown of the central features of cooperation that characterize multicellularity, including cheating in proliferation inhibition, cell death, division of labour, resource allocation and extracellular environment maintenance (which we term the five foundations of multicellularity). Cheating on division of labour, exhibited by a lack of differentiation and disorganized cell masses, has been observed in all forms of multicellularity. This suggests that deregulation of differentiation is a fundamental and universal aspect of carcinogenesis that may be underappreciated in cancer biology. Understanding cancer as a breakdown of multicellular cooperation provides novel insights into cancer hallmarks and suggests a set of assays and biomarkers that can be applied across species and characterize the fundamental requirements for generating a cancer. PMID:26056363

  5. Cancer across the tree of life: cooperation and cheating in multicellularity.

    PubMed

    Aktipis, C Athena; Boddy, Amy M; Jansen, Gunther; Hibner, Urszula; Hochberg, Michael E; Maley, Carlo C; Wilkinson, Gerald S

    2015-07-19

    Multicellularity is characterized by cooperation among cells for the development, maintenance and reproduction of the multicellular organism. Cancer can be viewed as cheating within this cooperative multicellular system. Complex multicellularity, and the cooperation underlying it, has evolved independently multiple times. We review the existing literature on cancer and cancer-like phenomena across life, not only focusing on complex multicellularity but also reviewing cancer-like phenomena across the tree of life more broadly. We find that cancer is characterized by a breakdown of the central features of cooperation that characterize multicellularity, including cheating in proliferation inhibition, cell death, division of labour, resource allocation and extracellular environment maintenance (which we term the five foundations of multicellularity). Cheating on division of labour, exhibited by a lack of differentiation and disorganized cell masses, has been observed in all forms of multicellularity. This suggests that deregulation of differentiation is a fundamental and universal aspect of carcinogenesis that may be underappreciated in cancer biology. Understanding cancer as a breakdown of multicellular cooperation provides novel insights into cancer hallmarks and suggests a set of assays and biomarkers that can be applied across species and characterize the fundamental requirements for generating a cancer.

  6. Generation of three-dimensional multiple spheroid model of olfactory ensheathing cells using floating liquid marbles

    PubMed Central

    Vadivelu, Raja K.; Ooi, Chin H.; Yao, Rebecca-Qing; Tello Velasquez, Johana; Pastrana, Erika; Diaz-Nido, Javier; Lim, Filip; Ekberg, Jenny A. K.; Nguyen, Nam-Trung; St John, James A.

    2015-01-01

    We describe a novel protocol for three-dimensional culturing of olfactory ensheathing cells (OECs), which can be used to understand how OECs interact with other cells in three dimensions. Transplantation of OECs is being trialled for repair of the paralysed spinal cord, with promising but variable results and thus the therapy needs improving. To date, studies of OEC behaviour in a multicellular environment have been hampered by the lack of suitable three-dimensional cell culture models. Here, we exploit the floating liquid marble, a liquid droplet coated with hydrophobic powder and placed on a liquid bath. The presence of the liquid bath increases the humidity and minimises the effect of evaporation. Floating liquid marbles allow the OECs to freely associate and interact to produce OEC spheroids with uniform shapes and sizes. In contrast, a sessile liquid marble on a solid surface suffers from evaporation and the cells aggregate with irregular shapes. We used floating liquid marbles to co-culture OECs with Schwann cells and astrocytes which formed natural structures without the confines of gels or bounding layers. This protocol can be used to determine how OECs and other cell types associate and interact while forming complex cell structures. PMID:26462469

  7. Generation of three-dimensional multiple spheroid model of olfactory ensheathing cells using floating liquid marbles

    NASA Astrophysics Data System (ADS)

    Vadivelu, Raja K.; Ooi, Chin H.; Yao, Rebecca-Qing; Tello Velasquez, Johana; Pastrana, Erika; Diaz-Nido, Javier; Lim, Filip; Ekberg, Jenny A. K.; Nguyen, Nam-Trung; St John, James A.

    2015-10-01

    We describe a novel protocol for three-dimensional culturing of olfactory ensheathing cells (OECs), which can be used to understand how OECs interact with other cells in three dimensions. Transplantation of OECs is being trialled for repair of the paralysed spinal cord, with promising but variable results and thus the therapy needs improving. To date, studies of OEC behaviour in a multicellular environment have been hampered by the lack of suitable three-dimensional cell culture models. Here, we exploit the floating liquid marble, a liquid droplet coated with hydrophobic powder and placed on a liquid bath. The presence of the liquid bath increases the humidity and minimises the effect of evaporation. Floating liquid marbles allow the OECs to freely associate and interact to produce OEC spheroids with uniform shapes and sizes. In contrast, a sessile liquid marble on a solid surface suffers from evaporation and the cells aggregate with irregular shapes. We used floating liquid marbles to co-culture OECs with Schwann cells and astrocytes which formed natural structures without the confines of gels or bounding layers. This protocol can be used to determine how OECs and other cell types associate and interact while forming complex cell structures.

  8. Transport by Collective Flagellar Beating Facilitates Evolutionary Transitions to Multicellularity

    NASA Astrophysics Data System (ADS)

    Short, Martin; Powers, Thomas

    2005-11-01

    A central problem underlying the evolution from single cells to multicellular organisms is the relationship between metabolic requirements and environmental metabolite exchange with increasing size. For organisms that form spherical colonies such as the volvocalean green algae, there is a bottleneck if diffusion alone governs nutrient uptake as they increase in size, for the diffusive flux is linear in the radius while the requirements of surface somatic cells grow quadratically. Using Volvox as a model organism, we examine experimentally and theoretically the role that advection of fluid by surface flagella plays in enhancing nutrient uptake. We show that the fluid flow driven by the coordinated beating of those flagella produces a boundary layer in the concentration of a diffusing solute which renders the metabolite exchange rate quadratic in the colony radius. This bypasses the diffusive bottleneck, facilitating evolutionary transitions to multicellularity which may be driven by other environmental factors. These results suggest that flagella may have evolved not only for motility, but also to enhance metabolite exchange.

  9. Emergent patterns of growth controlled by multicellular form and mechanics

    PubMed Central

    Nelson, Celeste M.; Jean, Ronald P.; Tan, John L.; Liu, Wendy F.; Sniadecki, Nathan J.; Spector, Alexander A.; Chen, Christopher S.

    2005-01-01

    Spatial patterns of cellular growth generate mechanical stresses that help to push, fold, expand, and deform tissues into their specific forms. Genetic factors are thought to specify patterns of growth and other behaviors to drive morphogenesis. Here, we show that tissue form itself can feed back to regulate patterns of proliferation. Using microfabrication to control the organization of sheets of cells, we demonstrated the emergence of stable patterns of proliferative foci. Regions of concentrated growth corresponded to regions of high tractional stress generated within the sheet, as predicted by a finite-element model of multicellular mechanics and measured directly by using a micromechanical force sensor array. Inhibiting actomyosin-based tension or cadherin-mediated connections between cells disrupted the spatial pattern of proliferation. These findings demonstrate the existence of patterns of mechanical forces that originate from the contraction of cells, emerge from their multicellular organization, and result in patterns of growth. Thus, tissue form is not only a consequence but also an active regulator of tissue growth. PMID:16049098

  10. THE SPLASH SURVEY: KINEMATICS OF ANDROMEDA's INNER SPHEROID

    SciTech Connect

    Dorman, Claire E.; Guhathakurta, Puragra; and others

    2012-06-20

    The combination of large size, high stellar density, high metallicity, and Sersic surface brightness profile of the spheroidal component of the Andromeda galaxy (M31) within R{sub proj} {approx} 20 kpc suggests that it is unlike any subcomponent of the Milky Way. In this work we capitalize on our proximity to and external view of M31 to probe the kinematical properties of this 'inner spheroid'. We employ a Markov chain Monte Carlo (MCMC) analysis of resolved stellar kinematics from Keck/DEIMOS spectra of 5651 red giant branch stars to disentangle M31's inner spheroid from its stellar disk. We measure the mean velocity and dispersion of the spheroid in each of five spatial bins after accounting for a locally cold stellar disk as well as the Giant Southern Stream and associated tidal debris. For the first time, we detect significant spheroid rotation (v{sub rot} {approx} 50 km s{sup -1}) beyond R{sub proj} {approx} 5 kpc. The velocity dispersion decreases from about 140 km s{sup -1} at R{sub proj} = 7 kpc to 120 km s{sup -1} at R{sub proj} = 14 kpc, consistent to 2{sigma} with existing measurements and models. We calculate the probability that a given star is a member of the spheroid and find that the spheroid has a significant presence throughout the spatial extent of our sample. Lastly, we show that the flattening of the spheroid is due to velocity anisotropy in addition to rotation. Though this suggests that the inner spheroid of M31 more closely resembles an elliptical galaxy than a typical spiral galaxy bulge, it should be cautioned that our measurements are much farther out (2-14r{sub eff}) than for the comparison samples.

  11. Mass Modelling of dwarf Spheroidal Galaxies

    NASA Astrophysics Data System (ADS)

    Klimentowski, Jarosław; Łokas, Ewa L.; Kazantzidis, Stelios; Prada, Francisco; Mayer, Lucio; Mamon, Gary A.

    2008-05-01

    We study the origin and properties of unbound stars in the kinematic samples of dwarf spheroidal galaxies. For this purpose we have run a high resolution N-body simulation of a two-component dwarf galaxy orbiting in a Milky Way potential. We create mock kinematic data sets by observing the dwarf in different directions. When the dwarf is observed along the tidal tails the kinematic samples are strongly contaminated by unbound stars from the tails. However, most of the unbound stars can be removed by the method of interloper rejection proposed by den Hartog & Katgert. We model the velocity dispersion profiles of the cleaned-up kinematic samples using solutions of the Jeans equation. We show that even for such a strongly stripped dwarf the Jeans analysis, when applied to cleaned samples, allows us to reproduce the mass and mass-to-light ratio of the dwarf with accuracy typically better than 25%.

  12. [Reparative and neoplastic spheroid cellular structures and their mathematical model].

    PubMed

    Kogan, E A; Namiot, V A; Demura, T A; Faĭzullina, N M; Sukhikh, G T

    2014-01-01

    Spheroid cell structures in the cell cultures have been described and are used for studying cell-cell and cell- matrix interactions. At the same time, spheroid cell structure participation in the repair and development of cancer in vivo remains unexplored. The aim of this study was to investigate the cellular composition of spherical structures and their functional significance in the repair of squamous epithelium in human papilloma virus-associated cervical pathology--chronic cervicitis and cervical intraepithelial neoplasia 1-3 degree, and also construct a mathematical model to explain the development and behavior of such spheroid cell structure.

  13. Nondestructive evaluation of spheroidized steel using magnetoacoustic and Barkhausen emission

    SciTech Connect

    Lo, C.C.H.; Jakubovics, J.P.; Scruby, C.B.

    1997-09-01

    Magnetoacoustic emission (MAE) and Barkhausen emission (BE) measurements have been made on pearlitic steel samples annealed to produce spheroidized microstructures with various sizes and distributions of carbides. It was found that spheroidization of the pearlitic structure and coarsening of carbide particles result in a decrease in the peak height ratio of the MAE profile and the formation of a second peak in the BE profile. These results can be interpreted from observations of the domain structure by Lorentz microscopy. The sensitivity of the MAE and BE profiles to the carbide size and morphology makes both techniques useful for monitoring the microstructure of spheroidized steels.

  14. An oxygen-permeable spheroid culture system for the prevention of central hypoxia and necrosis of spheroids.

    PubMed

    Anada, Takahisa; Fukuda, Junji; Sai, Yuko; Suzuki, Osamu

    2012-11-01

    Since oxygen is one of the critical limiting factors for maintaining cell viability and function, a great deal of effort is being focused on improving the oxygen supply to three-dimensional (3D) cellular constructs. Here, we report a technique to construct spheroids utilizing 3D culture chips with a rapid and simple method for the replication of the surface structures of a polydimethylsiloxane (PDMS) mold. The resultant spheroid culture chip made it possible to rapidly yield high numbers of the spheroids at a time as well as to obtain uniform spheroids with a narrow size distribution and to collect the spheroids easily and noninvasively. The most important feature of this spheroid culture chip is that it enables direct oxygen supply to the cells because the chip is made with only gas-permeable PDMS. When human hepatoma HepG2 cells were grown on the oxygen-permeable chips as a model for liver cells, the cellular growth was remarkably enhanced, and the anaerobic glycolysis was significantly reduced compared to the non-oxygen-permeable chips. Furthermore, the oxygen-permeable chip improved the albumin secretion rates compared to the conventional spheroid culture system after 10 days. Histochemical and immunohistochemical analyses demonstrated that the oxygen-permeable chip dramatically prevented hypoxia in the core of the spheroids and subsequent central necrosis. Surprisingly, the diameters of approximately 400 and 600 μm were estimated to be the threshold of the hypoxic and survival size, respectively, for the HepG2 spheroids in the oxygenated chip. These results indicate that this chip is useful for engineering 3D cellular constructs with high viability and functionality for tissue engineering.

  15. Coupling Gd-DTPA with a bispecific, recombinant protein anti-EGFR-iRGD complex improves tumor targeting in MRI

    PubMed Central

    XIN, XIAOYAN; SHA, HUIZI; SHEN, JINGTAO; ZHANG, BING; ZHU, BIN; LIU, BAORUI

    2016-01-01

    Recombinant anti-epidermal growth factor receptor-internalizing arginine-glycine-aspartic acid (anti-EGFR single-domain antibody fused with iRGD peptide) protein efficiently targets the EGFR extracellular domain and integrin αvβ/β5, and shows a high penetration into cells. Thus, this protein may improve penetration of conjugated drugs into the deep zone of gastric cancer multicellular 3D spheroids. In the present study, a novel tumor-targeting contrast agent for magnetic resonance imaging (MRI) was developed, by coupling gadolinium-diethylene triamine pentaacetate (Gd-DTPA) with the bispecific recombinant anti-EGFR-iRGD protein. The anti-EGFR-iRGD protein was extracted from Escherichia coli and Gd was loaded onto the recombinant protein by chelation using DTPA anhydride. Single-targeting agent anti-EGFR-DTPA-Gd, which served as the control, was also prepared. The results of the present study showed that anti-EGFR-iRGD-DTPA-Gd exhibited no significant cyto toxicity to human gastric carcinoma cells (BGC-823) under the experimental conditions used. Compared with a conventional contrast agent (Magnevist), anti-EGFR-iRGD-DTPA-Gd showed higher T1 relaxivity (10.157/mM/sec at 3T) and better tumor-targeting ability. In addition, the signal intensity and the area under curve for the enhanced signal time in tumor, in vivo, were stronger than Gd-DTPA alone or the anti-EGFR-Gd control. Thus, Gd-labelled anti-EGFR-iRGD has potential as a tumor-targeting contrast agent for improved MRI. PMID:27035336

  16. Cancer-Associated Fibroblasts Modify the Response of Prostate Cancer Cells to Androgen and Anti-Androgens in Three-Dimensional Spheroid Culture

    PubMed Central

    Eder, Theresa; Weber, Anja; Neuwirt, Hannes; Grünbacher, Georg; Ploner, Christian; Klocker, Helmut; Sampson, Natalie; Eder, Iris E.

    2016-01-01

    Androgen receptor (AR) targeting remains the gold standard treatment for advanced prostate cancer (PCa); however, treatment resistance remains a major clinical problem. To study the therapeutic effects of clinically used anti-androgens we characterized herein a tissue-mimetic three-dimensional (3D) in vitro model whereby PCa cells were cultured alone or with PCa-associated fibroblasts (CAFs). Notably, the ratio of PCa cells to CAFs significantly increased in time in favor of the tumor cells within the spheroids strongly mimicking PCa in vivo. Despite this loss of CAFs, the stromal cells, which were not sensitive to androgen and even stimulated by the anti-androgens, significantly influenced the sensitivity of PCa cells to androgen and to the anti-androgens bicalutamide and enzalutamide. In particular, DuCaP cells lost sensitivity to enzalutamide when co-cultured with CAFs. In LAPC4/CAF and LNCaP/CAF co-culture spheroids the impact of the CAFs was less pronounced. In addition, 3D spheroids exhibited a significant increase in E-cadherin and substantial expression of vimentin in co-culture spheroids, whereas AR levels remained unchanged or even decreased. In LNCaP/CAF spheroids we further found increased Akt signaling that could be inhibited by the phosphatidyl-inositol 3 kinase (PI3K) inhibitor LY294002, thereby overcoming the anti-androgen resistance of the spheroids. Our data show that CAFs influence drug response of PCa cells with varying impact and further suggest this spheroid model is a valuable in vitro drug testing tool. PMID:27598125

  17. Cancer-Associated Fibroblasts Modify the Response of Prostate Cancer Cells to Androgen and Anti-Androgens in Three-Dimensional Spheroid Culture.

    PubMed

    Eder, Theresa; Weber, Anja; Neuwirt, Hannes; Grünbacher, Georg; Ploner, Christian; Klocker, Helmut; Sampson, Natalie; Eder, Iris E

    2016-01-01

    Androgen receptor (AR) targeting remains the gold standard treatment for advanced prostate cancer (PCa); however, treatment resistance remains a major clinical problem. To study the therapeutic effects of clinically used anti-androgens we characterized herein a tissue-mimetic three-dimensional (3D) in vitro model whereby PCa cells were cultured alone or with PCa-associated fibroblasts (CAFs). Notably, the ratio of PCa cells to CAFs significantly increased in time in favor of the tumor cells within the spheroids strongly mimicking PCa in vivo. Despite this loss of CAFs, the stromal cells, which were not sensitive to androgen and even stimulated by the anti-androgens, significantly influenced the sensitivity of PCa cells to androgen and to the anti-androgens bicalutamide and enzalutamide. In particular, DuCaP cells lost sensitivity to enzalutamide when co-cultured with CAFs. In LAPC4/CAF and LNCaP/CAF co-culture spheroids the impact of the CAFs was less pronounced. In addition, 3D spheroids exhibited a significant increase in E-cadherin and substantial expression of vimentin in co-culture spheroids, whereas AR levels remained unchanged or even decreased. In LNCaP/CAF spheroids we further found increased Akt signaling that could be inhibited by the phosphatidyl-inositol 3 kinase (PI3K) inhibitor LY294002, thereby overcoming the anti-androgen resistance of the spheroids. Our data show that CAFs influence drug response of PCa cells with varying impact and further suggest this spheroid model is a valuable in vitro drug testing tool. PMID:27598125

  18. Sorodiplophrys stercorea: Another Novel Lineage of Sorocarpic Multicellularity.

    PubMed

    Tice, Alexander K; Silberman, Jeffrey D; Walthall, Austin C; Le, Khoa N D; Spiegel, Frederick W; Brown, Matthew W

    2016-09-01

    Sorodiplophrys stercorea is a sorocarpic organism that utilizes filose pseudopodia for locomotion and absorptive nutrition. It has traditionally been considered to be a member of the Labyrinthulae based on its morphology. Its closest relatives were thought to be species in the taxon Diplophrys. Since the genus Diplophrys has been shown to be paraphyletic and S. stercorea has pseudopodia similar to some members of Rhizaria, we examined its relationship with other eukaryotes. We obtained four isolates from the dung of cow and horse, brought each into monoeukaryotic culture, and sequenced their SSU rRNA gene for phylogenetic analysis. All our isolates were shown to form a monophyletic group in the Labyrinthulae, nested in the Amphifiloidea clade. Our results demonstrate that Sorodiplophrys is more closely related to species of the genus Amphifila than to Diplophrys and represents an additional independent origin of sorocarpic multicellularity among eukaryotes. This study represents the first confirmed sorocarpic lifestyle in the Stramenopiles.

  19. Sorodiplophrys stercorea: Another Novel Lineage of Sorocarpic Multicellularity.

    PubMed

    Tice, Alexander K; Silberman, Jeffrey D; Walthall, Austin C; Le, Khoa N D; Spiegel, Frederick W; Brown, Matthew W

    2016-09-01

    Sorodiplophrys stercorea is a sorocarpic organism that utilizes filose pseudopodia for locomotion and absorptive nutrition. It has traditionally been considered to be a member of the Labyrinthulae based on its morphology. Its closest relatives were thought to be species in the taxon Diplophrys. Since the genus Diplophrys has been shown to be paraphyletic and S. stercorea has pseudopodia similar to some members of Rhizaria, we examined its relationship with other eukaryotes. We obtained four isolates from the dung of cow and horse, brought each into monoeukaryotic culture, and sequenced their SSU rRNA gene for phylogenetic analysis. All our isolates were shown to form a monophyletic group in the Labyrinthulae, nested in the Amphifiloidea clade. Our results demonstrate that Sorodiplophrys is more closely related to species of the genus Amphifila than to Diplophrys and represents an additional independent origin of sorocarpic multicellularity among eukaryotes. This study represents the first confirmed sorocarpic lifestyle in the Stramenopiles. PMID:26940948

  20. Modeling collective & intelligent decision making of multi-cellular populations.

    PubMed

    Shin, Yong-Jun; Mahrou, Bahareh

    2014-01-01

    In the presence of unpredictable disturbances and uncertainties, cells intelligently achieve their goals by sharing information via cell-cell communication and making collective decisions, which are more reliable compared to individual decisions. Inspired by adaptive sensor network algorithms studied in communication engineering, we propose that a multi-cellular adaptive network can convert unreliable decisions by individual cells into a more reliable cell-population decision. It is demonstrated using the effector T helper (a type of immune cell) population, which plays a critical role in initiating immune reactions in response to invading foreign agents (e.g., viruses, bacteria, etc.). While each individual cell follows a simple adaptation rule, it is the combined coordination among multiple cells that leads to the manifestation of "self-organizing" decision making via cell-cell communication.

  1. Modeling a synthetic multicellular clock: Repressilators coupled by quorum sensing

    NASA Astrophysics Data System (ADS)

    Garcia-Ojalvo, Jordi; Elowitz, Michael B.; Strogatz, Steven H.

    2004-07-01

    Diverse biochemical rhythms are generated by thousands of cellular oscillators that somehow manage to operate synchronously. In fields ranging from circadian biology to endocrinology, it remains an exciting challenge to understand how collective rhythms emerge in multicellular structures. Using mathematical and computational modeling, we study the effect of coupling through intercell signaling in a population of Escherichia coli cells expressing a synthetic biological clock. Our results predict that a diverse and noisy community of such genetic oscillators interacting through a quorum-sensing mechanism should self-synchronize in a robust way, leading to a substantially improved global rhythmicity in the system. As such, the particular system of coupled genetic oscillators considered here might be a good candidate to provide the first quantitative example of a synchronization transition in a population of biological oscillators.

  2. Imaging Sensitivity of Quiescent Cancer Cells to Metabolic Perturbations in Bone Marrow Spheroids

    PubMed Central

    Cavnar, Stephen P.; Xiao, Annie; Gibbons, Anne E.; Rickelmann, Andrew D.; Neely, Taylor; Luker, Kathryn E.; Takayama, Shuichi; Luker, Gary D.

    2016-01-01

    Malignant cells from breast cancer and other common cancers such as prostate and melanoma may persist in bone marrow as quiescent, non-dividing cells that remain viable for years or even decades before resuming proliferation to cause recurrent disease. This phenomenon, referred to clinically as tumor dormancy, poses tremendous challenges to curing patients with breast cancer. Quiescent tumor cells resist chemotherapy drugs that predominantly target proliferating cells, limiting success of neo-adjuvant and adjuvant therapies. We recently developed a 3D spheroid model of quiescent breast cancer cells in bone marrow for mechanistic and drug testing studies. We combined this model with optical imaging methods for label-free detection of cells preferentially utilizing glycolysis versus oxidative metabolism to investigate the metabolic state of co-culture spheroids with different bone marrow stromal and breast cancer cells. Through imaging and biochemical assays, we identified different metabolic states of bone marrow stromal cells that control metabolic status and flexibilities of co-cultured breast cancer cells. We tested metabolic stresses and targeted inhibition of specific metabolic pathways to identify approaches to preferentially eliminate quiescent breast cancer cells from bone marrow environments. These studies establish an integrated imaging approach to analyze metabolism in complex tissue environments to identify new metabolically-targeted cancer therapies. PMID:27478871

  3. Nascent multicellular life and the emergence of individuality.

    PubMed

    De Monte, Silvia; Rainey, Paul B

    2014-04-01

    The evolution of multicellular organisms from unicellular ancestors involves a shift in the level at which selection operates. It is usual to think about this shift in terms of the emergence of traits that cause heritable differences in reproductive output at the level of nascent collectives. Defining these traits and the causes of their origin lies at the heart of understanding the evolution of multicellular life. In working toward a mechanistic, take-nothing-for-granted account, we begin by recognizing that the standard Lewontin formulation of properties necessary and sufficient for evolution by natural selection does not necessarily encompass Darwinian evolution in primitive collectives where parent-offspring relationships may have been poorly defined. This, we suggest, limits the ability to conceptualize and capture the earliest manifestations of Darwinian properties. By way of solution we propose a relaxed interpretation of Lewontin's conditions and present these in the form of a set of necessary requirements for evolution by natural selection based upon the establishment of genealogical connections between recurrences of collectives. With emphasis on genealogy - as opposed to reproduction - it is possible to conceive selection acting on collectives prior to any manifestation of heritable variance in fitness. Such possibility draws attention to the evolutionary emergence of traits that strengthen causal relationships between recurrences - traits likely to underpin the emergence of forms of multiplication that establish parent-offspring relationships. Application of this framework to collectives of marginal status, particularly those whose recurrence is not defined by genealogy, makes clear that change at the level of collectives need not arise from selection acting at the higher level. We conclude by outlining applicability of our framework to loosely defined collectives of cells, such as those comprising the slugs of social amoeba and microbes that constitute

  4. Nascent multicellular life and the emergence of individuality.

    PubMed

    De Monte, Silvia; Rainey, Paul B

    2014-04-01

    The evolution of multicellular organisms from unicellular ancestors involves a shift in the level at which selection operates. It is usual to think about this shift in terms of the emergence of traits that cause heritable differences in reproductive output at the level of nascent collectives. Defining these traits and the causes of their origin lies at the heart of understanding the evolution of multicellular life. In working toward a mechanistic, take-nothing-for-granted account, we begin by recognizing that the standard Lewontin formulation of properties necessary and sufficient for evolution by natural selection does not necessarily encompass Darwinian evolution in primitive collectives where parent-offspring relationships may have been poorly defined. This, we suggest, limits the ability to conceptualize and capture the earliest manifestations of Darwinian properties. By way of solution we propose a relaxed interpretation of Lewontin's conditions and present these in the form of a set of necessary requirements for evolution by natural selection based upon the establishment of genealogical connections between recurrences of collectives. With emphasis on genealogy - as opposed to reproduction - it is possible to conceive selection acting on collectives prior to any manifestation of heritable variance in fitness. Such possibility draws attention to the evolutionary emergence of traits that strengthen causal relationships between recurrences - traits likely to underpin the emergence of forms of multiplication that establish parent-offspring relationships. Application of this framework to collectives of marginal status, particularly those whose recurrence is not defined by genealogy, makes clear that change at the level of collectives need not arise from selection acting at the higher level. We conclude by outlining applicability of our framework to loosely defined collectives of cells, such as those comprising the slugs of social amoeba and microbes that constitute

  5. Calcium inhibits bap-dependent multicellular behavior in Staphylococcus aureus.

    PubMed

    Arrizubieta, María Jesús; Toledo-Arana, Alejandro; Amorena, Beatriz; Penadés, José R; Lasa, Iñigo

    2004-11-01

    Bap (biofilm-associated protein) is a 254-kDa staphylococcal surface protein implicated in formation of biofilms by staphylococci isolated from chronic mastitis infections. The presence of potential EF-hand motifs in the amino acid sequence of Bap prompted us to investigate the effect of calcium on the multicellular behavior of Bap-expressing staphylococci. We found that addition of millimolar amounts of calcium to the growth media inhibited intercellular adhesion of and biofilm formation by Bap-positive strain V329. Addition of manganese, but not addition of magnesium, also inhibited biofilm formation, whereas bacterial aggregation in liquid media was greatly enhanced by metal-chelating agents. In contrast, calcium or chelating agents had virtually no effect on the aggregation of Bap-deficient strain M556. The biofilm elicited by insertion of bap into the chromosome of a biofilm-negative strain exhibited a similar dependence on the calcium concentration, indicating that the observed calcium inhibition was an inherent property of the Bap-mediated biofilms. Site-directed mutagenesis of two of the putative EF-hand domains resulted in a mutant strain that was capable of forming a biofilm but whose biofilm was not inhibited by calcium. Our results indicate that Bap binds Ca2+ with low affinity and that Ca2+ binding renders the protein noncompetent for biofilm formation and for intercellular adhesion. The fact that calcium inhibition of Bap-mediated multicellular behavior takes place in vitro at concentrations similar to those found in milk serum supports the possibility that this inhibition is relevant to the pathogenesis and/or epidemiology of the bacteria in the mastitis process.

  6. Calcium Inhibits Bap-Dependent Multicellular Behavior in Staphylococcus aureus

    PubMed Central

    Arrizubieta, María Jesús; Toledo-Arana, Alejandro; Amorena, Beatriz; Penadés, José R.; Lasa, Iñigo

    2004-01-01

    Bap (biofilm-associated protein) is a 254-kDa staphylococcal surface protein implicated in formation of biofilms by staphylococci isolated from chronic mastitis infections. The presence of potential EF-hand motifs in the amino acid sequence of Bap prompted us to investigate the effect of calcium on the multicellular behavior of Bap-expressing staphylococci. We found that addition of millimolar amounts of calcium to the growth media inhibited intercellular adhesion of and biofilm formation by Bap-positive strain V329. Addition of manganese, but not addition of magnesium, also inhibited biofilm formation, whereas bacterial aggregation in liquid media was greatly enhanced by metal-chelating agents. In contrast, calcium or chelating agents had virtually no effect on the aggregation of Bap-deficient strain M556. The biofilm elicited by insertion of bap into the chromosome of a biofilm-negative strain exhibited a similar dependence on the calcium concentration, indicating that the observed calcium inhibition was an inherent property of the Bap-mediated biofilms. Site-directed mutagenesis of two of the putative EF-hand domains resulted in a mutant strain that was capable of forming a biofilm but whose biofilm was not inhibited by calcium. Our results indicate that Bap binds Ca2+ with low affinity and that Ca2+ binding renders the protein noncompetent for biofilm formation and for intercellular adhesion. The fact that calcium inhibition of Bap-mediated multicellular behavior takes place in vitro at concentrations similar to those found in milk serum supports the possibility that this inhibition is relevant to the pathogenesis and/or epidemiology of the bacteria in the mastitis process. PMID:15516560

  7. A conserved signalling pathway for amoebozoan encystation that was co-opted for multicellular development.

    PubMed

    Kawabe, Yoshinori; Schilde, Christina; Du, Qingyou; Schaap, Pauline

    2015-01-01

    The evolution of multicellularity required novel mechanisms for intercellular communication, but their origin is unclear. Dictyostelium cells exchange signals to position specialized cell types in multicellular spore-bearing structures. These signals activate complex pathways that converge on activation of cAMP-dependent protein kinase (PKA). Genes controlling PKA were detected in the Dictyostelid unicellular ancestors, which like most protists form dormant cysts when experiencing environmental stress. We deleted PKA and the adenylate cyclases AcrA and AcgA, which synthesize cAMP for PKA activation, in the intermediate species Polysphondylium, which can develop into either cysts or into multicellular structures. Loss of PKA prevented multicellular development, but also completely blocked encystation. Loss of AcrA and AcgA, both essential for sporulation in Dictyostelium, did not affect Polysphondylium sporulation, but prevented encystation. We conclude that multicellular cAMP signalling was co-opted from PKA regulation of protist encystation with progressive refunctionalization of pathway components.

  8. Green Algae and the Origins of Multicellularity in the Plant Kingdom

    PubMed Central

    Umen, James G.

    2014-01-01

    The green lineage of chlorophyte algae and streptophytes form a large and diverse clade with multiple independent transitions to produce multicellular and/or macroscopically complex organization. In this review, I focus on two of the best-studied multicellular groups of green algae: charophytes and volvocines. Charophyte algae are the closest relatives of land plants and encompass the transition from unicellularity to simple multicellularity. Many of the innovations present in land plants have their roots in the cell and developmental biology of charophyte algae. Volvocine algae evolved an independent route to multicellularity that is captured by a graded series of increasing cell-type specialization and developmental complexity. The study of volvocine algae has provided unprecedented insights into the innovations required to achieve multicellularity. PMID:25324214

  9. Plant architecture without multicellularity: quandaries over patterning and the soma-germline divide in siphonous algae

    PubMed Central

    Coneva, Viktoriya; Chitwood, Daniel H.

    2015-01-01

    Multicellularity has independently evolved numerous times throughout the major lineages of life. Often, multicellularity can enable complex, macroscopic organismal architectures but it is not required for the elaboration of morphology. Several alternative cellular strategies have arisen as solutions permitting exquisite forms. The green algae class Ulvophyceae, for example, contains truly multicellular organisms, as well as macroscopic siphonous cells harboring one or multiple nuclei, and siphonocladous species, which are multinucleate and multicellular. These diverse cellular organizations raise a number of questions about the evolutionary and molecular mechanisms underlying complex organismal morphology in the green plants. Importantly, how does morphological patterning arise in giant coenocytes, and do nuclei, analogous to cells in multicellular organisms, take on distinct somatic and germline identities? Here, we comparatively explore examples of patterning and differentiation in diverse coenocytic and single-cell organisms and discuss possible mechanisms of development and nuclear differentiation in the siphonous algae. PMID:25964794

  10. Green algae and the origins of multicellularity in the plant kingdom.

    PubMed

    Umen, James G

    2014-10-16

    The green lineage of chlorophyte algae and streptophytes form a large and diverse clade with multiple independent transitions to produce multicellular and/or macroscopically complex organization. In this review, I focus on two of the best-studied multicellular groups of green algae: charophytes and volvocines. Charophyte algae are the closest relatives of land plants and encompass the transition from unicellularity to simple multicellularity. Many of the innovations present in land plants have their roots in the cell and developmental biology of charophyte algae. Volvocine algae evolved an independent route to multicellularity that is captured by a graded series of increasing cell-type specialization and developmental complexity. The study of volvocine algae has provided unprecedented insights into the innovations required to achieve multicellularity.

  11. Electrostatic potential of point charges inside dielectric oblate spheroids

    PubMed Central

    Deng, Shaozhong

    2009-01-01

    As a sequel to a previous paper on electrostatic potential of point charges inside dielectric prolate spheroids [J. Electrostatics 66 (2008) 549-560], this note further presents the exact solution to the electrostatic problem of finding the electric potential of point charges inside a dielectric oblate spheroid that is embedded in a dissimilar dielectric medium. Numerical experiments have demonstrated the convergence of the proposed series solutions. PMID:20160934

  12. Cusp diffraction catastrophe from spheroids: generalized rainbows and inverse scattering.

    PubMed

    Marston, P L

    1985-12-01

    The angular location of the recently discovered cusp pattern in the far-field scattering from an oblate spheroid is calculated as a function of the aspect ratio D/H. The calculation assumes the diameter D > lambda and is limited to illumination perpendicular to the short axis of the spheroid. It agrees with observations for water drops in the range 1.22 < D/H < 1.37 with D ~ 1 mm. PMID:19730494

  13. Biological magnetic cellular spheroids as building blocks for tissue engineering

    PubMed Central

    Mattix, Brandon; Olsen, Timothy R.; Gu, Yu; Casco, Megan; Herbst, Austin; Simionescu, Dan T.; Visconti, Richard P.; Kornev, Konstantin G.; Alexis, Frank

    2016-01-01

    Magnetic nanoparticles (MNPs), primarily iron oxide nanoparticles, have been incorporated into cellular spheroids to allow for magnetic manipulation into desired shapes, patterns and 3-D tissue constructs using magnetic forces. However, the direct and long-term interaction of iron oxide nanoparticles with cells and biological systems can induce adverse effects on cell viability, phenotype and function, and remain a critical concern. Here we report the preparation of biological magnetic cellular spheroids containing magnetoferritin, a biological MNP, capable of serving as a biological alternative to iron oxide magnetic cellular spheroids as tissue engineered building blocks. Magnetoferritin NPs were incorporated into 3-D cellular spheroids with no adverse effects on cell viability up to 1 week. Additionally, cellular spheroids containing magnetoferritin NPs were magnetically patterned and fused into a tissue ring to demonstrate its potential for tissue engineering applications. These results present a biological approach that can serve as an alternative to the commonly used iron oxide magnetic cellular spheroids, which often require complex surface modifications of iron oxide NPs to reduce the adverse effects on cells. PMID:24176725

  14. Carbon stars in the outer spheroid of NGC 6822

    NASA Astrophysics Data System (ADS)

    Demers, S.; Battinelli, P.; Artigau, E.

    2006-09-01

    Context: .From a 2°× 2° of NGC 6822 survey we have previously established that this Local Group dwarf irregular galaxy possesses a huge spheroid having more than one degree in length. This spheroid is in rotation but its rotation curve is known only within ~ 15' from the center. It is therefore critical to identify bright stars belonging to the spheroid to characterize, as far as possible, its outer kinematics. Aims: .We use the new wide field near infrared imager CPAPIR, operated by the SMARTS consortium, to acquire J, Ks images of two 34.8'× 34.8' areas in the outer spheroid to search for C stars. Methods: .The colour diagram of the fields allows the identification of 192 C stars candidates but a study of the FWHM of the images permits the rejection of numerous non-stellar objects with colours similar to C stars. Results: . We are left with 75 new C stars, their mean Ks magnitude and mean colour are similar to the bulk of known NGC 6822 C stars. Conclusions: .This outer spheroid survey confirms that the intermediate-age AGB stars are a major contributor to the stellar populations of the spheroid. The discovery of some 50 C stars well beyond the limit of the previously known rotation curve calls for a promising spectroscopic follow-up to a major axis distance of 40'.

  15. Wave Dark Matter and Dwarf Spheroidal Galaxies

    NASA Astrophysics Data System (ADS)

    Parry, Alan R.

    We explore a model of dark matter called wave dark matter (also known as scalar field dark matter and boson stars) which has recently been motivated by a new geometric perspective by Bray. Wave dark matter describes dark matter as a scalar field which satisfies the Einstein-Klein-Gordon equations. These equations rely on a fundamental constant Upsilon (also known as the "mass term'' of the Klein-Gordon equation). Specifically, in this dissertation, we study spherically symmetric wave dark matter and compare these results with observations of dwarf spheroidal galaxies as a first attempt to compare the implications of the theory of wave dark matter with actual observations of dark matter. This includes finding a first estimate of the fundamental constant Upsilon. In the introductory Chapter 1, we present some preliminary background material to define and motivate the study of wave dark matter and describe some of the properties of dwarf spheroidal galaxies. In Chapter 2, we present several different ways of describing a spherically symmetric spacetime and the resulting metrics. We then focus our discussion on an especially useful form of the metric of a spherically symmetric spacetime in polar-areal coordinates and its properties. In particular, we show how the metric component functions chosen are extremely compatible with notions in Newtonian mechanics. We also show the monotonicity of the Hawking mass in these coordinates. Finally, we discuss how these coordinates and the metric can be used to solve the spherically symmetric Einstein-Klein-Gordon equations. In Chapter 3, we explore spherically symmetric solutions to the Einstein-Klein-Gordon equations, the defining equations of wave dark matter, where the scalar field is of the form f(t, r) = eiotF(r) for some constant o ∈ R and complex-valued function F(r). We show that the corresponding metric is static if and only if F( r) = h(r)eia for some constant alpha ∈ R and real-valued function h(r). We describe the

  16. Electron spin resonance microscopic imaging of oxygen concentration in cancer spheroids

    NASA Astrophysics Data System (ADS)

    Hashem, Mada; Weiler-Sagie, Michal; Kuppusamy, Periannan; Neufeld, Gera; Neeman, Michal; Blank, Aharon

    2015-07-01

    relationship between morphological and physiological alterations in the spheroids, which would help understand the oxygen metabolism in solid tumors and its correlation with the susceptibility of tumors to various oncologic treatments.

  17. Scattering of electromagnetic waves by spheroidal particles: a novel approach exploiting the scr(T) matrix computed in spheroidal coordinates

    SciTech Connect

    Schulz, F. Michael; Stamnes, Knut Stamnes, Jakob A.

    1998-11-01

    A method other than the extended-boundary-condition method (EBCM) to compute the {ital T} matrix for electromagnetic scattering is presented. The separation-of-variables method (SVM) is used to solve the electromagnetic scattering problem for a spheroidal particle and to derive its scr(T) matrix in spheroidal coordinates. A transformation is developed for transforming the scr(T) matrix in spheroidal coordinates into the corresponding T matrix in spherical coordinates. The T matrix so obtained can be used for analytical calculation of the optical properties of ensembles of randomly oriented spheroids of arbitrary shape by use of an existing method to average over orientational angles. The optical properties obtained with the SVM and the EBCM are compared for different test cases. For mildly aspherical particles the two methods yield indistinguishable results. Small differences appear for highly aspherical particles. The new approach can be used to compute optical properties for arbitrary values of the aspect ratio. To test the accuracy of the expansion coefficients of the spheroidal functions for arbitrary arguments, a new testing method based on the completeness relation of the spheroidal functions is developed. {copyright} 1998 Optical Society of America

  18. Analytic approximation to randomly oriented spheroid extinction

    NASA Astrophysics Data System (ADS)

    Evans, B. T. N.; Fournier, G. R.

    1993-12-01

    The estimation of electromagnetic extinction through dust or other nonspherical atmospheric aerosols and hydrosols is an essential first step in the evaluation of the performance of all electro-optic systems. Investigations were conducted to reduce the computational burden in calculating the extinction from nonspherical particles. An analytic semi-empirical approximation to the extinction efficiency Q(sub ext) for randomly oriented spheroids, based on an extension of the anomalous diffraction formula, is given and compared with the extended boundary condition or T-matrix method. This will allow for better and more general modeling of obscurants. Using this formula, Q(sub ext) can be evaluated over 10,000 times faster than with previous methods. This approximation has been verified for complex refractive indices m=n-ik, where n ranges from one to infinity and k from zero to infinity, and aspect ratios of 0.2 to 5. It is believed that the approximation is uniformly valid over all size parameters and aspect ratios. It has the correct Rayleigh, refractive index, and large particle asymptotic behaviors. The accuracy and limitations of this formula are extensively discussed.

  19. Testing Modified Gravity with Dwarf Spheroidal Galaxies

    NASA Astrophysics Data System (ADS)

    Haghi, Hosein; Amiri, Vahid

    2016-08-01

    The observed velocity dispersion of the classical dwarf spheroidal (dSph) galaxies of the Milky Way (MW) requires the Newtonian stellar mass-to-light (M★/L) ratios in the range of about 10 to more than 100 solar units, that are well outside the acceptable limit predicted by stellar population synthesis (SPS) models. Using Jeans analysis, we calculate the line-of-sight velocity dispersion (σ _ph {los}) of stars in eight MW dSphs in the context of the Modified Gravity (MOG) theory of Moffat, assuming a constant M★/L ratio without invoking the exotic cold dark matter. First, we use the weak field approximation of MOG and assume the two parameters α and μ of the theory to be constant as has already been inferred from fitting to the observed rotational data of the THINGS catalog of galaxies. We find that the derived M★/L ratios for almost all dSphs are too large to be explained by the stellar population values. In order to fit the line-of-sight velocity dispersions of the dSph with reasonable M★/L values we must vary α and μ on a case by case basis. A common pair of values cannot be found for all dSphs. Comparing with the values found from rotation curve fitting, it appears that μ correlates strongly with galaxy luminosity, shedding doubt on it as a universal constant.

  20. On Dark Matter in Dwarf Spheroidal Galaxies

    NASA Astrophysics Data System (ADS)

    Walker, M. G.

    2011-07-01

    The small (LV ≤ 107LV,⊙) baryonic components of the Milky Way's dwarf spheroidal (dSph) satellites contribute small "backgrounds" with regards to estimates of dSph dark matter density profiles and any related predictions of dark matter decay/annihilation signals. As dSphs gain attention from a broad range of physicists interested in dark matter, it becomes important to examine the robustness - with respect to details like modeling assumptions and observational uncertainties - of conclusions regarding the dark matter content of dSphs and the scaling relations derived therefrom. Indeed dSphs seem to contain copious amounts of dark matter. However, the standard Jeans analysis constrains the dark mass enclosed only within the luminous scale radius, and does not reveal the internal dark matter distribution in a model-indepdendent way. Furthermore, new observational results help to identify complexities in internal dSph kinematics and cast doubt on previously apparent similarities among dSph dark matter halos. Further progress in understsanding dark matter on small scales will require consideration of dSphs more as individual galaxies and less as laboratories.

  1. Human Cardiac Progenitor Spheroids Exhibit Enhanced Engraftment Potential

    PubMed Central

    Colangelo, Donato; Gregoletto, Luca; Reano, Simone; Pietronave, Stefano; Merlin, Simone; Talmon, Maria; Novelli, Eugenio; Diena, Marco; Nicoletti, Carmine; Musarò, Antonio; Filigheddu, Nicoletta; Follenzi, Antonia; Prat, Maria

    2015-01-01

    A major obstacle to an effective myocardium stem cell therapy has always been the delivery and survival of implanted stem cells in the heart. Better engraftment can be achieved if cells are administered as cell aggregates, which maintain their extra-cellular matrix (ECM). We have generated spheroid aggregates in less than 24 h by seeding human cardiac progenitor cells (hCPCs) onto methylcellulose hydrogel-coated microwells. Cells within spheroids maintained the expression of stemness/mesenchymal and ECM markers, growth factors and their cognate receptors, cardiac commitment factors, and metalloproteases, as detected by immunofluorescence, q-RT-PCR and immunoarray, and expressed a higher, but regulated, telomerase activity. Compared to cells in monolayers, 3D spheroids secreted also bFGF and showed MMP2 activity. When spheroids were seeded on culture plates, the cells quickly migrated, displaying an increased wound healing ability with or without pharmacological modulation, and reached confluence at a higher rate than cells from conventional monolayers. When spheroids were injected in the heart wall of healthy mice, some cells migrated from the spheroids, engrafted, and remained detectable for at least 1 week after transplantation, while, when the same amount of cells was injected as suspension, no cells were detectable three days after injection. Cells from spheroids displayed the same engraftment capability when they were injected in cardiotoxin-injured myocardium. Our study shows that spherical in vivo ready-to-implant scaffold-less aggregates of hCPCs able to engraft also in the hostile environment of an injured myocardium can be produced with an economic, easy and fast protocol. PMID:26375957

  2. Synchronization of Eukaryotic Flagella and the Evolution of Multicellularity

    NASA Astrophysics Data System (ADS)

    Goldstein, Raymond

    2009-03-01

    Flagella, among the most highly conserved structures in eukaryotes, are responsible for such tasks as fluid transport, motility and phototaxis, establishment of embryonic left-right asymmetry, and intercellular communication, and are thought to have played a key role in the development of multicellularity. These tasks are usually performed by the coordinated action of groups of flagella (from pairs to thousands), which display various types of spatio-temporal organization. The origin and quantitative characterization of flagellar synchronization has remained an important open problem, involving interplay between intracellular biochemistry and interflagellar mechanical/hydrodynamic coupling. The Volvocine green algae serve as useful model organisms for the study of these phenomena, as they form a lineage spanning from unicellular Chlamydomonas to germ-soma differentiated Volvox, having as many as 50,000 biflagellated surface somatic cells. In this talk I will describe extensive studies [1], using micromanipulation and high-speed imaging, of the flagellar synchronization of two key species - Chlamydomonas reinhardtii and Volvox carteri - over tens of thousands of cycles. With Chlamydomonas we find that the flagellar dynamics moves back and forth between a stochastic synchronized state consistent with a simple model of hydrodynamically coupled noisy oscillators, and a deterministic one driven by a large interflagellar frequency difference. These results reconcile previously contradictory studies, based on short observations, showing only one or the other of these two states, and, more importantly, show that the flagellar beat frequencies themselves are regulated by the cell. Moreover, high-resolution three-dimensional tracking of swimming cells provides strong evidence that these dynamical states are related to reorientation events in the trajectories, yielding a eukaryotic equivalent of the ``run and tumble'' motion of peritrichously flagellated bacteria. The degree

  3. Superhydrophobic chips for cell spheroids high-throughput generation and drug screening.

    PubMed

    Oliveira, Mariana B; Neto, Ana I; Correia, Clara R; Rial-Hermida, Maria Isabel; Alvarez-Lorenzo, Carmen; Mano, João F

    2014-06-25

    We suggest the use of biomimetic superhydrophobic patterned chips produced by a benchtop methodology as low-cost and waste-free platforms for the production of arrays of cell spheroids/microtissues by the hanging drop methodology. Cell spheroids have a wide range of applications in biotechnology fields. For drug screening, they allow studying 3D models in structures resembling real living tissues/tumors. In tissue engineering, they are suggested as building blocks of bottom-up fabricated tissues. We used the wettability contrast of the chips to fix cell suspension droplets in the wettable regions and evaluated on-chip drug screening in 3D environment. Cell suspensions were patterned in the wettable spots by three distinct methods: (1) by pipetting the cell suspension directly in each individual spot, (2) by the continuous dragging of a cell suspension on the chip, and (3) by dipping the whole chip in a cell suspension. These methods allowed working with distinct throughputs and degrees of precision. The platforms were robust, and we were able to have static or dynamic environments in each droplet. The access to cell culture media for exchange or addition/removal of components was versatile and opened the possibility of using each spot of the chip as a mini-bioreactor. The platforms' design allowed for samples visualization and high-content image-based analysis on-chip. The combinatorial analysis capability of this technology was validated by following the effect of doxorubicin at different concentrations on spheroids formed using L929 and SaOs-2 cells.

  4. Development of cell differentiation in the transition to multicellularity: a dynamical modeling approach

    PubMed Central

    Mora Van Cauwelaert, Emilio; Arias Del Angel, Juan A.; Benítez, Mariana; Azpeitia, Eugenio M.

    2015-01-01

    Multicellularity has emerged and continues to emerge in a variety of lineages and under diverse environmental conditions. In order to attain individuality and integration, multicellular organisms must exhibit spatial cell differentiation, which in turn allows cell aggregates to robustly generate traits and behaviors at the multicellular level. Nevertheless, the mechanisms that may lead to the development of cellular differentiation and patterning in emerging multicellular organisms remain unclear. We briefly review two conceptual frameworks that have addressed this issue: the cooperation-defection framework and the dynamical patterning modules (DPMs) framework. Then, situating ourselves in the DPM formalism first put forward by S. A. Newman and collaborators, we state a hypothesis for cell differentiation and arrangement in cellular masses of emerging multicellular organisms. Our hypothesis is based on the role of the generic cell-to-cell communication and adhesion patterning mechanisms, which are two fundamental mechanisms for the evolution of multicellularity, and whose molecules seem to be well-conserved in extant multicellular organisms and their unicellular relatives. We review some fundamental ideas underlying this hypothesis and contrast them with empirical and theoretical evidence currently available. Next, we use a mathematical model to illustrate how the mechanisms and assumptions considered in the hypothesis we postulate may render stereotypical arrangements of differentiated cells in an emerging cellular aggregate and may contribute to the variation and recreation of multicellular phenotypes. Finally, we discuss the potential implications of our approach and compare them to those entailed by the cooperation-defection framework in the study of cell differentiation in the transition to multicellularity. PMID:26157427

  5. Development of cell differentiation in the transition to multicellularity: a dynamical modeling approach.

    PubMed

    Mora Van Cauwelaert, Emilio; Arias Del Angel, Juan A; Benítez, Mariana; Azpeitia, Eugenio M

    2015-01-01

    Multicellularity has emerged and continues to emerge in a variety of lineages and under diverse environmental conditions. In order to attain individuality and integration, multicellular organisms must exhibit spatial cell differentiation, which in turn allows cell aggregates to robustly generate traits and behaviors at the multicellular level. Nevertheless, the mechanisms that may lead to the development of cellular differentiation and patterning in emerging multicellular organisms remain unclear. We briefly review two conceptual frameworks that have addressed this issue: the cooperation-defection framework and the dynamical patterning modules (DPMs) framework. Then, situating ourselves in the DPM formalism first put forward by S. A. Newman and collaborators, we state a hypothesis for cell differentiation and arrangement in cellular masses of emerging multicellular organisms. Our hypothesis is based on the role of the generic cell-to-cell communication and adhesion patterning mechanisms, which are two fundamental mechanisms for the evolution of multicellularity, and whose molecules seem to be well-conserved in extant multicellular organisms and their unicellular relatives. We review some fundamental ideas underlying this hypothesis and contrast them with empirical and theoretical evidence currently available. Next, we use a mathematical model to illustrate how the mechanisms and assumptions considered in the hypothesis we postulate may render stereotypical arrangements of differentiated cells in an emerging cellular aggregate and may contribute to the variation and recreation of multicellular phenotypes. Finally, we discuss the potential implications of our approach and compare them to those entailed by the cooperation-defection framework in the study of cell differentiation in the transition to multicellularity. PMID:26157427

  6. Cooperatively Generated Stresslet Flows Supply Fresh Fluid to Multicellular Choanoflagellate Colonies

    NASA Astrophysics Data System (ADS)

    Roper, Marcus; Dayel, Mark J.; Pepper, Rachel E.; Koehl, M. A. R.

    2013-05-01

    The flagellated protozoan Salpingoeca rosetta is one of the closest relatives of multicellular animals. Unicellular S. rosetta can be induced to form multicellular colonies, but colonies swim more slowly than individual cells so the advantages conferred by colony formation are uncertain. Here we use theoretical models to show that hydrodynamic cooperation between cells can increase the fluid supply to the colony, an important predictor of feeding rate. Our results suggest that hydrodynamic benefits may have been an important selective factor in the evolution of early multicellular animals.

  7. Novel species and expanded distribution of ellipsoidal multicellular magnetotactic prokaryotes.

    PubMed

    Chen, Yi-ran; Zhang, Wen-yan; Zhou, Ke; Pan, Hong-miao; Du, Hai-jian; Xu, Cong; Xu, Jian-hong; Pradel, Nathalie; Santini, Claire-Lise; Li, Jin-hua; Huang, Hui; Pan, Yong-xin; Xiao, Tian; Wu, Long-fei

    2016-04-01

    Multicellular magnetotactic prokaryotes (MMPs) are a peculiar group of magnetotactic bacteria, each comprising approximately 10-100 cells of the same phylotype. Two morphotypes of MMP have been identified, including several species of globally distributed spherical mulberry-like MMPs (s-MMPs), and two species of ellipsoidal pineapple-like MMPs (e-MMPs) from China (Qingdao and Rongcheng cities). We recently collected e-MMPs from Mediterranean Sea sediments (Six-Fours-les-Plages) and Drummond Island, in the South China Sea. Phylogenetic analysis revealed that the MMPs from Six-Fours-les-Plages and the previously reported e-MMP Candidatus Magnetananas rongchenensis have 98.5% sequence identity and are the same species, while the MMPs from Drummond Island appear to be a novel species, having > 7.1% sequence divergence from the most closely related e-MMP, Candidatus Magnetananas tsingtaoensis. Identification of the novel species expands the distribution of e-MMPs to Tropical Zone. Comparison of nine physical and chemical parameters revealed that sand grain size and the content of inorganic nitrogen (nitrate, ammonium and nitrite) in the sediments from Rongcheng City and Six-Fours-les-Plages were similar, and lower than found for sediments from the other two sampling sites. The results of the study reveal broad diversity and wide distribution of e-MMPs. PMID:26711721

  8. Robust finite time observer design for multicellular converters

    NASA Astrophysics Data System (ADS)

    Defoort, Michael; Djemai, Mohamed; Floquet, Thierry; Perruquetti, Wilfrid

    2011-11-01

    In this article, a nonlinear finite time observer is designed for multicellular converters. The aim is to estimate the capacitor voltages by taking into account the hybrid behaviour of the converter. This article extends the validity of the strong Lyapunov function, proposed in Moreno and Osorio (Moreno, J., and Osorio, M. (2008), 'A Lyapunov Approach to Second Order Sliding Mode Controllers and Observers', in Proceedings of the IEEE Conference on Decision and Control, New Orleans, USA, pp. 2856-2861), in order to deeply study the reaching time estimation and robustness of the homogeneous finite time observer given in Perruquetti et al. (Perruquetti, W., Floquet, T., and Moulay, E. (2008), 'Finite Time Observers: Application to Secure Communication', IEEE Transactions on Automatic Control, 53, 356-360). The proposed approach enables the stabilisation of the observation errors in spite of the presence of perturbations and uncertainties. Some simulations and comparisons with the super-twisting sliding mode observer highlight the efficiency of the proposed strategy.

  9. Origins of multicellular complexity: Volvox and the volvocine algae.

    PubMed

    Herron, Matthew D

    2016-03-01

    The collection of evolutionary transformations known as the 'major transitions' or 'transitions in individuality' resulted in changes in the units of evolution and in the hierarchical structure of cellular life. Volvox and related algae have become an important model system for the major transition from unicellular to multicellular life, which touches on several fundamental questions in evolutionary biology. The Third International Volvox Conference was held at the University of Cambridge in August 2015 to discuss recent advances in the biology and evolution of this group of algae. Here, I highlight the benefits of integrating phylogenetic comparative methods and experimental evolution with detailed studies of developmental genetics in a model system with substantial genetic and genomic resources. I summarize recent research on Volvox and its relatives and comment on its implications for the genomic changes underlying major evolutionary transitions, evolution and development of complex traits, evolution of sex and sexes, evolution of cellular differentiation and the biophysics of motility. Finally, I outline challenges and suggest future directions for research into the biology and evolution of the volvocine algae. PMID:26822195

  10. Multicellularity and the Functional Interdependence of Motility and Molecular Transport

    NASA Astrophysics Data System (ADS)

    Solari, C.; Ganguly, S.; Kessler, J. O.; Michod, R.; Goldstein, R. E.

    2006-03-01

    Benefits, costs and requirements accompany the transition from motile totipotent unicellular organisms to multicellular organisms having cells specialized into reproductive (germ) and vegetative (sterile soma) functions such as motility. In flagellated colonial organisms such as the volvocalean green algae, organized beating by the somatic cells' flagella yields propulsion important in phototaxis and chemotaxis. It has not been generally appreciated that for the larger colonies, flagellar stirring of boundary layers and remote transport are fundamental for maintaining a sufficient rate of metabolite turnover, one not attainable by diffusive transport alone. We describe experiments that quantify the role of advective dynamics in enhancing productivity in germ-soma differentiated colonies. First, experiments with suspended deflagellated colonies of Volvox carteri show that forced advection improves productivity. Second, Particle Imaging Velocimetry of fluid motion around colonies reveals flow fields with very large characteristic velocities U extending to length scales comparable to the colony radius R. For a typical metabolite diffusion constant D, the Peclet number Pe=2UR/D 1, indicative of the dominance of advection over diffusion, with striking augmentation at the cell division stage.

  11. MicroRNAs in a multicellular green alga Volvox carteri.

    PubMed

    Li, Jingrui; Wu, Yang; Qi, Yijun

    2014-01-01

    microRNAs (miRNAs) have emerged as key components in the eukaryotic gene regulatory network. We and others have previously identified many miRNAs in a unicellular green alga, Chlamydomonas reinhardtii. To investigate whether miRNA-mediated gene regulation is a general mechanism in green algae and how miRNAs have been evolved in the green algal lineage, we examined small RNAs in Volvox carteri, a multicellular species in the same family with Chlamydomonas reinhardtii. We identified 174 miRNAs in Volvox, with many of them being highly enriched in gonidia or somatic cells. The targets of the miRNAs were predicted and many of them were subjected to miRNA-mediated cleavage in vivo, suggesting that miRNAs play regulatory roles in the biology of green algae. Our catalog of miRNAs and their targets provides a resource for further studies on the evolution, biological functions, and genomic properties of miRNAs in green algae. PMID:24369344

  12. Biocellion: accelerating computer simulation of multicellular biological system models

    PubMed Central

    Kang, Seunghwa; Kahan, Simon; McDermott, Jason; Flann, Nicholas; Shmulevich, Ilya

    2014-01-01

    Motivation: Biological system behaviors are often the outcome of complex interactions among a large number of cells and their biotic and abiotic environment. Computational biologists attempt to understand, predict and manipulate biological system behavior through mathematical modeling and computer simulation. Discrete agent-based modeling (in combination with high-resolution grids to model the extracellular environment) is a popular approach for building biological system models. However, the computational complexity of this approach forces computational biologists to resort to coarser resolution approaches to simulate large biological systems. High-performance parallel computers have the potential to address the computing challenge, but writing efficient software for parallel computers is difficult and time-consuming. Results: We have developed Biocellion, a high-performance software framework, to solve this computing challenge using parallel computers. To support a wide range of multicellular biological system models, Biocellion asks users to provide their model specifics by filling the function body of pre-defined model routines. Using Biocellion, modelers without parallel computing expertise can efficiently exploit parallel computers with less effort than writing sequential programs from scratch. We simulate cell sorting, microbial patterning and a bacterial system in soil aggregate as case studies. Availability and implementation: Biocellion runs on x86 compatible systems with the 64 bit Linux operating system and is freely available for academic use. Visit http://biocellion.com for additional information. Contact: seunghwa.kang@pnnl.gov PMID:25064572

  13. Multicellular contractility contributes to the emergence of mesothelioma nodules

    NASA Astrophysics Data System (ADS)

    Czirok, Andras

    Malignant pleural mesothelioma (MPM) nodules arise from the mesothelial lining of the pleural cavity by a poorly understood mechanism. We demonstrate that macroscopic multicellular aggregates, reminiscent of the MPM nodules found in patients, develop when MPM cell lines are cultured at high cell densities for several weeks. Surprisingly, the nodule-like aggregates do not arise by excessive local cell proliferation, but by myosin II-driven cell contractility. Contractile nodules contain prominent actin cables that can span several cells. Several features of the in vitro MPM nodule development can be explained by a computational model that assumes uniform and steady intercellular contractile forces within a monolayer of cells, and a mechanical load-dependent lifetime of cell-cell contacts. The model behaves as a self-tensioned Maxwell fluid and exhibits an instability that leads to pattern formation. Altogether, our findings suggest that inhibition of the actomyosin system may provide a hitherto not utilized therapeutic approach to affect MPM growth. NIH R01-GM102801.

  14. Origins of multicellular complexity: Volvox and the volvocine algae.

    PubMed

    Herron, Matthew D

    2016-03-01

    The collection of evolutionary transformations known as the 'major transitions' or 'transitions in individuality' resulted in changes in the units of evolution and in the hierarchical structure of cellular life. Volvox and related algae have become an important model system for the major transition from unicellular to multicellular life, which touches on several fundamental questions in evolutionary biology. The Third International Volvox Conference was held at the University of Cambridge in August 2015 to discuss recent advances in the biology and evolution of this group of algae. Here, I highlight the benefits of integrating phylogenetic comparative methods and experimental evolution with detailed studies of developmental genetics in a model system with substantial genetic and genomic resources. I summarize recent research on Volvox and its relatives and comment on its implications for the genomic changes underlying major evolutionary transitions, evolution and development of complex traits, evolution of sex and sexes, evolution of cellular differentiation and the biophysics of motility. Finally, I outline challenges and suggest future directions for research into the biology and evolution of the volvocine algae.

  15. Novel species and expanded distribution of ellipsoidal multicellular magnetotactic prokaryotes.

    PubMed

    Chen, Yi-ran; Zhang, Wen-yan; Zhou, Ke; Pan, Hong-miao; Du, Hai-jian; Xu, Cong; Xu, Jian-hong; Pradel, Nathalie; Santini, Claire-Lise; Li, Jin-hua; Huang, Hui; Pan, Yong-xin; Xiao, Tian; Wu, Long-fei

    2016-04-01

    Multicellular magnetotactic prokaryotes (MMPs) are a peculiar group of magnetotactic bacteria, each comprising approximately 10-100 cells of the same phylotype. Two morphotypes of MMP have been identified, including several species of globally distributed spherical mulberry-like MMPs (s-MMPs), and two species of ellipsoidal pineapple-like MMPs (e-MMPs) from China (Qingdao and Rongcheng cities). We recently collected e-MMPs from Mediterranean Sea sediments (Six-Fours-les-Plages) and Drummond Island, in the South China Sea. Phylogenetic analysis revealed that the MMPs from Six-Fours-les-Plages and the previously reported e-MMP Candidatus Magnetananas rongchenensis have 98.5% sequence identity and are the same species, while the MMPs from Drummond Island appear to be a novel species, having > 7.1% sequence divergence from the most closely related e-MMP, Candidatus Magnetananas tsingtaoensis. Identification of the novel species expands the distribution of e-MMPs to Tropical Zone. Comparison of nine physical and chemical parameters revealed that sand grain size and the content of inorganic nitrogen (nitrate, ammonium and nitrite) in the sediments from Rongcheng City and Six-Fours-les-Plages were similar, and lower than found for sediments from the other two sampling sites. The results of the study reveal broad diversity and wide distribution of e-MMPs.

  16. Coulomb energy of uniformly charged spheroidal shell systems

    NASA Astrophysics Data System (ADS)

    Jadhao, Vikram; Yao, Zhenwei; Thomas, Creighton K.; de la Cruz, Monica Olvera

    2015-03-01

    We provide exact expressions for the electrostatic energy of uniformly charged prolate and oblate spheroidal shells. We find that uniformly charged prolate spheroids of eccentricity greater than 0.9 have lower Coulomb energy than a sphere of the same area. For the volume-constrained case, we find that a sphere has the highest Coulomb energy among all spheroidal shells. Further, we derive the change in the Coulomb energy of a uniformly charged shell due to small, area-conserving perturbations on the spherical shape. Our perturbation calculations show that buckling-type deformations on a sphere can lower the Coulomb energy. Finally, we consider the possibility of counterion condensation on the spheroidal shell surface. We employ a Manning-Oosawa two-state model approximation to evaluate the renormalized charge and analyze the behavior of the equilibrium free energy as a function of the shell's aspect ratio for both area-constrained and volume-constrained cases. Counterion condensation is seen to favor the formation of spheroidal structures over a sphere of equal area for high values of shell volume fractions.

  17. Coulomb energy of uniformly charged spheroidal shell systems.

    PubMed

    Jadhao, Vikram; Yao, Zhenwei; Thomas, Creighton K; de la Cruz, Monica Olvera

    2015-03-01

    We provide exact expressions for the electrostatic energy of uniformly charged prolate and oblate spheroidal shells. We find that uniformly charged prolate spheroids of eccentricity greater than 0.9 have lower Coulomb energy than a sphere of the same area. For the volume-constrained case, we find that a sphere has the highest Coulomb energy among all spheroidal shells. Further, we derive the change in the Coulomb energy of a uniformly charged shell due to small, area-conserving perturbations on the spherical shape. Our perturbation calculations show that buckling-type deformations on a sphere can lower the Coulomb energy. Finally, we consider the possibility of counterion condensation on the spheroidal shell surface. We employ a Manning-Oosawa two-state model approximation to evaluate the renormalized charge and analyze the behavior of the equilibrium free energy as a function of the shell's aspect ratio for both area-constrained and volume-constrained cases. Counterion condensation is seen to favor the formation of spheroidal structures over a sphere of equal area for high values of shell volume fractions. PMID:25871108

  18. The transitional wake behind an inclined prolate spheroid

    NASA Astrophysics Data System (ADS)

    Jiang, Fengjian; Gallardo, José P.; Andersson, Helge I.; Zhang, Zhiguo

    2015-09-01

    The wake behind a 6:1 prolate spheroid at 45° incidence has been studied by means of direct numerical simulations (DNSs). The Reynolds number based on the minor axis of the spheroid was 3000 as compared to 1000 in our preceding study [Jiang et al., "The laminar wake behind a 6:1 prolate spheroid at 45° incidence angle," Phys. Fluids 26, 113602 (2014)]. The resulting wake is no longer laminar and the transitional wake is fundamentally unsteady and highly asymmetric from the very beginning. A substantial side force resulted from the asymmetric pressure field. No signs of vortex shedding could be observed. The forces and the flow field around the spheroid exhibited a dominant periodicity with a surprisingly low Strouhal number of 0.0733. One part of the counter-rotating vortex pair which dominated the near-wake broke down into small-scale vortices as soon as the vortex left the shadow behind the spheroid. The other part appeared as a helical vortex inside which the mechanical energy was conserved over a substantial length. The axial flow within this vortex tube experienced a sudden change from having maximum to minimum at the vortex center while maintaining the sign of the circulation. The severe asymmetry of the wake is ascribed to a global instability and may impact on submarine maneuverability.

  19. [Characteristics of biodegradation of triphenyltin by Rhodopseudomonos spheroids].

    PubMed

    Chen, Shuo-Na; Ye, Jin-Shao; Yin, Hu; Peng, Hui; Zhang, Na; He, Bao-Yan

    2011-02-01

    The biodegradation of triphenyltin (TPT) by Rhodopseudomonos spheroids was investigated in this study. The results illuminated that R. spheroids was an effective strain for the biodegradation of TPT. The maximum removal ratio was attained when the growth temperature of R. spheroids was 30 degrees C. After treating for 3 hours, the removal ratios of 3 mg x L(-1) TPT were 13.82% to 47.29% using 0.49 g x L(-1) (based on dry weight) biomass of R. spheroids. The experiments on biodegradation of TPT were carried out in double-distilled water, simulated seawater,culture medium and river water, respectively. The results demonstrated that river water was optimal for the biodegradation since the indigenous microorganisms in water synergistically increased the removal ratios of TPT. Extracellular enzyme produced by R. spheroids was also effective on the degradation of TPT, and 71.64% of TPT was degraded by this way within 24 hours. The experiments also revealed that the biodegradation process of TPT included biosorption by cell wall, TPT entering cells, and initial degradation by intracellular enzyme, then the TPT and intermediate products backing out of cells to be degraded by extracellular enzyme. PMID:21528580

  20. An epithelial tissue in Dictyostelium challenges the traditional origin of metazoan multicellularity.

    PubMed

    Dickinson, Daniel J; Nelson, W James; Weis, William I

    2012-10-01

    We hypothesize that aspects of animal multicellularity originated before the divergence of metazoans from fungi and social amoebae. Polarized epithelial tissues are a defining feature of metazoans and contribute to the diversity of animal body plans. The recent finding of a polarized epithelium in the non-metazoan social amoeba Dictyostelium discoideum demonstrates that epithelial tissue is not a unique feature of metazoans, and challenges the traditional paradigm that multicellularity evolved independently in social amoebae and metazoans. An alternative view, presented here, is that the common ancestor of social amoebae, fungi, and animals spent a portion of its life cycle in a multicellular state and possessed molecular machinery necessary for forming an epithelial tissue. Some descendants of this ancestor retained multicellularity, while others reverted to unicellularity. This hypothesis makes testable predictions regarding tissue organization in close relatives of metazoans and provides a novel conceptual framework for studies of early animal evolution. PMID:22930590

  1. High relatedness is necessary and sufficient to maintain multicellularity in Dictyostelium.

    PubMed

    Kuzdzal-Fick, Jennie J; Fox, Sara A; Strassmann, Joan E; Queller, David C

    2011-12-16

    Most complex multicellular organisms develop clonally from a single cell. This should limit conflicts between cell lineages that could threaten the extensive cooperation of cells within multicellular bodies. Cellular composition can be manipulated in the social amoeba Dictyostelium discoideum, which allows us to test and confirm the two key predictions of this theory. Experimental evolution at low relatedness favored cheating mutants that could destroy multicellular development. However, under high relatedness, the forces of mutation and within-individual selection are too small for these destructive cheaters to spread, as shown by a mutation accumulation experiment. Thus, we conclude that the single-cell bottleneck is a powerful stabilizer of cellular cooperation in multicellular organisms. PMID:22174251

  2. Phylogeny of Opisthokonta and the evolution of multicellularity and complexity in Fungi and Metazoa

    NASA Astrophysics Data System (ADS)

    Medina, Mónica; Collins, Allen G.; Taylor, John W.; Valentine, James W.; Lipps, Jere H.; Amaral-Zettler, Linda; Sogin, Mitchell L.

    2003-07-01

    While early eukaryotic life must have been unicellular, multicellular lifeforms evolved multiple times from protistan ancestors in diverse eukaryotic lineages. The origins of multicellularity are of special interest because they require evolutionary transitions towards increased levels of complexity. We have generated new sequence data from the nuclear large subunit ribosomal DNA gene (LSU rDNA) and the SSU rDNA gene of several unicellular opisthokont protists - a nucleariid amoeba (Nuclearia simplex) and four choanoflagellates (Codosiga gracilis, Choanoeca perplexa, Proterospongia choanojuncta and Stephanoeca diplocostata) to provide the basis for re-examining relationships among several unicellular lineages and their multicellular relatives (animals and fungi). Our data indicate that: (1) choanoflagellates are a monophyletic rather than a paraphyletic assemblage that independently gave rise to animals and fungi as suggested by some authors and (2) the nucleariid filose amoebae are the likely sister group to Fungi. We also review published information regarding the origin of multicellularity in the opisthokonts.

  3. Multicellular group formation in response to predators in the alga Chlorella vulgaris.

    PubMed

    Fisher, R M; Bell, T; West, S A

    2016-03-01

    A key step in the evolution of multicellular organisms is the formation of cooperative multicellular groups. It has been suggested that predation pressure may promote multicellular group formation in some algae and bacteria, with cells forming groups to lower their chance of being eaten. We use the green alga Chlorella vulgaris and the protist Tetrahymena thermophila to test whether predation pressure can initiate the formation of colonies. We found that: (1) either predators or just predator exoproducts promote colony formation; (2) higher predator densities cause more colonies to form; and (3) colony formation in this system is facultative, with populations returning to being unicellular when the predation pressure is removed. These results provide empirical support for the hypothesis that predation pressure promotes multicellular group formation. The speed of the reversion of populations to unicellularity suggests that this response is due to phenotypic plasticity and not evolutionary change. PMID:26663204

  4. Potential impact of gene regulatory mechanisms on the evolution of multicellularity in the volvocine algae.

    PubMed

    Kianianmomeni, Arash

    2015-01-01

    A fundamental question in biology is how multicellular organisms can arise from their single-celled precursors. The evolution of multicellularity requires the adoption of new traits in unicellular ancestors that allows the generation of form by, for example, increasing the size and developing new cell types. But what are the genetic, cellular and biochemical bases underlying the evolution of multicellularity? Recent advances in evolutionary developmental biology suggest that the regulation of gene expression by cis-regulatory factors, gene duplication and alternative splicing contribute to phenotypic evolution. These mechanisms enable different degrees of phenotypic divergence and complexity with variation in traits from genomes with similar gene contents. In addition, signaling pathways specific to cell types are developed to guarantee the modulation of cellular and developmental processes matched to the cell types as well as the maintenance of multicellularity. PMID:26479715

  5. Response of multicell spheroids to 1-MHz ultrasonic irradiation: cavitation-related damage

    SciTech Connect

    Sacks, P.G.; Miller, M.W.; Sutherland, R.M.

    1983-03-01

    The response of EMT6/Ro single cells and multicell spheroids (150-800 ..mu..m diameter) to 1-MHz ultrasonic irradiation was determined for intensities of 0-5W/cm/sup 2/ for 1-5 min. Damage was assessed by evaluating both lysis and survival of individual spheroid cells, growth of whole spheroids, and the physical integrity of whole spheroids by histological and scanning electron microscopic techniques. The surviving fraction of spheroids cells was higher than that of single cells, and several of the intensity-duration combinations showed a statistically significant correlation between resistace to ultrasound and increased spheroid size. This resistance with size was also found in spheroid growth experiments. Histological sections showed that both peripheral and internal damage occur. The relationship of spheroid damage to studies of effects on tissues and possible therapeutic use of nonthermal ultrasound are discussed.

  6. A microfluidic device for uniform-sized cell spheroids formation, culture, harvesting and flow cytometry analysis.

    PubMed

    Patra, Bishnubrata; Chen, Ying-Hua; Peng, Chien-Chung; Lin, Shiang-Chi; Lee, Chau-Hwang; Tung, Yi-Chung

    2013-01-01

    Culture of cells as three-dimensional (3D) aggregates, named spheroids, possesses great potential to improve in vitro cell models for basic biomedical research. However, such cell spheroid models are often complicated, cumbersome, and expensive compared to conventional Petri-dish cell cultures. In this work, we developed a simple microfluidic device for cell spheroid formation, culture, and harvesting. Using this device, cells could form uniformly sized spheroids due to strong cell-cell interactions and the spatial confinement of microfluidic culture chambers. We demonstrated cell spheroid formation and culture in the designed devices using embryonic stem cells, carcinoma cells, and fibroblasts. We further scaled up the device capable of simultaneously forming and culturing 5000 spheroids in a single chip. Finally, we demonstrated harvesting of the cultured spheroids from the device with a simple setup. The harvested spheroids possess great integrity, and the cells can be exploited for further flow cytometry assays due to the ample cell numbers. PMID:24396525

  7. Detachment energies of spheroidal particles from fluid-fluid interfaces

    NASA Astrophysics Data System (ADS)

    Davies, Gary B.; Krüger, Timm; Coveney, Peter V.; Harting, Jens

    2014-10-01

    The energy required to detach a single particle from a fluid-fluid interface is an important parameter for designing certain soft materials, for example, emulsions stabilised by colloidal particles, colloidosomes designed for targeted drug delivery, and bio-sensors composed of magnetic particles adsorbed at interfaces. For a fixed particle volume, prolate and oblate spheroids attach more strongly to interfaces because they have larger particle-interface areas. Calculating the detachment energy of spheroids necessitates the difficult measurement of particle-liquid surface tensions, in contrast with spheres, where the contact angle suffices. We develop a simplified detachment energy model for spheroids which depends only on the particle aspect ratio and the height of the particle centre of mass above the fluid-fluid interface. We use lattice Boltzmann simulations to validate the model and provide quantitative evidence that the approach can be applied to simulate particle-stabilized emulsions, and highlight the experimental implications of this validation.

  8. Optical caustics observed in light scattered by an oblate spheroid.

    PubMed

    Lock, James A; Xu, Feng

    2010-03-10

    The electromagnetic fields scattered when a plane wave is incident on an oblate spheroid in the side-on orientation may be calculated using a generalization of Mie theory, and the results may be decomposed in a Debye series expansion. A number of optical caustics are observed in the computed scattered intensity for the one internal reflection portion of the Debye series for scattering angles in the vicinity of the first-order rainbow, and are analyzed in terms of the rainbow, transverse cusp, and hyperbolic umbilic caustics of catastrophe optics. The specific features of these three caustics are described, as is their assembly into the global structure of the observed caustics for spheroid scattering. It is found that, for a spheroid whose radius is an order of magnitude larger than the wavelength of the incident light, the interference structure accompanying the transverse cusp and hyperbolic umbilic caustics is only partially formed.

  9. Spheroidization of glass powders for glass ionomer cements.

    PubMed

    Gu, Y W; Yap, A U J; Cheang, P; Kumar, R

    2004-08-01

    Commercial angular glass powders were spheroidized using both the flame spraying and inductively coupled radio frequency plasma spraying techniques. Spherical powders with different particle size distributions were obtained after spheroidization. The effects of spherical glass powders on the mechanical properties of glass ionomer cements (GICs) were investigated. Results showed that the particle size distribution of the glass powders had a significant influence on the mechanical properties of GICs. Powders with a bimodal particle size distribution ensured a high packing density of glass ionomer cements, giving relatively high mechanical properties of GICs. GICs prepared by flame-spheroidized powders showed low strength values due to the loss of fine particles during flame spraying, leading to a low packing density and few metal ions reacting with polyacrylic acid to form cross-linking. GICs prepared by the nano-sized powders showed low strength because of the low bulk density of the nano-sized powders and hence low powder/liquid ratio of GICs.

  10. A numerical study of separation on a spheroid at incidence

    NASA Technical Reports Server (NTRS)

    Rosenfeld, M.; Israeli, M.; Wolfshtein, M.

    1986-01-01

    The three-dimensional incompressible, steady and laminar flow field around a prolate spheroid at incidence is considered. The parabolized Navier-Stokes equations are solved numerically. The method can handle vortex types as well as bubble type flow separation because the pressure is one of the dependent variables. Here, the distribution of the skin friction is reported for two test cases. The first test case is a prolate spheroid of aspect ratio of 4:1 at 6 degrees incidence and Reynolds number of 1 million (based on half the major axis). The second case is a spheroid with a 6:1 aspect ratio at 10 degrees incidence and Reynolds number of 0.8 x 1 million. The properties of the flow field near the body are discussed on the basis of the pattern of the skin friction lines, and the shape of the separation lines. Favorable agreement with experimental results is obtained.

  11. Liquid-based three-dimensional tumor models for cancer research and drug discovery

    PubMed Central

    Ham, Stephanie L; Joshi, Ramila; Thakuri, Pradip S

    2016-01-01

    Tumors are three-dimensional tissues where close contacts between cancer cells, intercellular interactions between cancer and stromal cells, adhesion of cancer cells to the extracellular matrix, and signaling of soluble factors modulate functions of cancer cells and their response to therapeutics. Three-dimensional cultures of cancer cells overcome limitations of traditionally used monolayer cultures and recreate essential characteristics of tumors such as spatial gradients of oxygen, growth factors, and metabolites and presence of necrotic, hypoxic, quiescent, and proliferative cells. As such, three-dimensional tumor models provide a valuable tool for cancer research and oncology drug discovery. Here, we describe different tumor models and primarily focus on a model known as tumor spheroid. We summarize different technologies of spheroid formation, and discuss the use of spheroids to address the influence of stromal fibroblasts and immune cells on cancer cells in tumor microenvironment, study cancer stem cells, and facilitate compound screening in the drug discovery process. We review major techniques for quantification of cellular responses to drugs and discuss challenges ahead to enable broad utility of tumor spheroids in research laboratories, integrate spheroid models into drug development and discovery pipeline, and use primary tumor cells for drug screening studies to realize personalized cancer treatment. PMID:27072562

  12. Liquid-based three-dimensional tumor models for cancer research and drug discovery.

    PubMed

    Ham, Stephanie L; Joshi, Ramila; Thakuri, Pradip S; Tavana, Hossein

    2016-05-01

    Tumors are three-dimensional tissues where close contacts between cancer cells, intercellular interactions between cancer and stromal cells, adhesion of cancer cells to the extracellular matrix, and signaling of soluble factors modulate functions of cancer cells and their response to therapeutics. Three-dimensional cultures of cancer cells overcome limitations of traditionally used monolayer cultures and recreate essential characteristics of tumors such as spatial gradients of oxygen, growth factors, and metabolites and presence of necrotic, hypoxic, quiescent, and proliferative cells. As such, three-dimensional tumor models provide a valuable tool for cancer research and oncology drug discovery. Here, we describe different tumor models and primarily focus on a model known as tumor spheroid. We summarize different technologies of spheroid formation, and discuss the use of spheroids to address the influence of stromal fibroblasts and immune cells on cancer cells in tumor microenvironment, study cancer stem cells, and facilitate compound screening in the drug discovery process. We review major techniques for quantification of cellular responses to drugs and discuss challenges ahead to enable broad utility of tumor spheroids in research laboratories, integrate spheroid models into drug development and discovery pipeline, and use primary tumor cells for drug screening studies to realize personalized cancer treatment.

  13. Modeling light scattering by mineral dust particles using spheroids

    NASA Astrophysics Data System (ADS)

    Merikallio, Sini; Nousiainen, Timo

    Suspended dust particles have a considerable influence on light scattering in both terrestrial and planetary atmospheres and can therefore have a large effect on the interpretation of remote sensing measurements. Assuming dust particles to be spherical is known to produce inaccurate results when modeling optical properties of real mineral dust particles. Yet this approximation is widely used for its simplicity. Here, we simulate light scattering by mineral dust particles using a distribution of model spheroids. This is done by comparing scattering matrices calculated from a dust optical database of Dubovik et al. [2006] with those measured in the laboratory by Volten et al. [2001]. Wavelengths of 441,6 nm and 632,8 nm and refractive indexes of Re = 1.55 -1.7 and Im = 0.001i -0.01i were adopted in this study. Overall, spheroids are found to fit the measurements significantly better than Mie spheres. Further, we confirm that the shape distribution parametrization developed in Nousiainen et al. (2006) significantly improves the accuracy of simulated single-scattering for small mineral dust particles. The spheroid scheme should therefore yield more reliable interpretations of remote sensing data from dusty planetary atmospheres. While the spheroidal scheme is superior to spheres in remote sensing applications, its performance is far from perfect especially for samples with large particles. Thus, additional advances are clearly possible. Further studies of the Martian atmosphere are currently under way. Dubovik et al. (2006) Application of spheroid models to account for aerosol particle nonspheric-ity in remote sensing of desert dust, JGR, Vol. 111, D11208 Volten et al. (2001) Scattering matrices of mineral aerosol particles at 441.6 nm and 632.8 nm, JGR, Vol. 106, No. D15, pp. 17375-17401 Nousiainen et al. (2006) Light scattering modeling of small feldspar aerosol particles using polyhedral prisms and spheroids, JQSRT 101, pp. 471-487

  14. Optical properties of core-mantle spheroidal particles

    NASA Astrophysics Data System (ADS)

    Somsikov, Vadim V.; Farafonov, Victor G.

    1994-12-01

    The new solution of the problem of light scattering by coated spheroids was used to calculate the optical properties of prolate and oblate particles. The solution was obtained by the method of separation of variables for confocal spheroids. We consider the silicate core ice mantle particles and present the extinction cross-sections for prolate and oblate spheroids with the refractive indices mcore equals 1.7 + Oi, 1.7 + 0.1i and mmantle equals 1.3, the aspect ratio (a/b)mantle equals 2 and various volume ratios Vcore/Vtotal. The results are plotted for different size parameters xv equals 2(pi) rv/(lambda) , where rv is the radius of equivolume sphere and (lambda) is the wavelength of incident radiation. The main conclusions are: (a) if Vcore/Vtotal equals 0.5, the optical properties of a core-mantle particle are determined mainly by its core: for prolate non-absorbing spheroids when xv spheroids when xv 10, for oblate absorbing and non-absorbing spheroids when xv

  15. Development of ichthyosporeans sheds light on the origin of metazoan multicellularity.

    PubMed

    Suga, Hiroshi; Ruiz-Trillo, Iñaki

    2013-05-01

    To understand the mechanisms involved in the transition from protists to multicellular animals (metazoans), studying unicellular relatives of metazoans is as important as studying metazoans themselves. However, investigations remain poor on the closest unicellular (or colonial) relatives of Metazoa, i.e., choanoflagellates, filastereans and ichthyosporeans. Molecular-level analyses on these protists have been severely limited by the lack of transgenesis tools. Their genomes, however, contain several key genes encoding proteins important for metazoan development and multicellularity, including those involved in cell-cell communication, cell proliferation, cell differentiation, and tissue growth control. Tools to analyze their functions in a molecular level are awaited. Here we report techniques of cell transformation and gene silencing developed for the first time in a close relative of metazoans, the ichthyosporean Creolimax fragrantissima. We propose C. fragrantissima as a model organism to investigate the origin of metazoan multicellularity. By transgenesis, we demonstrate that its colony develops from a fully-grown multinucleate syncytium, in which nuclear divisions are strictly synchronized. It has been hypothesized that metazoan multicellular development initially occurred in the course of evolution through successive rounds of cell division, which were not necessarily be synchronized, or alternatively through cell aggregation. Our findings point to another possible mechanism for the evolution of animal multicellularity, namely, cellularization of a syncytium in which nuclear divisions are synchronized. We believe that further studies on the development of ichthyosporeans by the use of our methodologies will provide novel insights into the origin of metazoan multicellularity.

  16. Evolution of multicellularity coincided with increased diversification of cyanobacteria and the Great Oxidation Event.

    PubMed

    Schirrmeister, Bettina E; de Vos, Jurriaan M; Antonelli, Alexandre; Bagheri, Homayoun C

    2013-01-29

    Cyanobacteria are among the most diverse prokaryotic phyla, with morphotypes ranging from unicellular to multicellular filamentous forms, including those able to terminally (i.e., irreversibly) differentiate in form and function. It has been suggested that cyanobacteria raised oxygen levels in the atmosphere around 2.45-2.32 billion y ago during the Great Oxidation Event (GOE), hence dramatically changing life on the planet. However, little is known about the temporal evolution of cyanobacterial lineages, and possible interplay between the origin of multicellularity, diversification of cyanobacteria, and the rise of atmospheric oxygen. We estimated divergence times of extant cyanobacterial lineages under Bayesian relaxed clocks for a dataset of 16S rRNA sequences representing the entire known diversity of this phylum. We tested whether the evolution of multicellularity overlaps with the GOE, and whether multicellularity is associated with significant shifts in diversification rates in cyanobacteria. Our results indicate an origin of cyanobacteria before the rise of atmospheric oxygen. The evolution of multicellular forms coincides with the onset of the GOE and an increase in diversification rates. These results suggest that multicellularity could have played a key role in triggering cyanobacterial evolution around the GOE.

  17. Evolution of multicellularity coincided with increased diversification of cyanobacteria and the Great Oxidation Event

    PubMed Central

    Schirrmeister, Bettina E.; de Vos, Jurriaan M.; Antonelli, Alexandre; Bagheri, Homayoun C.

    2013-01-01

    Cyanobacteria are among the most diverse prokaryotic phyla, with morphotypes ranging from unicellular to multicellular filamentous forms, including those able to terminally (i.e., irreversibly) differentiate in form and function. It has been suggested that cyanobacteria raised oxygen levels in the atmosphere around 2.45–2.32 billion y ago during the Great Oxidation Event (GOE), hence dramatically changing life on the planet. However, little is known about the temporal evolution of cyanobacterial lineages, and possible interplay between the origin of multicellularity, diversification of cyanobacteria, and the rise of atmospheric oxygen. We estimated divergence times of extant cyanobacterial lineages under Bayesian relaxed clocks for a dataset of 16S rRNA sequences representing the entire known diversity of this phylum. We tested whether the evolution of multicellularity overlaps with the GOE, and whether multicellularity is associated with significant shifts in diversification rates in cyanobacteria. Our results indicate an origin of cyanobacteria before the rise of atmospheric oxygen. The evolution of multicellular forms coincides with the onset of the GOE and an increase in diversification rates. These results suggest that multicellularity could have played a key role in triggering cyanobacterial evolution around the GOE. PMID:23319632

  18. Three dimensional spheroid cell culture for nanoparticle safety testing.

    PubMed

    Sambale, Franziska; Lavrentieva, Antonina; Stahl, Frank; Blume, Cornelia; Stiesch, Meike; Kasper, Cornelia; Bahnemann, Detlef; Scheper, Thomas

    2015-07-10

    Nanoparticles are widely employed for many applications and the number of consumer products, incorporating nanotechnology, is constantly increasing. A novel area of nanotechnology is the application in medical implants. The widespread use of nanoparticles leads to their higher prevalence in our environment. This, in turn, raises concerns regarding potential risks to humans. Previous studies have shown possible hazardous effects of some nanoparticles on mammalian cells grown in two-dimensional (2D) cultures. However, 2D in vitro cell cultures display several disadvantages such as changes in cell shape, cell function, cell responses and lack of cell-cell contacts. For this reason, the development of better models for mimicking in vivo conditions is essential. In the present work, we cultivated A549 cells and NIH-3T3 cells in three-dimensional (3D) spheroids and investigated the effects of zinc oxide (ZnO-NP) and titanium dioxide nanoparticles (TiO2-NP). The results were compared to cultivation in 2D monolayer culture. A549 cells in 3D cell culture formed loose aggregates which were more sensitive to the toxicity of ZnO-NP in comparison to cells grown in 2D monolayers. In contrast, NIH-3T3 cells showed a compact 3D spheroid structure and no differences in the sensitivity of the NIH-3T3 cells to ZnO-NP were observed between 2D and 3D cultures. TiO2-NP were non-toxic in 2D cultures but affected cell-cell interaction during 3D spheroid formation of A549 and NIH-3T3 cells. When TiO2-NP were directly added during spheroid formation in the cultures of the two cell lines tested, several smaller spheroids were formed instead of a single spheroid. This effect was not observed if the nanoparticles were added after spheroid formation. In this case, a slight decrease in cell viability was determined only for A549 3D spheroids. The obtained results demonstrate the importance of 3D cell culture studies for nanoparticle safety testing, since some effects cannot be revealed in 2D

  19. Polarization of cosmic dust simulated with the rough spheroid model

    NASA Astrophysics Data System (ADS)

    Kolokolova, Ludmilla; Das, Himadri Sekhar; Dubovik, Oleg; Lapyonok, Tatyana; Yang, Ping

    2015-10-01

    Cosmic dust is a polydisperse mixture of irregular, often aggregated, particles. Previous attempts have tried to simulate polarimetric properties of this dust using aggregate dust models, but it has not been possible to consider particle sizes larger than a couple of microns due to limitations of computer memory and processing power. Attempts have also been made to replace aggregates by polydisperse regular particles (spheres, spheroids, cylinders), but those models could not consistently reproduce the observed photopolarimetric characteristics. In this study, we introduce to the astronomical community the software package developed by Dubovik et al. (2006) for modeling light scattering by a polydisperse mixture of randomly oriented smooth and rough spheroids of a variety of aspect ratios. The roughness of spheroids is defined by a normal distribution of the surface slopes, and its degree depends on the standard deviation of the distribution (which is zero for smooth surface and greater than zero for rough surface). The pre-calculated kernels in the software package allow for fast, accurate, and flexible modeling of different size and shape distributions. We present our results of a systematic investigation of polarization obtained with the rough and smooth spheroid models; we study differences in their phase angle dependence and how those differences change with the particle size distribution. We found that the difference between smooth and rough particles increases with increasing effective size parameter and affects mainly the value and position of the maximum polarization. Negative polarization was found to be typical only for silicate-like refractive indexes and only when the particles have size parameters within 2.5-25. As an example of an application of the rough spheroid model, we made computations for rough spheroids that have a size distribution and composition typical for cometary dust. We found that a mixture of porous rough spheroids made of absorbing

  20. Symbiotic Cell Differentiation and Cooperative Growth in Multicellular Aggregates

    PubMed Central

    Yamagishi, Jumpei F; Saito, Nen; Kaneko, Kunihiko

    2016-01-01

    As cells grow and divide under a given environment, they become crowded and resources are limited, as seen in bacterial biofilms and multicellular aggregates. These cells often show strong interactions through exchanging chemicals, as evident in quorum sensing, to achieve mutualism and division of labor. Here, to achieve stable division of labor, three characteristics are required. First, isogenous cells differentiate into several types. Second, this aggregate of distinct cell types shows better growth than that of isolated cells without interaction and differentiation, by achieving division of labor. Third, this cell aggregate is robust with respect to the number distribution of differentiated cell types. Indeed, theoretical studies have thus far considered how such cooperation is achieved when the ability of cell differentiation is presumed. Here, we address how cells acquire the ability of cell differentiation and division of labor simultaneously, which is also connected with the robustness of a cell society. For this purpose, we developed a dynamical-systems model of cells consisting of chemical components with intracellular catalytic reaction dynamics. The reactions convert external nutrients into internal components for cellular growth, and the divided cells interact through chemical diffusion. We found that cells sharing an identical catalytic network spontaneously differentiate via induction from cell-cell interactions, and then achieve division of labor, enabling a higher growth rate than that in the unicellular case. This symbiotic differentiation emerged for a class of reaction networks under the condition of nutrient limitation and strong cell-cell interactions. Then, robustness in the cell type distribution was achieved, while instability of collective growth could emerge even among the cooperative cells when the internal reserves of products were dominant. The present mechanism is simple and general as a natural consequence of interacting cells with

  1. Multicellular spheroids of bone marrow stromal cells: a three-dimensional in vitro culture system for the study of hematopoietic cell migration.

    PubMed

    Rossi, M I D; Barros, A P D N; Baptista, L S; Garzoni, L R; Meirelles, M N; Takiya, C M; Pascarelli, B M O; Dutra, H S; Borojevic, R

    2005-10-01

    Cell fate decisions are governed by a complex interplay between cell-autonomous signals and stimuli from the surrounding tissue. In vivo cells are connected to their neighbors and to the extracellular matrix forming a complex three-dimensional (3-D) microenvironment that is not reproduced in conventional in vitro systems. A large body of evidence indicates that mechanical tension applied to the cytoskeleton controls cell proliferation, differentiation and migration, suggesting that 3-D in vitro culture systems that mimic the in vivo situation would reveal biological subtleties. In hematopoietic tissues, the microenvironment plays a crucial role in stem and progenitor cell survival, differentiation, proliferation, and migration. In adults, hematopoiesis takes place inside the bone marrow cavity where hematopoietic cells are intimately associated with a specialized three 3-D scaffold of stromal cell surfaces and extracellular matrix that comprise specific niches. The relationship between hematopoietic cells and their niches is highly dynamic. Under steady-state conditions, hematopoietic cells migrate within the marrow cavity and circulate in the bloodstream. The mechanisms underlying hematopoietic stem/progenitor cell homing and mobilization have been studied in animal models, since conventional two-dimensional (2-D) bone marrow cell cultures do not reproduce the complex 3-D environment. In this review, we will highlight some of the mechanisms controlling hematopoietic cell migration and 3-D culture systems.

  2. Hepatocyte-derived cultured cells with unusual cytoplasmic keratin-rich spheroid bodies

    SciTech Connect

    Delavalle, Pierre-Yves; Alsaleh, Khaled; Pillez, Andre; Cocquerel, Laurence; Allet, Cecile; Dumont, Patrick; Loyens, Anne; Leteurtre, Emmanuelle; Omary, M. Bishr; Dubuisson, Jean; Rouille, Yves; Wychowski, Czeslaw

    2011-11-01

    Cytoplasmic inclusions are found in a variety of diseases that are characteristic morphological features of several hepatic, muscular and neurodegenerative disorders. They display a predominantly filamentous ultrastructure that is also observed in malignant rhabdoid tumor (MRT). A cellular clone containing an intracytoplasmic body was isolated from hepatocyte cell culture, and in the present study we examined whether this body might be related or not to Mallory-Denk body (MDB), a well characterized intracytoplasmic inclusion, or whether this cellular clone was constituted by malignant rhabdoid tumor cells. The intracytoplasmic body was observed in electron microscopy (EM), confocal immunofluorescence microscopy and several proteins involved in the formation of its structure were identified. Using light microscopy, a spheroid body (SB) described as a single regular-shaped cytoplasmic body was observed in cells. During cytokinesis, the SB was disassembled and reassembled in a way to reconstitute a unique SB in each progeny cell. EM examination revealed that the SB was not surrounded by a limiting membrane. However, cytoplasmic filaments were concentrated in a whorled array. These proteins were identified as keratins 8 and 18 (K8/K18), which formed the central core of the SB surrounded by a vimentin cage-like structure. This structure was not related to Mallory-Denk body or aggresome since no aggregated proteins were located in SB. Moreover, the structure of SB was not due to mutations in the primary sequence of K8/K18 and vimentin since no difference was observed in the mRNA sequence of their genes, isolated from Huh-7 and Huh-7w7.3 cells. These data suggested that cellular factor(s) could be responsible for the SB formation process. Aggregates of K18 were relocated in the SB when a mutant of K18 inducing disruption of K8/K18 IF network was expressed in the cellular clone. Furthermore, the INI1 protein, a remodeling-chromatin factor deficient in rhabdoid cells, which

  3. Which Way Is Jerusalem? Navigating on a Spheroid

    ERIC Educational Resources Information Center

    Schechter, Murray

    2007-01-01

    Given two points on a spheroidal planet, what is the direction from the first to the second? The answer depends, of course, on what path you take. This paper compares two paths which suggest themselves, namely, the loxodrome, which is the path in which the direction stays constant, and the geodesic, which is the shortest path. The geodesic does…

  4. Glycine decarboxylase in Rhodopseudomonas spheroides and in rat liver mitochondria

    PubMed Central

    Tait, G. H.

    1970-01-01

    1. Glycine decarboxylase and glycine–bicarbonate exchange activities were detected in extracts of Rhodopseudomonas spheroides and in rat liver mitochondria and their properties were studied. 2. The glycine decarboxylase activity from both sources is stimulated when glyoxylate is added to the assay system. 3. Several proteins participate in these reactions and a heat-stable low-molecular-weight protein was purified from both sources. 4. These enzyme activities increase markedly when R. spheroides is grown in the presence of glycine, glyoxylate, glycollate, oxalate or serine. 5. All the enzymes required to catalyse the conversion of glycine into acetyl-CoA via serine and pyruvate were detected in extracts of R. spheroides; of these glycine decarboxylase has the lowest activity. 6. The increase in the activity of glycine decarboxylase on illumination of R. spheroides in a medium containing glycine, and the greater increase when ATP is also present in the medium, probably accounts for the increased incorporation of the methylene carbon atom of glycine into fatty acids found previously under these conditions (Gajdos, Gajdos-Török, Gorchein, Neuberger & Tait, 1968). 7. The results are compared with those obtained by other workers on the glycine decarboxylase and glycine–bicarbonate exchange activities in other systems. PMID:5476725

  5. X-Ray Sources in the Dwarf Spheroidal Galaxy DRACO

    NASA Astrophysics Data System (ADS)

    Sonbas, E.; Dhuga, K.; Rangelov, B.; Kargaltsev, O.

    2016-06-01

    We present the results of a spectral analysis of X - ray sources in Draco, a nearby dwarf spheroidal galaxy recently observed by XMM-Newton. While most of the sources exhibit properties consistent with AGN, few of them possess characteristics of LMXBs and CVs. We also discuss the possibility of the existence of a central IMBH in Draco.

  6. Chromosome Conformation of Human Fibroblasts Grown in 3-Dimensional Spheroids

    PubMed Central

    Chen, Haiming; Comment, Nicholas; Chen, Jie; Ronquist, Scott; Hero, Alfred; Ried, Thomas; Rajapakse, Indika

    2015-01-01

    In the study of interphase chromosome organization, genome-wide chromosome conformation capture (Hi-C) maps are often generated using 2-dimensional (2D) monolayer cultures. These 2D cells have morphological deviations from cells that exist in 3-dimensional (3D) tissues in vivo, and may not maintain the same chromosome conformation. We used Hi-C maps to test the extent of differences in chromosome conformation between human fibroblasts grown in 2D cultures and those grown in 3D spheroids. Significant differences in chromosome conformation were found between 2D cells and those grown in spheroids. Intra-chromosomal interactions were generally increased in spheroid cells, with a few exceptions, while inter-chromosomal interactions were generally decreased. Overall, chromosomes located closer to the nuclear periphery had increased intra-chromosomal contacts in spheroid cells, while those located more centrally had decreased interactions. This study highlights the necessity to conduct studies on the topography of the interphase nucleus under conditions that mimic an in vivo environment. PMID:25738643

  7. Void nucleation in spheroidized steels during tensile deformation

    SciTech Connect

    Fisher, Jr, J R

    1980-04-01

    An investigation was conducted to determine the effects of various mechanical and material parameters on void formation at cementite particles in axisymmetric tensile specimens of spheroidized plain carbon steels. Desired microstructures for each of three steel types were obtained. Observations of void morphology with respect to various microstructural features were made using optical and scanning electron microscopy.

  8. Improvement of Mechanical Properties of Spheroidized 1045 Steel by Induction Heat Treatment

    NASA Astrophysics Data System (ADS)

    Kim, Minwook; Shin, Jung-Ho; Choi, Young; Lee, Seok-Jae

    2016-04-01

    The effects of induction heat treatment on the formation of carbide particles and mechanical properties of spheroidized 1045 steel were investigated by means of microstructural analysis and tensile testing. The induction spheroidization accelerated the formation of spherical cementite particles and effectively softened the steel. The volume fraction of cementite was found to be a key factor that affected the mechanical properties of spheroidized steels. Further tests showed that sequential spheroidization by induction and furnace heat treatments enhanced elongation within a short spheroidization time, resulting in better mechanical properties. This was due to the higher volume fraction of spherical cementite particles that had less diffusion time for particle coarsening.

  9. Plasma spheroidization of iron powders in a non-transferred DC thermal plasma jet

    SciTech Connect

    Kumar, S. Selvarajan, V

    2008-06-15

    In this paper, the results of plasma spheroidization of iron powders using a DC non-transferred plasma spray torch are presented. The morphology of the processed powders was characterized through scanning electron microscopy (SEM) and optical microscopy (OM). The percentages of spheroidized powders were calculated by the shape factors such as the Irregularity Parameter (IP) and Roundness (RN). A maximum of 83% of spheroidization can be achieved. The spheroidization results are compared with the theoretical estimation and they are found to be in good agreement. The phase composition of the spheroidized powder was analyzed by XRD. The effect of plasma jet temperature and plasma gas flow rate on spheroidization is discussed. At low plasma gas flow rates and at high plasma jet temperatures, the percentage of spheroidization is high.

  10. A general allometric and life-history model for cellular differentiation in the transition to multicellularity.

    PubMed

    Solari, Cristian A; Kessler, John O; Goldstein, Raymond E

    2013-03-01

    The transition from unicellular, to colonial, to larger multicellular organisms has benefits, costs, and requirements. Here we present a model inspired by the volvocine green algae that explains the dynamics involved in the unicellular-multicellular transition using life-history theory and allometry. We model the two fitness components (fecundity and viability) and compare the fitness of hypothetical colonies of different sizes with varying degrees of cellular differentiation to understand the general principles that underlie the evolution of multicellularity. We argue that germ-soma separation may have evolved to counteract the increasing costs and requirements of larger multicellular colonies. The model shows that the cost of investing in soma decreases with size. For lineages such as the Volvocales, as reproduction costs increase with size for undifferentiated colonies, soma specialization benefits the colony indirectly by decreasing such costs and directly by helping reproductive cells acquire resources for their metabolic needs. Germ specialization is favored once soma evolves and takes care of vegetative functions. To illustrate the model, we use some allometric relationships measured in Volvocales. Our analysis shows that the cost of reproducing an increasingly larger group has likely played an important role in the transition to multicellularity and cellular differentiation.

  11. 'Candidatus Magnetoglobus multicellularis', a multicellular, magnetotactic prokaryote from a hypersaline environment.

    PubMed

    Abreu, Fernanda; Martins, Juliana Lopes; Silveira, Thaís Souza; Keim, Carolina Neumann; de Barros, Henrique G P Lins; Filho, Frederico J Gueiros; Lins, Ulysses

    2007-06-01

    Phylogenetic analysis and phenotypic characterization were used to assign a multicellular magnetotactic prokaryote the name 'Candidatus Magnetoglobus multicellularis'. 'Candidatus Magnetoglobus multicellularis' lives in a large hypersaline coastal lagoon from Brazil and has properties that are unique among prokaryotes. It consists of a compact assembly or aggregate of flagellated bacterial cells, highly organized in a sphere, that swim in either helical or straight trajectories. The life cycle of 'Candidatus Magnetoglobus multicellularis' is completely multicellular, in which one aggregate grows by enlarging the size of its cells and approximately doubling the volume of the whole organism. Cells then divide synchronously, maintaining the spherical arrangement; finally the cells separate into two identical aggregates. Phylogenetic 16S rRNA gene sequence analysis showed that 'Candidatus Magnetoglobus multicellularis' is related to the dissimilatory sulfate-reducing bacteria within the Deltaproteobacteria and to other previously described, but not yet well characterized, multicellular magnetotactic prokaryotes.

  12. Cell cycle arrest or survival signaling through αv integrins, activation of PKC and ERK1/2 lead to anoikis resistance of ovarian cancer spheroids.

    PubMed

    Carduner, Ludovic; Picot, Cédric R; Leroy-Dudal, Johanne; Blay, Lyvia; Kellouche, Sabrina; Carreiras, Franck

    2014-01-15

    Ovarian cancer is the most lethal gynecologic cancer mainly due to spheroids organization of cancer cells that disseminate within the peritoneal cavity. We have investigated the molecular mechanisms by which ovarian cancer spheroids resist anoikis, choosing as models the 2 well-characterized human ovarian cancer cell lines IGROV1 and SKOV3. These cell lines have the propensity to float as clusters, and were isolated from tumor tissue and ascites, respectively. To form spheroids, IGROV1 and SKOV3 ovarian adenocarcinoma cells were maintained under anchorage-independent culture conditions, in which both lines survive at least a week. A short apoptotic period prior to a survival signaling commitment was observed for IGROV1 cells whereas SKOV3 cells entered G0/G1 phase of the cell cycle. This difference in behavior was due to different signals. With regard to SKOV3 cells, activation of p38 and an increase in p130/Rb occurred once anchorage-independent culture was established. Analyses of the survival signaling pathway switched on by IGROV1 cells showed that activation of ERK1/2 was required to evade apoptosis, an effect partly dependent on PKC activation and αv integrins. αv-integrin expression is essential for survival through activation of ERK1/2 phosphorylation. The above data indicate that ovarian cancer cells can resist anoikis in the spheroid state by arrest in the cell cycle or through activation of αv-integrin-ERK-mediated survival signals. Such signaling might result in the selection of resistant cells within disseminating spheroids, favoring further relapse in ovarian cancers. PMID:24291221

  13. Manipulation of cellular spheroid composition and the effects on vascular tissue fusion

    PubMed Central

    Olsen, T.R.; Mattix, B.; Casco, M.; Herbst, A.; Williams, C.; Tarasidis, A.; Simionescu, D.; Visconti, R.P.; Alexis, F.

    2016-01-01

    Cellular spheroids were investigated as tissue-engineered building blocks that can be fused to form functional tissue constructs. While spheroids can be assembled using passive contacts for the fusion of complex tissues, physical forces can be used to promote active contacts to improve tissue homogeneity and accelerate tissue fusion. Understanding the mechanisms affecting the fusion of spheroids is critical to fabricating tissues. Here, manipulation of the spheroid composition was used to accelerate the fusion process mediated by magnetic forces. The Janus structure of magnetic cellular spheroids spatially controls iron oxide magnetic nanoparticles (MNPs) to form two distinct domains: cells and extracellular MNPs. Studies were performed to evaluate the influence of extracellular matrix (ECM) content and cell number on the fusion of Janus magnetic cellular spheroids (JMCSs). Results showed that the integration of iron oxide MNPs into spheroids increased the production of collagen over time when compared to spheroids without MNPs. The results also showed that ring tissues composed of JMCSs with high ECM concentrations and high cell numbers fused together, but exhibited less contraction when compared to their lower concentration counterparts. Results from spheroid fusion in capillary tubes showed that low ECM concentrations and high cell numbers experienced more fusion and cellular intermixing over time when compared to their higher counterparts. These findings indicate that cell–cell and cell–matrix interactions play an important role in regulating fusion, and this understanding sets the rationale of spheroid composition to fabricate larger and more complex tissue-engineered constructs. PMID:25463485

  14. Implantable Three-Dimensional Salivary Spheroid Assemblies Demonstrate Fluid and Protein Secretory Responses to Neurotransmitters

    PubMed Central

    Pradhan-Bhatt, Swati; Harrington, Daniel A.; Duncan, Randall L.; Jia, Xinqiao; Witt, Robert L.

    2013-01-01

    Radiation treatment in patients with head and neck tumors commonly results in hyposalivation and xerostomia due to the loss of fluid-secreting salivary acinar cells. Patients develop susceptibility to oral infections, dental caries, impaired speech and swallowing, reducing the quality of life. Clinical management is largely unsatisfactory. The development of a tissue-engineered, implantable salivary gland will greatly benefit patients suffering from xerostomia. This report compares the ability of a 2.5-dimensional (2.5D) and a three-dimensional (3D) hyaluronic acid (HA)-based culture system to support functional salivary units capable of producing fluid and phenotypic proteins. Parotid cells seeded on 2.5D, as well as those encapsulated in 3D HA hydrogels, self-assembled into acini-like structures and expressed functional neurotransmitter receptors. Structures in 3D hydrogels merged to form organized 50 μm spheroids that could be maintained in culture for over 100 days and merged to form structures over 500 μm in size. Treatment of acini-like structures with the β-adrenergic agonists norepinephrine or isoproterenol increased granule production and α-amylase staining in treated structures, demonstrating regain of protein secretion. Upon treatment with the M3 muscarinic agonist acetylcholine, acini-like structures activated the fluid production pathway by increasing intracellular calcium levels. The increase in intracellular calcium seen in structures in the 3D hydrogel culture system was more robust and prolonged than that in 2.5D. To compare the long-term survival and retention of acini-like structures in vivo, cell-seeded 2.5D and 3D hydrogels were implanted into an athymic rat model. Cells in 2.5D failed to maintain organized acini-like structures and dispersed in the surrounding tissue. Encapsulated cells in 3D retained their spheroid structure and structural integrity, along with the salivary biomarkers and maintained viability for over 3 weeks in vivo

  15. Spheroid and Tissue Assembly via Click Chemistry in Microfluidic Flow.

    PubMed

    O'Brien, Paul J; Luo, Wei; Rogozhnikov, Dmitry; Chen, Jean; Yousaf, Muhammad N

    2015-09-16

    Proper cell-cell contact and communication are essential for the correct development and survival of higher order organisms. In order to study complex cell interactions that occur in vivo, model systems that are able to recapitulate 3D cell-cell interactions in vitro are key to advancing new biotechnologies, therapeutics, and tissue engineering applications. Herein, we show a new strategy to rapidly and efficiently generate complex multiple cell line containing spheroids and tissues in microfluidic flow without the use of scaffolds, molecular biology, or metabolic biosynthesis. The method relies on the integration of microfluidics, liposome fusion, bio-orthogonal chemistry, and cell surface engineering to rapidly click coculture cell assemblies in flow. We demonstrate this strategy by assembling various combinations of cell types with an interfacial cell to cell click chemistry in microfluidic flow to generate a range of spheroid types and oriented tissue multilayers.

  16. The Dark Halo - Spheroid Conspiracy Reloaded: Evolution with Redshift

    NASA Astrophysics Data System (ADS)

    Remus, Rhea-Silvia; Dolag, Klaus; Burkert, Andreas

    2015-04-01

    The total density profiles of elliptical galaxies can be fit by a single power law, i.e., ρtot ~ r γ with γ ~ -2. While strong lensing observations show a tendency for the slopes to become flatter with increasing redshift, simulations indicate an opposite trend. To understand this discrepancy, we study a set of simulated spheroids formed within the cosmological framework. From our simulations we find that the steepness of the total density slope correlates with the compactness of the stellar component within the half-mass radius, and that spheroidal galaxies tend to be more compact at high redshifts than their present-day counterparts. While both these results are in agreement with observations, the observed trend of the total density slope with redshift remains in contradiction to the results from simulations.

  17. A method of smoothed particle hydrodynamics using spheroidal kernels

    NASA Technical Reports Server (NTRS)

    Fulbright, Michael S.; Benz, Willy; Davies, Melvyn B.

    1995-01-01

    We present a new method of three-dimensional smoothed particle hydrodynamics (SPH) designed to model systems dominated by deformation along a preferential axis. These systems cause severe problems for SPH codes using spherical kernels, which are best suited for modeling systems which retain rough spherical symmetry. Our method allows the smoothing length in the direction of the deformation to evolve independently of the smoothing length in the perpendicular plane, resulting in a kernel with a spheroidal shape. As a result the spatial resolution in the direction of deformation is significantly improved. As a test case we present the one-dimensional homologous collapse of a zero-temperature, uniform-density cloud, which serves to demonstrate the advantages of spheroidal kernels. We also present new results on the problem of the tidal disruption of a star by a massive black hole.

  18. Adhesive Wear Behavior of Heat Treated Spheroidal Graphite Cast Iron

    NASA Astrophysics Data System (ADS)

    Salim, M.; Shama, S.; Mozumder, Y. H.; Behera, R. K.; P, Sindhoora L.; Pattanaik, A.; Mishra, S. C.; Sen, S.

    2015-02-01

    Spheroidal graphite cast iron is one of the most favorable materials in cast iron family due to its wide range of mechanical and tribological properties. In the current research priority is given towards the investigation of wear system response of spheroidal graphite cast iron subjected to various heat treatment conditions. Pearlitic/ferritic and upper bainitic matrix was obtained through normalizing and austempering treatment for an austenitizing temperature of 1000°C. Dry sliding wear test was performed at 10N, 20N, 30N for a sliding distance of 7.54m. With increase in load weight loss was observed for as-cast specimen whereas normalized specimen showed very less weight loss with increasing load. On the other hand the austempered specimen observed to lose weight when operated at 10N, whereas at 20N and 30N drastic gain in weight was observed.

  19. Two step continuous method to synthesize colloidal spheroid gold nanorods.

    PubMed

    Chandra, S; Doran, J; McCormack, S J

    2015-12-01

    This research investigated a two-step continuous process to synthesize colloidal suspension of spheroid gold nanorods. In the first step; gold precursor was reduced to seed-like particles in the presence of polyvinylpyrrolidone and ascorbic acid. In continuous second step; silver nitrate and alkaline sodium hydroxide produced various shape and size Au nanoparticles. The shape was manipulated through weight ratio of ascorbic acid to silver nitrate by varying silver nitrate concentration. The specific weight ratio of 1.35-1.75 grew spheroid gold nanorods of aspect ratio ∼1.85 to ∼2.2. Lower weight ratio of 0.5-1.1 formed spherical nanoparticle. The alkaline medium increased the yield of gold nanorods and reduced reaction time at room temperature. The synthesized gold nanorods retained their shape and size in ethanol. The surface plasmon resonance was red shifted by ∼5 nm due to higher refractive index of ethanol than water.

  20. Numerical Simulations of the Metallicity Distribution in Dwarf Spheroidal Galaxies

    SciTech Connect

    Ripamonti, Emanuele; Tolstoy, E.; Helmi, A.; Battaglia, G.; Abel, T.; /KIPAC, Menlo Park

    2006-12-12

    Recent observations show that the number of stars with very low metallicities in the dwarf spheroidal satellites of the Milky Way is low, despite the low average metallicities of stars in these systems. We undertake numerical simulations of star formation and metal enrichment of dwarf galaxies in order to verify whether this result can be reproduced with ''standard'' assumptions. The answer is likely to be negative, unless some selection bias against very low metallicity stars is present in the observations.

  1. Structure of molecular liquids: closure relations for hard spheroids.

    PubMed

    Cheung, David L; Anton, Lucian; Allen, Michael P; Masters, Andrew J

    2007-10-01

    We present the results of Monte Carlo simulations of hard spheroids of revolution of different elongations. Both prolate and oblate shapes are examined. A systematic study of the bridge function b(1,2), and direct comparison with the indirect correlation function gamma(1,2)=h(1,2)-c(1,2) at densities spanning the isotropic fluid range, allow us to evaluate the accuracy of various proposed closure relations for integral equations.

  2. Star Formation History of the Fornax Dwarf Spheroidal Galaxy

    NASA Astrophysics Data System (ADS)

    Del Pino, A.; Aparicio, A.; Gallart, C.; Hidalgo, S.

    2011-07-01

    We present color-magnitude diagrams (CMD) reaching the oldest main sequence turn offs for three fields in the Fornax dwarf spheroidal galaxy, obtained with FORS1 at the VLT. One of them is situated at the center of the galaxy while the other two are located at a distance of 10' form the center. We determine the full star formation history (SFH) of the central field, extending to the first star formation events.

  3. VERITAS deep observations of the dwarf spheroidal galaxy Segue 1

    NASA Astrophysics Data System (ADS)

    Aliu, E.; Archambault, S.; Arlen, T.; Aune, T.; Beilicke, M.; Benbow, W.; Bouvier, A.; Bradbury, S. M.; Buckley, J. H.; Bugaev, V.; Byrum, K.; Cannon, A.; Cesarini, A.; Christiansen, J. L.; Ciupik, L.; Collins-Hughes, E.; Connolly, M. P.; Cui, W.; Decerprit, G.; Dickherber, R.; Dumm, J.; Errando, M.; Falcone, A.; Feng, Q.; Ferrer, F.; Finley, J. P.; Finnegan, G.; Fortson, L.; Furniss, A.; Galante, N.; Gall, D.; Godambe, S.; Griffin, S.; Grube, J.; Gyuk, G.; Hanna, D.; Holder, J.; Huan, H.; Hughes, G.; Humensky, T. B.; Kaaret, P.; Karlsson, N.; Kertzman, M.; Khassen, Y.; Kieda, D.; Krawczynski, H.; Krennrich, F.; Lee, K.; Madhavan, A. S.; Maier, G.; Majumdar, P.; McArthur, S.; McCann, A.; Moriarty, P.; Mukherjee, R.; Ong, R. A.; Orr, M.; Otte, A. N.; Park, N.; Perkins, J. S.; Pohl, M.; Prokoph, H.; Quinn, J.; Ragan, K.; Reyes, L. C.; Reynolds, P. T.; Roache, E.; Rose, H. J.; Ruppel, J.; Saxon, D. B.; Schroedter, M.; Sembroski, G. H.; Şentürk, G. D.; Skole, C.; Smith, A. W.; Staszak, D.; Telezhinsky, I.; Tešić, G.; Theiling, M.; Thibadeau, S.; Tsurusaki, K.; Varlotta, A.; Vassiliev, V. V.; Vincent, S.; Vivier, M.; Wagner, R. G.; Wakely, S. P.; Ward, J. E.; Weekes, T. C.; Weinstein, A.; Weisgarber, T.; Williams, D. A.; Zitzer, B.

    2012-03-01

    The VERITAS array of Cherenkov telescopes has carried out a deep observational program on the nearby dwarf spheroidal galaxy Segue 1. We report on the results of nearly 48 hours of good quality selected data, taken between January 2010 and May 2011. No significant γ-ray emission is detected at the nominal position of Segue 1, and upper limits on the integrated flux are derived. According to recent studies, Segue 1 is the most dark matter-dominated dwarf spheroidal galaxy currently known. We derive stringent bounds on various annihilating and decaying dark matter particle models. The upper limits on the velocity-weighted annihilation cross-section are ⟨σv⟩95%CL≲10-23cm3s-1, improving our limits from previous observations of dwarf spheroidal galaxies by at least a factor of 2 for dark matter particle masses mχ≳300GeV. The lower limits on the decay lifetime are at the level of τ95%CL≳1024s. Finally, we address the interpretation of the cosmic ray lepton anomalies measured by ATIC and PAMELA in terms of dark matter annihilation, and show that the VERITAS observations of Segue 1 disfavor such a scenario.

  4. Mergers and the outside-in formation of dwarf spheroidals

    NASA Astrophysics Data System (ADS)

    Benítez-Llambay, A.; Navarro, J. F.; Abadi, M. G.; Gottlöber, S.; Yepes, G.; Hoffman, Y.; Steinmetz, M.

    2016-02-01

    We use a cosmological simulation of the formation of the Local Group to explore the origin of age and metallicity gradients in dwarf spheroidal galaxies. We find that a number of simulated dwarfs form `outside-in', with an old, metal-poor population that surrounds a younger, more concentrated metal-rich component, reminiscent of dwarf spheroidals like Sculptor or Sextans. We focus on a few examples where stars form in two populations distinct in age in order to elucidate the origin of these gradients. The spatial distributions of the two components reflect their diverse origin; the old stellar component is assembled through mergers, but the young population forms largely in situ. The older component results from a first episode of star formation that begins early but is quickly shut off by the combined effects of stellar feedback and reionization. The younger component forms when a late accretion event adds gas and reignites star formation. The effect of mergers is to disperse the old stellar population, increasing their radius and decreasing their central density relative to the young population. We argue that dwarf-dwarf mergers offer a plausible scenario for the formation of systems with multiple distinct populations and, more generally, for the origin of age and metallicity gradients in dwarf spheroidals.

  5. Random sequential adsorption of spheroidal particles: Kinetics and jamming limit

    NASA Astrophysics Data System (ADS)

    Adamczyk, Zbigniew; Weroński, Paweł

    1996-10-01

    Localized adsorption of hard (noninteracting) spheroidal particles on homogeneous interfaces was analyzed theoretically. In contrast to previous studies concentrated on flat (side on) adsorption in the present approach an unoriented (quasi-three-dimensional) adsorption of prolate and oblate spheroids was considered. By applying the random sequential adsorption (RSA) approach asymptotic analytic expressions were derived for the available surface function (surface blocking parameter) and adsorption kinetics in the limit of low and moderate surface concentrations. The range of validity of the approximate analytical results was determined by numerical simulations of adsorption kinetics performed using the Monte Carlo RSA technique. It was revealed by this comparison that the analytical approximation can be used with a good accuracy for the dimensionless adsorption time τ smaller than two. The numerical calculations also enabled us to determine the maximum (jamming) surface concentrations for unoriented adsorption of spheroids as a function of the elongation or flattening parameter A. It was demonstrated that these jamming concentrations θ∞ are approached for long adsorption times as τ-1/4, therefore deviating considerably from the Langmuir model used often in the literature.

  6. Centrifugal force induced by relativistically rotating spheroids and cylinders

    NASA Astrophysics Data System (ADS)

    Katz, Joseph; Lynden Bell, Donald; Bičák, Jiří

    2011-03-01

    Starting from the gravitational potential of a Newtonian spheroidal shell we discuss electrically charged rotating prolate spheroidal shells in the Maxwell theory. In particular we consider two confocal charged shells which rotate oppositely in such a way that there is no magnetic field outside the outer shell. In the Einstein theory we solve the Ernst equations in the region where the long prolate spheroids are almost cylindrical; in equatorial regions the exact Lewis 'rotating cylindrical' solution is so derived by a limiting procedure from a spatially bound system. In the second part we analyze two cylindrical shells rotating in opposite directions in such a way that the static Levi-Civita metric is produced outside and no angular momentum flux escapes to infinity. The rotation of the local inertial frames in flat space inside the inner cylinder is thus exhibited without any approximation or interpretational difficulties within this model. A test particle within the inner cylinder kept at rest with respect to axes that do not rotate as seen from infinity experiences a centrifugal force. Although in suitably chosen axes the spacetime there is exactly Minkowskian out to the inner cylinder, nevertheless, those inertial frame axes rotate with respect to infinity, so relative to the inertial frame inside the inner cylinder a test particle is traversing a circular orbit.

  7. Ixmyelocel-T, an expanded multicellular therapy, contains a unique population of M2-like macrophages

    PubMed Central

    2013-01-01

    Introduction M2 macrophages promote tissue repair and regeneration through various mechanisms including immunomodulation and scavenging of tissue debris. Delivering increased numbers of these cells to ischemic tissues may limit tissue injury and promote repair. Ixmyelocel-T is an expanded, autologous multicellular therapy cultured from bone-marrow mononuclear cells (BMMNCs). The purpose of this study was to characterize further a unique expanded population of M2-like macrophages, generated in ixmyelocel-T therapy. Methods Approximately 50 ml of whole bone marrow was obtained from healthy donors and shipped overnight. BMMNCs were produced by using density-gradient separation and cultured for approximately 12 days to generate ixmyelocel-T. CD14+ cells were isolated from ixmyelocel-T with positive selection for analysis. Cell-surface phenotype was examined with flow cytometry and immunofluorescence, and expression of cytokines and chemokines was analyzed with enzyme-linked immunosorbent assay (ELISA). Quantitative real-time PCR was used to analyze expression of genes in BMMNCs, ixmyelocel-T, the CD14+ population from ixmyelocel-T, and M1 and M2 macrophages. Ixmyelocel-T was cultured with apoptotic BMMNCs, and then visualized under fluorescence microscopy to assess efferocytosis. Results Macrophages in ixmyelocel-T therapy expressed surface markers of M2 macrophages, CD206, and CD163. These cells were also found to express several M2 markers, and few to no M1 markers. After stimulation with lipopolysaccharide (LPS), they showed minimal secretion of the proinflammatory cytokines interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α) compared with M1 and M2 macrophages. Ixmyelocel-T macrophages efficiently ingested apoptotic BMMNCs. Conclusions Ixmyelocel-T therapy contains a unique population of M2-like macrophages that are characterized by expression of M2 markers, decreased secretion of proinflammatory cytokines after inflammatory stimuli, and efficient

  8. Magnetic Flattening of Stem-Cell Spheroids Indicates a Size-Dependent Elastocapillary Transition

    NASA Astrophysics Data System (ADS)

    Mazuel, Francois; Reffay, Myriam; Du, Vicard; Bacri, Jean-Claude; Rieu, Jean-Paul; Wilhelm, Claire

    2015-03-01

    Cellular aggregates (spheroids) are widely used in biophysics and tissue engineering as model systems for biological tissues. In this Letter we propose novel methods for molding stem-cell spheroids, deforming them, and measuring their interfacial and elastic properties with a single method based on cell tagging with magnetic nanoparticles and application of a magnetic field gradient. Magnetic molding yields spheroids of unprecedented sizes (up to a few mm in diameter) and preserves tissue integrity. On subjecting these spheroids to magnetic flattening (over 150 g ), we observed a size-dependent elastocapillary transition with two modes of deformation: liquid-drop-like behavior for small spheroids, and elastic-sphere-like behavior for larger spheroids, followed by relaxation to a liquidlike drop.

  9. Sound scattering by rigid oblate spheroids, with implication to pressure gradient microphones

    NASA Technical Reports Server (NTRS)

    Maciulaitis, A.; Seiner, J.; Norum, T. D.

    1976-01-01

    The frequency limit below which sound scattering by a microphone body is sufficiently small to permit accurate pressure gradient measurements was determined. The sound pressure was measured at various points on the surface of a rigid oblate spheroid illuminated by spherical waves generated by a point source at a large distance from the spheroid, insuring an essentially plane sound field. The measurements were made with small pressure microphones flush mounted from the inside of the spheroid model. Numerical solutions were obtained for a variety of spheroid shapes, including that of the experimental model. Very good agreement was achieved between the experimental and theoretical results. It was found that scattering effects are insignificant if the ratio of the major circumference of the spheroid to the wavelength of the incident sound is less than about 0.7, this number being dependent upon the shape of the spheroid. This finding can be utilized in the design of pressure gradient microphones.

  10. The Gonium pectorale genome demonstrates co-option of cell cycle regulation during the evolution of multicellularity.

    PubMed

    Hanschen, Erik R; Marriage, Tara N; Ferris, Patrick J; Hamaji, Takashi; Toyoda, Atsushi; Fujiyama, Asao; Neme, Rafik; Noguchi, Hideki; Minakuchi, Yohei; Suzuki, Masahiro; Kawai-Toyooka, Hiroko; Smith, David R; Sparks, Halle; Anderson, Jaden; Bakarić, Robert; Luria, Victor; Karger, Amir; Kirschner, Marc W; Durand, Pierre M; Michod, Richard E; Nozaki, Hisayoshi; Olson, Bradley J S C

    2016-01-01

    The transition to multicellularity has occurred numerous times in all domains of life, yet its initial steps are poorly understood. The volvocine green algae are a tractable system for understanding the genetic basis of multicellularity including the initial formation of cooperative cell groups. Here we report the genome sequence of the undifferentiated colonial alga, Gonium pectorale, where group formation evolved by co-option of the retinoblastoma cell cycle regulatory pathway. Significantly, expression of the Gonium retinoblastoma cell cycle regulator in unicellular Chlamydomonas causes it to become colonial. The presence of these changes in undifferentiated Gonium indicates extensive group-level adaptation during the initial step in the evolution of multicellularity. These results emphasize an early and formative step in the evolution of multicellularity, the evolution of cell cycle regulation, one that may shed light on the evolutionary history of other multicellular innovations and evolutionary transitions. PMID:27102219

  11. Multicellularity arose several times in the evolution of eukaryotes (response to DOI 10.1002/bies.201100187).

    PubMed

    Parfrey, Laura Wegener; Lahr, Daniel J G

    2013-04-01

    The cellular slime mold Dictyostelium has cell-cell connections similar in structure, function, and underlying molecular mechanisms to animal epithelial cells. These similarities form the basis for the proposal that multicellularity is ancestral to the clade containing animals, fungi, and Amoebozoa (including Dictyostelium): Amorphea (formerly "unikonts"). This hypothesis is intriguing and if true could precipitate a paradigm shift. However, phylogenetic analyses of two key genes reveal patterns inconsistent with a single origin of multicellularity. A single origin in Amorphea would also require loss of multicellularity in each of the many unicellular lineages within this clade. Further, there are numerous other origins of multicellularity within eukaryotes, including three within Amorphea, that are not characterized by these structural and mechanistic similarities. Instead, convergent evolution resulting from similar selective pressures for forming multicellular structures with motile and differentiated cells is the most likely explanation for the observed similarities between animal and dictyostelid cell-cell connections. PMID:23315654

  12. The Gonium pectorale genome demonstrates co-option of cell cycle regulation during the evolution of multicellularity

    PubMed Central

    Hanschen, Erik R.; Marriage, Tara N.; Ferris, Patrick J.; Hamaji, Takashi; Toyoda, Atsushi; Fujiyama, Asao; Neme, Rafik; Noguchi, Hideki; Minakuchi, Yohei; Suzuki, Masahiro; Kawai-Toyooka, Hiroko; Smith, David R.; Sparks, Halle; Anderson, Jaden; Bakarić, Robert; Luria, Victor; Karger, Amir; Kirschner, Marc W.; Durand, Pierre M.; Michod, Richard E.; Nozaki, Hisayoshi; Olson, Bradley J. S. C.

    2016-01-01

    The transition to multicellularity has occurred numerous times in all domains of life, yet its initial steps are poorly understood. The volvocine green algae are a tractable system for understanding the genetic basis of multicellularity including the initial formation of cooperative cell groups. Here we report the genome sequence of the undifferentiated colonial alga, Gonium pectorale, where group formation evolved by co-option of the retinoblastoma cell cycle regulatory pathway. Significantly, expression of the Gonium retinoblastoma cell cycle regulator in unicellular Chlamydomonas causes it to become colonial. The presence of these changes in undifferentiated Gonium indicates extensive group-level adaptation during the initial step in the evolution of multicellularity. These results emphasize an early and formative step in the evolution of multicellularity, the evolution of cell cycle regulation, one that may shed light on the evolutionary history of other multicellular innovations and evolutionary transitions. PMID:27102219

  13. Scattering of Gaussian Beam by a Conducting Spheroidal Particle with Confocal Dielectric Coating

    NASA Astrophysics Data System (ADS)

    Sun, Xianming; Wang, Haihua; Zhang, Huayong

    2010-09-01

    An analytic solution to the scattering by a coated spheroidal particle, for arbitrary incidence of a Gaussian beam, is constructed by expanding the incident and scattered electromagnetic fields in terms of spheroidal vector wave functions. The unknown expansion coefficients are determined by a system of linear equations derived from the appropriate boundary conditions. Numerical results of the normalized differential scattering cross section of the conducting and coated spheroidal particle are evaluated, and the scattering characteristics are discussed concisely.

  14. Spheroidal models of the exterior gravitational field of Asteroids Bennu and Castalia

    NASA Astrophysics Data System (ADS)

    Sebera, Josef; Bezděk, Aleš; Pešek, Ivan; Henych, Tomáš

    2016-07-01

    Gravitational field of small bodies can be modeled e.g. with mascons, a polyhedral model or in terms of harmonic functions. If the shape of a body is close to the spheroid, it is advantageous to employ the spheroidal basis functions for expressing the gravitational field. Spheroidal harmonic models, similarly to the spherical ones, may be used in navigation and geophysical tasks. We focus on modeling the exterior gravitational field of oblate-like Asteroid (101955) Bennu and prolate-like Asteroid (4769) Castalia with spheroidal harmonics. Using the Gauss-Legendre quadrature and the spheroidal basis functions, we converted the gravitational potential of a particular polyhedral model of a constant density into the spheroidal harmonics. The results consist of (i) spheroidal harmonic coefficients of the exterior gravitational field for the Asteroids Bennu and Castalia, (ii) spherical harmonic coefficients for Bennu, and (iii) the first and second-order Cartesian derivatives in the local spheroidal South-East-Up frame for both bodies. The spheroidal harmonics offer biaxial flexibility (compared with spherical harmonics) and low computational costs that allow high-degree expansions (compared with ellipsoidal harmonics). The obtained spheroidal models for Bennu and Castalia represent the exterior gravitational field valid on and outside the Brillouin spheroid but they can be used even under this surface. For Bennu, 5 m above the surface the agreement with point-wise integration was 1% or less, while it was about 10% for Castalia due to its more irregular shape. As the shape models may produce very high frequencies, it was crucial to use higher maximum degree to reduce the aliasing. We have used the maximum degree 360 to achieve 9-10 common digits (in RMS) when reconstructing the input (the gravitational potential) from the spheroidal coefficients. The physically meaningful maximum degree may be lower (≪ 360) but its particular value depends on the distance and/or on the

  15. Experimental evolution reveals that high relatedness protects multicellular cooperation from cheaters.

    PubMed

    Bastiaans, Eric; Debets, Alfons J M; Aanen, Duur K

    2016-01-01

    In multicellular organisms, there is a potential risk that cheating mutants gain access to the germline. Development from a single-celled zygote resets relatedness among cells to its maximum value each generation, which should accomplish segregation of cheating mutants from non-cheaters and thereby protect multicellular cooperation. Here we provide the crucial direct comparison between high- and low-relatedness conditions to test this hypothesis. We allow two variants of the fungus Neurospora crassa to evolve, one with and one without the ability to form chimeras with other individuals, thus generating two relatedness levels. While multicellular cooperation remains high in the high-relatedness lines, it significantly decreases in all replicate low-relatedness lines, resulting in an average threefold decrease in spore yield. This reduction is caused by cheating mutants with reduced investment in somatic functions, but increased competitive success when fusing with non-cheaters. Our experiments demonstrate that high genetic relatedness is crucial to sustain multicellular cooperation. PMID:27139112

  16. Experimental evolution reveals that high relatedness protects multicellular cooperation from cheaters

    PubMed Central

    Bastiaans, Eric; Debets, Alfons J. M.; Aanen, Duur K.

    2016-01-01

    In multicellular organisms, there is a potential risk that cheating mutants gain access to the germline. Development from a single-celled zygote resets relatedness among cells to its maximum value each generation, which should accomplish segregation of cheating mutants from non-cheaters and thereby protect multicellular cooperation. Here we provide the crucial direct comparison between high- and low-relatedness conditions to test this hypothesis. We allow two variants of the fungus Neurospora crassa to evolve, one with and one without the ability to form chimeras with other individuals, thus generating two relatedness levels. While multicellular cooperation remains high in the high-relatedness lines, it significantly decreases in all replicate low-relatedness lines, resulting in an average threefold decrease in spore yield. This reduction is caused by cheating mutants with reduced investment in somatic functions, but increased competitive success when fusing with non-cheaters. Our experiments demonstrate that high genetic relatedness is crucial to sustain multicellular cooperation. PMID:27139112

  17. A Novel Laboratory Activity for Teaching about the Evolution of Multicellularity

    ERIC Educational Resources Information Center

    Ratcliff, William C.; Raney, Allison; Westreich, Sam; Cotner, Sehoya

    2014-01-01

    The evolution of complexity remains one of the most challenging topics in biology to teach effectively. We present a novel laboratory activity, modeled on a recent experimental breakthrough, in which students experimentally evolve simple multicellularity using single-celled yeast ("Saccharomyces cerevisiae"). By simply selecting for…

  18. Transcription factor evolution in eukaryotes and the assembly of the regulatory toolkit in multicellular lineages.

    PubMed

    de Mendoza, Alex; Sebé-Pedrós, Arnau; Šestak, Martin Sebastijan; Matejcic, Marija; Torruella, Guifré; Domazet-Loso, Tomislav; Ruiz-Trillo, Iñaki

    2013-12-10

    Transcription factors (TFs) are the main players in transcriptional regulation in eukaryotes. However, it remains unclear what role TFs played in the origin of all of the different eukaryotic multicellular lineages. In this paper, we explore how the origin of TF repertoires shaped eukaryotic evolution and, in particular, their role into the emergence of multicellular lineages. We traced the origin and expansion of all known TFs through the eukaryotic tree of life, using the broadest possible taxon sampling and an updated phylogenetic background. Our results show that the most complex multicellular lineages (i.e., those with embryonic development, Metazoa and Embryophyta) have the most complex TF repertoires, and that these repertoires were assembled in a stepwise manner. We also show that a significant part of the metazoan and embryophyte TF toolkits evolved earlier, in their respective unicellular ancestors. To gain insights into the role of TFs in the development of both embryophytes and metazoans, we analyzed TF expression patterns throughout their ontogeny. The expression patterns observed in both groups recapitulate those of the whole transcriptome, but reveal some important differences. Our comparative genomics and expression data reshape our view on how TFs contributed to eukaryotic evolution and reveal the importance of TFs to the origins of multicellularity and embryonic development. PMID:24277850

  19. The evo-devo of multinucleate cells, tissues, and organisms, and an alternative route to multicellularity.

    PubMed

    Niklas, Karl J; Cobb, Edward D; Crawford, David R

    2013-01-01

    Multinucleate cells, tissues, or organisms occur in 60 families of land plants and in five otherwise diverse algal lineages (Rhodophyceae, Xanthophyceae, Chlorophyceae, Ulvophyceae, and Charophyceae). Inspection of a morphospace constructed out of eight developmental processes reveals a large number of possible variants of multinucleate cells and organisms that, with two exceptions, are represented by one or more plant species in one or more clades. Thus, most of these permutations of developmental processes exist in nature. Inspection of the morphospace also shows how the siphonous body plan (a multinucleate cell with the capacity for indeterminate growth in size) can theoretically serve as the direct progenitor of a multicellular organism by a process similar to segregative cell division observed in siphonocladean algae. Using molecular phylogenies of algal clades, different evolutionary scenarios are compared to see how the multicellular condition may have evolved from a multinucleate unicellular progenitor. We also show that the siphonous progenitor of a multicellular organism has previously passed through the alignment-of-fitness phase (in which genetic similarity among cells/nuclei minimizes internal genomic conflict) and the export-of-fitness phase (in which genetically similar cells/nuclei collaborate to achieve a reproductively integrated multicellular organism). All that is theoretically required is the evolutionary acquisition of the capacity to compartmentalize its cytoplasm. PMID:24261447

  20. Processing and characterization of multi-cellular monolithic bioceramics for bone regenerative scaffolds

    NASA Astrophysics Data System (ADS)

    Ari-Wahjoedi, Bambang; Ginta, Turnad Lenggo; Parman, Setyamartana; Abustaman, Mohd Zikri Ahmad

    2014-10-01

    Multicellular monolithic ceramic body is a ceramic material which has many gas or liquid passages partitioned by thin walls throughout the bulk material. There are many currently known advanced industrial applications of multicellular ceramics structures i.e. as supports for various catalysts, electrode support structure for solid oxide fuel cells, refractories, electric/electronic materials, aerospace vehicle re-entry heat shields and biomaterials for dental as well as orthopaedic implants by naming only a few. Multicellular ceramic bodies are usually made of ceramic phases such as mullite, cordierite, aluminum titanate or pure oxides such as silica, zirconia and alumina. What make alumina ceramics is excellent for the above functions are the intrinsic properties of alumina which are hard, wear resistant, excellent dielectric properties, resists strong acid and alkali attacks at elevated temperatures, good thermal conductivities, high strength and stiffness as well as biocompatible. In this work the processing technology leading to truly multicellular monolithic alumina ceramic bodies and their characterization are reported. Ceramic slip with 66 wt.% solid loading was found to be optimum as impregnant to the polyurethane foam template. Mullitic ceramic composite of alumina-sodium alumino disilicate-Leucite-like phases with bulk and true densities of 0.852 and 1.241 g cm-3 respectively, pore linear density of ±35 cm-1, linear and bulk volume shrinkages of 7-16% and 32 vol.% were obtained. The compressive strength and elastic modulus of the bioceramics are ≈0.5-1.0 and ≈20 MPa respectively.

  1. Bioelectrical Signals and Ion Channels in the Modeling of Multicellular Patterns and Cancer Biophysics.

    PubMed

    Cervera, Javier; Alcaraz, Antonio; Mafe, Salvador

    2016-02-04

    Bioelectrical signals and ion channels are central to spatial patterns in cell ensembles, a problem of fundamental interest in positional information and cancer processes. We propose a model for electrically connected cells based on simple biological concepts: i) the membrane potential of a single cell characterizes its electrical state; ii) the long-range electrical coupling of the multicellular ensemble is realized by a network of gap junction channels between neighboring cells; and iii) the spatial distribution of an external biochemical agent can modify the conductances of the ion channels in a cell membrane and the multicellular electrical state. We focus on electrical effects in small multicellular ensembles, ignoring slow diffusional processes. The spatio-temporal patterns obtained for the local map of cell electric potentials illustrate the normalization of regions with abnormal cell electrical states. The effects of intercellular coupling and blocking of specific channels on the electrical patterns are described. These patterns can regulate the electrically-induced redistribution of charged nanoparticles over small regions of a model tissue. The inclusion of bioelectrical signals provides new insights for the modeling of cancer biophysics because collective multicellular states show electrical coupling mechanisms that are not readily deduced from biochemical descriptions at the individual cell level.

  2. Bioelectrical Signals and Ion Channels in the Modeling of Multicellular Patterns and Cancer Biophysics

    PubMed Central

    Cervera, Javier; Alcaraz, Antonio; Mafe, Salvador

    2016-01-01

    Bioelectrical signals and ion channels are central to spatial patterns in cell ensembles, a problem of fundamental interest in positional information and cancer processes. We propose a model for electrically connected cells based on simple biological concepts: i) the membrane potential of a single cell characterizes its electrical state; ii) the long-range electrical coupling of the multicellular ensemble is realized by a network of gap junction channels between neighboring cells; and iii) the spatial distribution of an external biochemical agent can modify the conductances of the ion channels in a cell membrane and the multicellular electrical state. We focus on electrical effects in small multicellular ensembles, ignoring slow diffusional processes. The spatio-temporal patterns obtained for the local map of cell electric potentials illustrate the normalization of regions with abnormal cell electrical states. The effects of intercellular coupling and blocking of specific channels on the electrical patterns are described. These patterns can regulate the electrically-induced redistribution of charged nanoparticles over small regions of a model tissue. The inclusion of bioelectrical signals provides new insights for the modeling of cancer biophysics because collective multicellular states show electrical coupling mechanisms that are not readily deduced from biochemical descriptions at the individual cell level. PMID:26841954

  3. Sponge cell reaggregation: Cellular structure and morphogenetic potencies of multicellular aggregates.

    PubMed

    Lavrov, Andrey I; Kosevich, Igor A

    2016-02-01

    Sponges (phylum Porifera) are one of the most ancient extant multicellular animals and can provide valuable insights into origin and early evolution of Metazoa. High plasticity of cell differentiations and anatomical structure is characteristic feature of sponges. Present study deals with sponge cell reaggregation after dissociation as the most outstanding case of sponge plasticity. Dynamic of cell reaggregation and structure of multicellular aggregates of three demosponge species (Halichondria panicea (Pallas, 1766), Haliclona aquaeductus (Sсhmidt, 1862), and Halisarca dujardinii Johnston, 1842) were studied. Sponge tissue dissociation was performed mechanically. Resulting cell suspensions were cultured at 8-10°C for at least 5 days. Structure of multicellular aggregates was studied by light, transmission and scanning electron microscopy. Studied species share common stages of cell reaggregation-primary multicellular aggregates, early-stage primmorphs and primmorphs, but the rate of reaggregation varies considerably among species. Only cells of H. dujardinii are able to reconstruct functional and viable sponge after primmorphs formation. Sponge reconstruction in this species occurs due to active cell locomotion. Development of H. aquaeductus and H. panicea cells ceases at the stages of early primmorphs and primmorphs, respectively. Development of aggregates of these species is most likely arrested due to immobility of the majority of cells inside them. However, the inability of certain sponge species to reconstruct functional and viable individuals during cell reaggregation may be not a permanent species-specific characteristic, but depends on various factors, including the stage of the life cycle and experimental conditions. PMID:26863993

  4. The evo-devo of multinucleate cells, tissues, and organisms, and an alternative route to multicellularity.

    PubMed

    Niklas, Karl J; Cobb, Edward D; Crawford, David R

    2013-01-01

    Multinucleate cells, tissues, or organisms occur in 60 families of land plants and in five otherwise diverse algal lineages (Rhodophyceae, Xanthophyceae, Chlorophyceae, Ulvophyceae, and Charophyceae). Inspection of a morphospace constructed out of eight developmental processes reveals a large number of possible variants of multinucleate cells and organisms that, with two exceptions, are represented by one or more plant species in one or more clades. Thus, most of these permutations of developmental processes exist in nature. Inspection of the morphospace also shows how the siphonous body plan (a multinucleate cell with the capacity for indeterminate growth in size) can theoretically serve as the direct progenitor of a multicellular organism by a process similar to segregative cell division observed in siphonocladean algae. Using molecular phylogenies of algal clades, different evolutionary scenarios are compared to see how the multicellular condition may have evolved from a multinucleate unicellular progenitor. We also show that the siphonous progenitor of a multicellular organism has previously passed through the alignment-of-fitness phase (in which genetic similarity among cells/nuclei minimizes internal genomic conflict) and the export-of-fitness phase (in which genetically similar cells/nuclei collaborate to achieve a reproductively integrated multicellular organism). All that is theoretically required is the evolutionary acquisition of the capacity to compartmentalize its cytoplasm.

  5. Bioelectrical Signals and Ion Channels in the Modeling of Multicellular Patterns and Cancer Biophysics.

    PubMed

    Cervera, Javier; Alcaraz, Antonio; Mafe, Salvador

    2016-01-01

    Bioelectrical signals and ion channels are central to spatial patterns in cell ensembles, a problem of fundamental interest in positional information and cancer processes. We propose a model for electrically connected cells based on simple biological concepts: i) the membrane potential of a single cell characterizes its electrical state; ii) the long-range electrical coupling of the multicellular ensemble is realized by a network of gap junction channels between neighboring cells; and iii) the spatial distribution of an external biochemical agent can modify the conductances of the ion channels in a cell membrane and the multicellular electrical state. We focus on electrical effects in small multicellular ensembles, ignoring slow diffusional processes. The spatio-temporal patterns obtained for the local map of cell electric potentials illustrate the normalization of regions with abnormal cell electrical states. The effects of intercellular coupling and blocking of specific channels on the electrical patterns are described. These patterns can regulate the electrically-induced redistribution of charged nanoparticles over small regions of a model tissue. The inclusion of bioelectrical signals provides new insights for the modeling of cancer biophysics because collective multicellular states show electrical coupling mechanisms that are not readily deduced from biochemical descriptions at the individual cell level. PMID:26841954

  6. Sponge cell reaggregation: Cellular structure and morphogenetic potencies of multicellular aggregates.

    PubMed

    Lavrov, Andrey I; Kosevich, Igor A

    2016-02-01

    Sponges (phylum Porifera) are one of the most ancient extant multicellular animals and can provide valuable insights into origin and early evolution of Metazoa. High plasticity of cell differentiations and anatomical structure is characteristic feature of sponges. Present study deals with sponge cell reaggregation after dissociation as the most outstanding case of sponge plasticity. Dynamic of cell reaggregation and structure of multicellular aggregates of three demosponge species (Halichondria panicea (Pallas, 1766), Haliclona aquaeductus (Sсhmidt, 1862), and Halisarca dujardinii Johnston, 1842) were studied. Sponge tissue dissociation was performed mechanically. Resulting cell suspensions were cultured at 8-10°C for at least 5 days. Structure of multicellular aggregates was studied by light, transmission and scanning electron microscopy. Studied species share common stages of cell reaggregation-primary multicellular aggregates, early-stage primmorphs and primmorphs, but the rate of reaggregation varies considerably among species. Only cells of H. dujardinii are able to reconstruct functional and viable sponge after primmorphs formation. Sponge reconstruction in this species occurs due to active cell locomotion. Development of H. aquaeductus and H. panicea cells ceases at the stages of early primmorphs and primmorphs, respectively. Development of aggregates of these species is most likely arrested due to immobility of the majority of cells inside them. However, the inability of certain sponge species to reconstruct functional and viable individuals during cell reaggregation may be not a permanent species-specific characteristic, but depends on various factors, including the stage of the life cycle and experimental conditions.

  7. Scattering of a focused Laguerre-Gaussian beam by a spheroidal particle

    NASA Astrophysics Data System (ADS)

    Jiang, Yuesong; Shao, Yuwei; Qu, Xiaosheng; Ou, Jun; Hua, Houqiang

    2012-12-01

    A focused Laguerre-Gaussian beam scattered by a homogeneous prolate spheroidal particle is studied for on-axis incidence. An approach to expanding a focused Laguerre-Gaussian beam in terms of the spheroidal wavefunctions in spheroidal coordinates is presented. By using the localized approximations method, the beam-shape coefficients are evaluated and the results agree with the cases of on-axis incidence. Calculations of the far-field scattering intensity are performed to study the shaped beam scattered by a spheroid, which has different size parameters and eccentricities.

  8. Methods: Using Three-Dimensional Culture (Spheroids) as an In Vitro Model of Tumour Hypoxia.

    PubMed

    Leek, Russell; Grimes, David Robert; Harris, Adrian L; McIntyre, Alan

    2016-01-01

    Regions of hypoxia in tumours can be modelled in vitro in 2D cell cultures with a hypoxic chamber or incubator in which oxygen levels can be regulated. Although this system is useful in many respects, it disregards the additional physiological gradients of the hypoxic microenvironment, which result in reduced nutrients and more acidic pH. Another approach to hypoxia modelling is to use three-dimensional spheroid cultures. In spheroids, the physiological gradients of the hypoxic tumour microenvironment can be inexpensively modelled and explored. In addition, spheroids offer the advantage of more representative modelling of tumour therapy responses compared with 2D culture. Here, we review the use of spheroids in hypoxia tumour biology research and highlight the different methodologies for spheroid formation and how to obtain uniformity. We explore the challenge of spheroid analyses and how to determine the effect on the hypoxic versus normoxic components of spheroids. We discuss the use of high-throughput analyses in hypoxia screening of spheroids. Furthermore, we examine the use of mathematical modelling of spheroids to understand more fully the hypoxic tumour microenvironment. PMID:27325267

  9. Galaxy and Mass Assembly (GAMA): the stellar mass budget of galaxy spheroids and discs

    NASA Astrophysics Data System (ADS)

    Moffett, Amanda J.; Lange, Rebecca; Driver, Simon P.; Robotham, Aaron S. G.; Kelvin, Lee S.; Alpaslan, Mehmet; Andrews, Stephen K.; Bland-Hawthorn, Joss; Brough, Sarah; Cluver, Michelle E.; Colless, Matthew; Davies, Luke J. M.; Holwerda, Benne W.; Hopkins, Andrew M.; Kafle, Prajwal R.; Liske, Jochen; Meyer, Martin

    2016-11-01

    We build on a recent photometric decomposition analysis of 7506 Galaxy and Mass Assembly (GAMA) survey galaxies to derive stellar mass function fits to individual spheroid and disc component populations down to a lower mass limit of log(M*/M⊙) = 8. We find that the spheroid/disc mass distributions for individual galaxy morphological types are well described by single Schechter function forms. We derive estimates of the total stellar mass densities in spheroidsspheroid = 1.24 ± 0.49 × 108 M⊙ Mpc -3h0.7) and discs (ρdisc = 1.20 ± 0.45 × 108 M⊙ Mpc -3h0.7), which translates to approximately 50 per cent of the local stellar mass density in spheroids and 48 per cent in discs. The remaining stellar mass is found in the dwarf `little blue spheroid' class, which is not obviously similar in structure to either classical spheroid or disc populations. We also examine the variation of component mass ratios across galaxy mass and group halo mass regimes, finding the transition from spheroid to disc mass dominance occurs near galaxy stellar mass ˜1011 M⊙ and group halo mass ˜1012.5 M⊙h-1. We further quantify the variation in spheroid-to-total mass ratio with group halo mass for central and satellite populations as well as the radial variation of this ratio within groups.

  10. The GEA method for light scattering by dielectric spheroids and ellipsoids with fixed and random orientations

    NASA Astrophysics Data System (ADS)

    Yang, Lei Ming

    The GEA method is employed to study light scattering by dielectric spheroids and ellipsoids with either fixed or random orientations. A simple formula is obtained for a dielectric ellipsoid. The results from a dielectric spheroid with fixed or random orientations are compared numerically and found to agree well with the T-matrix method for small angle scattering. The numerical results for an ellipsoid are obtained. The validity region for the equal-volume sphere method for a dielectric spheroid and equal-volume spheroid method for a dielectric ellipsoid are also discussed.

  11. Establishment and Characterization of a Tumor Stem Cell-Based Glioblastoma Invasion Model

    PubMed Central

    Jensen, Stine Skov; Meyer, Morten; Halle, Bo; Rosager, Ann Mari; Aaberg-Jessen, Charlotte; Thomassen, Mads; Burton, Mark; Kruse, Torben A.; Kristensen, Bjarne Winther

    2016-01-01

    Aims Glioblastoma is the most frequent and malignant brain tumor. Recurrence is inevitable and most likely connected to tumor invasion and presence of therapy resistant stem-like tumor cells. The aim was therefore to establish and characterize a three-dimensional in vivo-like in vitro model taking invasion and tumor stemness into account. Methods Glioblastoma stem cell-like containing spheroid (GSS) cultures derived from three different patients were established and characterized. The spheroids were implanted in vitro into rat brain slice cultures grown in stem cell medium and in vivo into brains of immuno-compromised mice. Invasion was followed in the slice cultures by confocal time-lapse microscopy. Using immunohistochemistry, we compared tumor cell invasion as well as expression of proliferation and stem cell markers between the models. Results We observed a pronounced invasion into brain slice cultures both by confocal time-lapse microscopy and immunohistochemistry. This invasion closely resembled the invasion in vivo. The Ki-67 proliferation indexes in spheroids implanted into brain slices were lower than in free-floating spheroids. The expression of stem cell markers varied between free-floating spheroids, spheroids implanted into brain slices and tumors in vivo. Conclusion The established invasion model kept in stem cell medium closely mimics tumor cell invasion into the brain in vivo preserving also to some extent the expression of stem cell markers. The model is feasible and robust and we suggest the model as an in vivo-like model with a great potential in glioma studies and drug discovery. PMID:27454178

  12. Evaluation of The Combined Effects of Hyperthermia, Cobalt-60 Gamma Rays and IUdR on Cultured Glioblastoma Spheroid Cells and Dosimetry Using TLD-100

    PubMed Central

    Neshasteh-Riz, Ali; Rahdani, Rozhin; Mostaar, Ahmad

    2014-01-01

    Objective In radiation treatment, the irradiation which is effective enough to control the tumors far exceeds normal-tissues tolerance. Thus to avoid such unfavourable outcomes, some methods sensitizing the tumor cells to radiation are used. Iododeoxyuridine (IUdR) is a halogenated thymidine analogue that known to be effective as a radiosensitizer in human cancer therapy. Improving the potential efficacy of radiation therapy after combining to hyperthermia depends on the magnitude of the differential sensitization of the hyperthermic effects or on the differential cytotoxicity of the radiation effects on the tumor cells. In this study, we evaluated the combined effects of IUdR, hyperthermia and gamma rays of 60Co on human glioblastoma spheroids culture. Materials and Methods In this experimental study,the cultured spheroids with 100µm diameter were treated by 1 µM IUdR, 43°C hyperthermia for an hour and 2 Gy gamma rays, respectively. The DNA damages induced in cells were compared using alkaline comet assay method, and dosimetry was then performed by TLD-100. Comet scores were calculated as mean ± standard error of mean (SEM) using one-way ANOVA. Results Comparison of DNA damages induced by IUdR and hyperthermia + gamma treatment showed 2.67- and 1.92-fold enhancement, respectively, as compared to the damages induced by radiation alone or radiation combined IUdR. Dosimetry results showed the accurate dose delivered to cells. Conclusion Analysis of the comet tail moments of spheroids showed that the radiation treatments combined with hyperthermia and IUdR caused significant radiosensitization when compared to related results of irradiation alone or of irradiation with IUdR. These results suggest a potential clinical advantage of combining radiation with hyperthermia and indicate effectiveness of hyperthermia treatment in inducing cytotoxicity of tumor cells. PMID:24611138

  13. Enhanced angiogenic effect of adipose-derived stromal cell spheroid with low-level light therapy in hindlimb ischemia mice

    NASA Astrophysics Data System (ADS)

    Park, In-Su; Ahn, Jin-Chul; Chung, Phil-Sang

    2014-02-01

    Adipose-derived stromal cells (ASCs) are attractive cell source for tissue engineering. However, one obstacle to this approach is that the transplanted ASC population can decline rapidly in the recipient tissue. The aim of this study was to investigate the effects of low-level laser therapy (LLLT) on transplanted human ASCs (hASCs) spheroid in a hindlimb ischemia animal model. LLLT, hASCs spheroid and hASCs spheroid transplantation with LLLT (spheroid + LLLT) were applied to the ischemic hindlimbs in athymic mice. The survival, differentiation and secretion of vascular endothelial growth (VEGF) of spheroid ASCs were evaluated by immunohistochemistry. The spheroid + LLLT group enhanced the tissue regeneration, including angiogenesis, compared with other groups. The spheroid contributed tissue regeneration via differentiation and secretion of growth factors. In the spheroid + LLLT group, the survival of spheroid hASCs was increased by the decreased apoptosis of spheroid hASCs in the ischemic hindlimb. The secretion of growth factors was stimulated in the spheroid + LLLT group compared with the ASCs group and spheroid group. These data suggest that LLLT is an effective biostimulator of spheroid hASCs in tissue regeneration that enhances the survival of ASCs and stimulates the secretion of growth factors in the ischemic hindlimb.

  14. Direct Measurements of Oxygen Gradients in Spheroid Culture System Using Electron Parametric Resonance Oximetry

    PubMed Central

    Langan, Laura M.; Dodd, Nicholas J. F.; Owen, Stewart F.; Purcell, Wendy M.; Jackson, Simon K.; Jha, Awadhesh N.

    2016-01-01

    Advanced in vitro culture from tissues of different origin includes three-dimensional (3D) organoid micro structures that may mimic conditions in vivo. One example of simple 3D culture is spheroids; ball shaped structures typically used as liver and tumour models. Oxygen is critically important in physiological processes, but is difficult to quantify in 3D culture: and the question arises, how small does a spheroid have to be to have minimal micro-environment formation? This question is of particular importance in the growing field of 3D based models for toxicological assessment. Here, we describe a simple non-invasive approach modified for the quantitative measurement and subsequent evaluation of oxygen gradients in spheroids developed from a non-malignant fish cell line (i.e. RTG-2 cells) using Electron Paramagnetic Resonance (EPR) oximetry. Sonication of the paramagnetic probe Lithium phthalocyanine (LiPc) allows for incorporation of probe particulates into spheroid during its formation. Spectra signal strength after incorporation of probe into spheroid indicated that a volume of 20 μl of probe (stock solution: 0.10 mg/mL) is sufficient to provide a strong spectra across a range of spheroid sizes. The addition of non-toxic probes (that do not produce or consume oxygen) report on oxygen diffusion throughout the spheroid as a function of size. We provide evidence supporting the use of this model over a range of initial cell seeding densities and spheroid sizes with the production of oxygen distribution as a function of these parameters. In our spheroid model, lower cell seeding densities (∼2,500 cells/spheroid) and absolute size (118±32 μm) allow control of factors such as pre-existing stresses (e.g. ∼ 2% normoxic/hypoxic interface) for more accurate measurement of treatment response. The applied methodology provides an elegant, widely applicable approach to directly characterize spheroid (and other organoid) cultures in biomedical and toxicological

  15. Spheroid formation and enhanced cardiomyogenic potential of adipose-derived stem cells grown on chitosan.

    PubMed

    Liu, Bing-Hsien; Yeh, Hsi-Yi; Lin, Yu-Chun; Wang, Min-Hsiung; Chen, David C; Lee, Bo-Hua; Hsu, Shan-Hui

    2013-02-01

    Mesenchymal stem cells may differentiate into cardiomyocytes and participate in local tissue repair after heart injury. In the current study, rat adipose-derived adult stem cells (ASCs) grown on chitosan membranes were observed to form cell spheroids after 3 days. The cell seeding density and surface modification of chitosan with Arg-Gly-Asp-containing peptide had an influence on the sizes of ASC spheroids. In the absence of induction, these spheroids showed an increased level of cardiac marker gene expression (Gata4, Nkx2-5, Myh6, and Tnnt2) more than 20-fold versus cells on the tissue culture polystyrene (TCPS) dish. Induction by 5-azacytidine or p38 MAP kinase inhibitor (SB202190) did not further increase the cardiac marker gene expression of these spheroids. Moreover, the enhanced cardiomyogenic potential of the spheroids was highly associated with the chitosan substrates. When ASC spheroids were plated onto TCPS with either basal or cardiac induction medium for 9 days, the spheroids spread into a monolayer and the positive effect on cardiomyogenic marker gene expression disappeared. The possible role of calcium ion and the up-regulation of adhesion molecule P-selectin and chemokine receptor Cxcr4 were demonstrated in ASC spheroids. Applying these spheroids to the chronic myocardial infarction animal model showed better functional recovery versus single cells after 12 weeks. Taken together, this study suggested that the ASC spheroids on chitosan may form as a result of calcium ion signaling, and the transplantation of these spheroids may offer a simple method to enhance the efficiency of stem cell-based therapy in myocardial infarction. PMID:23514754

  16. Enhanced transfection of brain tumor suppressor genes by photochemical internalization

    NASA Astrophysics Data System (ADS)

    Chou, Chih H.; Sun, Chung-Ho; Zhou, Yi-Hong; Madsen, Steen J.; Hirschberg, Henry

    2011-03-01

    One of many limitations for cancer gene therapy is the inability of the therapeutic gene to transfect a sufficient number of tumor cells. Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. The utility of PCI for the delivery of a tumor suppressor gene (PAX-6) was investigated in monolayers and spheroids consisting of F98 rat glioma cells.

  17. A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay.

    PubMed

    Wen, Z; Liao, Q; Hu, Y; You, L; Zhou, L; Zhao, Y

    2013-07-01

    Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D) models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer.

  18. Leo I - The youngest Milky Way dwarf spheroidal galaxy?

    NASA Technical Reports Server (NTRS)

    Lee, Myung G.; Freedman, Wendy; Mateo, Mario; Thompson, Ian; Roth, Miguel; Ruiz, Maria-Teresa

    1993-01-01

    Deep CCD photometry of about 16,000 stars in the Milky Way's Leo I spheroidal galaxy satellite is reported. An account is given of the features observed in the color-magnitude diagrams (CMDs) derived therefrom. A very blue and well-defined red giant branch (RGB) is noted. The CMDs of Leo I shows about 50 anomalous Cepheid candidates; there are another 50 or so asymptotic giant branch stars above the tip of the RGB, including 15 known carbon stars. The mean color of the RGB is estimated at M sub I = -3.5 mag.

  19. Hyperbolic umbilic diffraction catastrophe and rainbow scattering from spheroidal drops

    NASA Technical Reports Server (NTRS)

    Marston, P. L.; Trinh, E. H.

    1984-01-01

    Short-wavelength scattering from drops whose shapes closely approximate that of an oblate spheroid with a vertical short or symmetry axis have been studied. The drops were illuminated by a horizontally propagating gaussian beam with a wavelength of 633 nm. The drops were observed to scatter in the horizontal rainbow region with patterns like those of hyperbolic-umblic (classification D4(+)) diffraction catastrophes. Visible D4(+) diffraction patterns observed previously include light transmitted by frosted glass surfaces and by liquid lenses clinging to tilted glass plates.

  20. VARIATIONS IN A UNIVERSAL DARK MATTER PROFILE FOR DWARF SPHEROIDALS

    SciTech Connect

    Jardel, John R.; Gebhardt, Karl

    2013-09-20

    Using a newly developed modeling technique, we present orbit-based dynamical models of the Carina, Draco, Fornax, Sculptor, and Sextans dwarf spheroidal (dSph) galaxies. These models calculate the dark matter profiles non-parametrically without requiring any assumptions to be made about their profile shapes. By lifting this restriction, we discover a host of dark matter profiles in the dSphs that are different from the typical profiles suggested by both theorists and observers. However, when we scale these profiles appropriately and plot them on a common axis, they appear to follow an approximate r {sup –1} power law with considerable scatter.

  1. Note: 3D printed spheroid for uniform magnetic field generation

    NASA Astrophysics Data System (ADS)

    Öztürk, Y.; Aktaş, B.

    2016-10-01

    This article is focused on a novel and practical production method for a uniform magnetic field generator. The method involves building of a surface coil template using a desktop 3D printer and winding of a conducting wire onto the structure using surface grooves as a guide. Groove pattern was based on the parametric spheroidal helical coil formula. The coil was driven by a current source and the magnetic field inside was measured using a Hall probe placed into the holes on the printed structure. The measurements are found to be in good agreement with our finite element analysis results and indicate a fairly uniform field inside.

  2. WFPC2 Observations of the URSA Minor Dwarf Spheroidal Galaxy

    NASA Technical Reports Server (NTRS)

    Mighell, Kenneth J.; Burke, Christopher J.

    1999-01-01

    We present our analysis of archival Hubble Space Telescope Wide Field Planetary Camera 2 (WFPC2) observations in F555W (approximately V) and F814W (approximately I) of the central region of the Ursa Minor dwarf spheroidal galaxy. The V versus V - I color-magnitude diagram features a sparsely populated blue horizontal branch, a steep thin red giant branch, and a narrow subgiant branch. The main sequence reaches approximately 2 magnitudes below the main-sequence turnoff (V(sup UMi, sub TO) approximately equals 23.27 +/- 0.11 mag) of the median stellar population. We compare the fiducial sequence of the Galactic globular cluster M92 (NGC 6341). The excellent match between Ursa Minor and M92 confirms that the median stellar population of the UMi dSph galaxy is metal poor ([Fe/H](sub UMi) approximately equals [Fe/H](sub M92) approximately equals -2.2 dex) and ancient (age(sub UMi)approximately equalsage(sub M92) approximately equals 14 Gyr). The B - V reddening and the absorption in V are estimated to be E(B - V) = 0.03 +/- 0.01 mag and A(sup UMi, sub V) = 0.09 +/- 0.03 mag. A new estimate of the distance modulus of Ursa Minor, (m - M)(sup UMi, sub 0) = 19.18 +/- 0.12 mag, has been derived based on fiducial-sequence fitting M92 [DELTA.V(sub UMi - M92) = 4.60 +/- 0.03 mag and DELTA(V - I)(sub UMi - M92) = 0.010 +/- 0.005 mag] and the adoption of the apparent V distance modulus for M92 of (m - M)(sup M92, sub V) = 14.67 +/- 0.08 mag (Pont et al. 1998, A&A, 329, 87). The Ursa Minor dwarf spheroidal galaxy is then at a distance of 69 +/- 4 kpc from the Sun. These HST observations indicate that Ursa Minor has had a very simple star formation history consisting mainly of a single major burst of star formation about 14 Gyr ago which lasted approximately < 2 Gyr. While we may have missed minor younger stellar populations due to the small field-of-view of the WFPC2 instrument, these observations clearly show that most of the stars in the central region Ursa Minor dwarf

  3. [Direct cell-cell communications and social behavior of cells in mammals, protists, and bacteria. Possible causes of multicellularity].

    PubMed

    Brodskiĭ, V Ia

    2009-01-01

    Comparison of current data on direct cell-cell communications in mammals, protists, and bacteria suggests that the emergence of the signaling systems of self-organization underlay the emergence of multicellular organisms. Biogenic amines, regulators of coordinated behavior and aggregation in bacteria, have been found in protists and multicellular organisms. In metazoans, biogenic amines have become specific neurotransmitters. At the same time, the studies on synchronization of protein synthesis rhythm in mammalian cell cultures demonstrated that noradrenaline and serotonin have conserved their ancient function of cell-cell cooperation in mammals, which is manifested as coordinated social behavior of cells in population in the case of bacteria and multicellular organisms.

  4. An integrative microfluidically supported in vitro model of an endothelial barrier combined with cortical spheroids simulates effects of neuroinflammation in neocortex development.

    PubMed

    Raasch, Martin; Rennert, Knut; Jahn, Tobias; Gärtner, Claudia; Schönfelder, Gilbert; Huber, Otmar; Seiler, Andrea E M; Mosig, Alexander S

    2016-07-01

    The development of therapeutic substances to treat diseases of the central nervous system is hampered by the tightness and selectivity of the blood-brain barrier. Moreover, testing of potential drugs is time-consuming and cost-intensive. Here, we established a new microfluidically supported, biochip-based model of the brain endothelial barrier in combination with brain cortical spheroids suitable to detect effects of neuroinflammation upon disruption of the endothelial layer in response to inflammatory signals. Unilateral perfusion of the endothelial cell layer with a cytokine mix comprising tumor necrosis factor, IL-1β, IFNγ, and lipopolysaccharide resulted in a loss of endothelial von Willebrand factor and VE-cadherin expression accompanied with an increased leakage of the endothelial layer and diminished endothelial cell viability. In addition, cytokine treatment caused a loss of neocortex differentiation markers Tbr1, Tbr2, and Pax6 in the cortical spheroids concomitant with reduced cell viability and spheroid integrity. From these observations, we conclude that our endothelial barrier/cortex model is suitable to specifically reflect cytokine-induced effects on barrier integrity and to uncover damage and impairment of cortical tissue development and viability. With all its limitations, the model represents a novel tool to study cross-communication between the brain endothelial barrier and underlying cortical tissue that can be utilized for toxicity and drug screening studies focusing on inflammation and neocortex formation. PMID:27478526

  5. Trigonometric potentials arising from the spheroidal equation: Supersymmetric partners and integral formulas

    NASA Astrophysics Data System (ADS)

    Schulze-Halberg, Axel

    2016-06-01

    We construct supersymmetric partners of a quantum system featuring a class of trigonometric potentials that emerge from the spheroidal equation. Examples of both standard and confluent supersymmetric transformations are presented. Furthermore, we use integral formulas arising from the confluent supersymmetric formalism to derive new representations for single and multiple integrals of spheroidal functions.

  6. The Clinical Significance and Molecular Features of the Spatial Tumor Shapes in Breast Cancers

    PubMed Central

    Jeong, Seongmun; Lee, Minju; Moon, HyunHye; Kim, Jongjin; Yoo, Tae-Kyung; Lee, Han-Byoel; Kim, Jisun; Noh, Dong-Young; Han, Wonshik

    2015-01-01

    Each breast cancer has its unique spatial shape, but the clinical importance and the underlying mechanism for the three-dimensional tumor shapes are mostly unknown. We collected the data on the three-dimensional tumor size and tumor volume data of invasive breast cancers from 2,250 patients who underwent surgery between Jan 2000 and Jul 2007. The degree of tumor eccentricity was estimated by using the difference between the spheroid tumor volume and ellipsoid tumor volume (spheroid-ellipsoid discrepancy, SED). In 41 patients, transcriptome and exome sequencing data obtained. Estimation of more accurate tumor burden by calculating ellipsoid tumor volumes did not improve the outcome prediction when compared to the traditional longest diameter measurement. However, the spatial tumor eccentricity, which was measured by SED, showed significant variation between the molecular subtypes of breast cancer. Additionally, the degree of tumor eccentricity was associated with well-known prognostic factors of breast cancer such as tumor size and lymph node metastasis. Transcriptome data from 41 patients showed significant association between MMP13 and spatial tumor shapes. Network analysis and analysis of TCGA gene expression data suggest that MMP13 is regulated by ERBB2 and S100A7A. The present study validates the usefulness of the current tumor size method in determining tumor stages. Furthermore, we show that the tumors with high eccentricity are more likely to have aggressive tumor characteristics. Genes involved in the extracellular matrix remodeling can be candidate regulators of the spatial tumor shapes in breast cancer. PMID:26669540

  7. Switchable water-adhesive, superhydrophobic palladium-layered silicon nanowires potentiate the angiogenic efficacy of human stem cell spheroids.

    PubMed

    Seo, Jungmok; Lee, Jung Seung; Lee, Kihong; Kim, Dayeong; Yang, Kisuk; Shin, Sera; Mahata, Chandreswar; Jung, Hwae Bong; Lee, Wooyoung; Cho, Seung-Woo; Lee, Taeyoon

    2014-11-01

    A switchable water-adhesive, super-hydrophobic nanowire surface is developed for the formation of functional stem cell spheroids. The sizes of hADSC spheroids are readily controllable on the surface. Our surface increases cell-cell and cell-matrix interaction, which improves viability and paracrine secretion of the spheroids. Accordingly, the hADSC spheroids produced on the surface exhibit significantly enhanced angiogenic efficacy.

  8. Multiplexing Spheroid Volume, Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres

    PubMed Central

    Ivanov, Delyan P.; Parker, Terry L.; Walker, David A.; Alexander, Cameron; Ashford, Marianne B.; Gellert, Paul R.; Garnett, Martin C.

    2014-01-01

    Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money. PMID:25119185

  9. Scattering of an oblique incident focused Gaussian beam by a spheroidal particle

    NASA Astrophysics Data System (ADS)

    Zhao, Wenjuan; Han, Yiping; Han, Lu

    2014-09-01

    Based on the expansion of a Gaussian beam in spheroidal coordinates in general case of oblique illumination, a theoretical procedure to determine the scattered fields of spheroid obliquely illuminated by a Gaussian beam is presented. Specific attention is paid to the study of scattering properties of a spheroidal particle from an obliquely incident Gaussian beam. The calculated results for spheroid are compared with those from the surface integral equation method, and very good agreements are observed. Numerical results concerning the influences of shaped beam parameters (beam waist radius, incident angle) as well as spheroid parameters (major axis, minor axis, refractive index, size parameter) on the scattering properties are presented. These results can be used as a reference for other numerical methods to analyze the light scattering by non-spherical particles illuminated by Gaussian beam.

  10. Development of controlled release spheroids using natural polysaccharide as release modifier.

    PubMed

    Kulkarni, Giriraj T; Gowthamarajan, K; Dhobe, Rohan R; Yohanan, Fenni; Suresh, B

    2005-01-01

    A polysaccharide hydrogel was isolated from the seeds of Tamarindus indica (tamarind) and was used as release modifier for the preparation of diclofenac sodium spheroids, using extrusion-spheronization technique. The process was studied for the effect of variables to arrive at spheroids with satisfactory particle shape, size and size-distribution. The prepared spheroids were characterized for surface morphology, qualitative surface porosity, friability, bulk density, and flow properties. The in vitro release studies exhibited a zero-order release kinetics that was confirmed by Higuchi's and Peppas' models. A credible correlation was obtained among swelling index, viscosity, surface roughness of the polysaccharide, and in vitro dissolution profile of the spheroids. In the comparative bioavailability study, we found that the developed spheroids were able to sustain the drug release over 8 hr and could improve the extent of absorption and bioavailability of the drug. PMID:16036714

  11. Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy.

    PubMed

    Fu, Qinyi; Martin, Benjamin L; Matus, David Q; Gao, Liang

    2016-01-01

    Despite the progress made in selective plane illumination microscopy, high-resolution 3D live imaging of multicellular specimens remains challenging. Tiling light-sheet selective plane illumination microscopy (TLS-SPIM) with real-time light-sheet optimization was developed to respond to the challenge. It improves the 3D imaging ability of SPIM in resolving complex structures and optimizes SPIM live imaging performance by using a real-time adjustable tiling light sheet and creating a flexible compromise between spatial and temporal resolution. We demonstrate the 3D live imaging ability of TLS-SPIM by imaging cellular and subcellular behaviours in live C. elegans and zebrafish embryos, and show how TLS-SPIM can facilitate cell biology research in multicellular specimens by studying left-right symmetry breaking behaviour of C. elegans embryos. PMID:27004937

  12. Bacterial Stigmergy: An Organising Principle of Multicellular Collective Behaviours of Bacteria

    PubMed Central

    Gloag, Erin S.; Whitchurch, Cynthia B.

    2015-01-01

    The self-organisation of collective behaviours often manifests as dramatic patterns of emergent large-scale order. This is true for relatively “simple” entities such as microbial communities and robot “swarms,” through to more complex self-organised systems such as those displayed by social insects, migrating herds, and many human activities. The principle of stigmergy describes those self-organised phenomena that emerge as a consequence of indirect communication between individuals of the group through the generation of persistent cues in the environment. Interestingly, despite numerous examples of multicellular behaviours of bacteria, the principle of stigmergy has yet to become an accepted theoretical framework that describes how bacterial collectives self-organise. Here we review some examples of multicellular bacterial behaviours in the context of stigmergy with the aim of bringing this powerful and elegant self-organisation principle to the attention of the microbial research community. PMID:25653882

  13. Evolution of oxygen utilization in multicellular organisms and implications for cell signalling in tissue engineering

    PubMed Central

    Stamati, Katerina; Mudera, Vivek

    2011-01-01

    Oxygen is one of the critically defining elements resulting in the existence of eukaryotic life on this planet. The rise and fall of this element can be tracked through time and corresponds with the evolution of diverse life forms, development of efficient energy production (oxidative phosphorylation) in single cell organisms, the evolution of multicellular organisms and the regulation of complex cell phenotypes. By understanding these events, we can plot the effect of oxygen on evolution and its direct influence on different forms of life today, from the whole organism to specific cells within multicellular organisms. In the emerging field of tissue engineering, understanding the role of different levels of oxygen for normal cell function as well as control of complex signalling cascades is paramount to effectively build 3D tissues in vitro and their subsequent survival when implanted. PMID:22292107

  14. Noise-plasticity correlations of gene expression in the multicellular organism Arabidopsis thaliana.

    PubMed

    Hirao, Koudai; Nagano, Atsushi J; Awazu, Akinori

    2015-12-21

    Gene expression levels exhibit stochastic variations among genetically identical organisms under the same environmental conditions (called gene expression "noise" or phenotype "fluctuation"). In yeast and Escherichia coli, positive correlations have been found between such gene expression noise and "plasticity" with environmental variations. To determine the universality of such correlations in both unicellular and multicellular organisms, we focused on the relationships between gene expression "noise" and "plasticity" in Arabidopsis thaliana, a multicellular model organism. In recent studies on yeast and E. coli, only some gene groups with specific properties of promoter architecture, average expression levels, and functions exhibited strong noise-plasticity correlations. However, we found strong noise-plasticity correlations for most gene groups in Arabidopsis; additionally, promoter architecture, functional essentiality of genes, and circadian rhythm appeared to have only a weak influence on the correlation strength. The differences in the characteristics of noise-plasticity correlations may result from three-dimensional chromosomal structures and/or circadian rhythm.

  15. Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy

    PubMed Central

    Fu, Qinyi; Martin, Benjamin L.; Matus, David Q.; Gao, Liang

    2016-01-01

    Despite the progress made in selective plane illumination microscopy, high-resolution 3D live imaging of multicellular specimens remains challenging. Tiling light-sheet selective plane illumination microscopy (TLS-SPIM) with real-time light-sheet optimization was developed to respond to the challenge. It improves the 3D imaging ability of SPIM in resolving complex structures and optimizes SPIM live imaging performance by using a real-time adjustable tiling light sheet and creating a flexible compromise between spatial and temporal resolution. We demonstrate the 3D live imaging ability of TLS-SPIM by imaging cellular and subcellular behaviours in live C. elegans and zebrafish embryos, and show how TLS-SPIM can facilitate cell biology research in multicellular specimens by studying left-right symmetry breaking behaviour of C. elegans embryos. PMID:27004937

  16. Noise-plasticity correlations of gene expression in the multicellular organism Arabidopsis thaliana.

    PubMed

    Hirao, Koudai; Nagano, Atsushi J; Awazu, Akinori

    2015-12-21

    Gene expression levels exhibit stochastic variations among genetically identical organisms under the same environmental conditions (called gene expression "noise" or phenotype "fluctuation"). In yeast and Escherichia coli, positive correlations have been found between such gene expression noise and "plasticity" with environmental variations. To determine the universality of such correlations in both unicellular and multicellular organisms, we focused on the relationships between gene expression "noise" and "plasticity" in Arabidopsis thaliana, a multicellular model organism. In recent studies on yeast and E. coli, only some gene groups with specific properties of promoter architecture, average expression levels, and functions exhibited strong noise-plasticity correlations. However, we found strong noise-plasticity correlations for most gene groups in Arabidopsis; additionally, promoter architecture, functional essentiality of genes, and circadian rhythm appeared to have only a weak influence on the correlation strength. The differences in the characteristics of noise-plasticity correlations may result from three-dimensional chromosomal structures and/or circadian rhythm. PMID:26431771

  17. The Dynamic Regulatory Genome of Capsaspora and the Origin of Animal Multicellularity.

    PubMed

    Sebé-Pedrós, Arnau; Ballaré, Cecilia; Parra-Acero, Helena; Chiva, Cristina; Tena, Juan J; Sabidó, Eduard; Gómez-Skarmeta, José Luis; Di Croce, Luciano; Ruiz-Trillo, Iñaki

    2016-05-19

    The unicellular ancestor of animals had a complex repertoire of genes linked to multicellular processes. This suggests that changes in the regulatory genome, rather than in gene innovation, were key to the origin of animals. Here, we carry out multiple functional genomic assays in Capsaspora owczarzaki, the unicellular relative of animals with the largest known gene repertoire for transcriptional regulation. We show that changing chromatin states, differential lincRNA expression, and dynamic cis-regulatory sites are associated with life cycle transitions in Capsaspora. Moreover, we demonstrate conservation of animal developmental transcription-factor networks and extensive network interconnection in this premetazoan organism. In contrast, however, Capsaspora lacks animal promoter types, and its regulatory sites are small, proximal, and lack signatures of animal enhancers. Overall, our results indicate that the emergence of animal multicellularity was linked to a major shift in genome cis-regulatory complexity, most notably the appearance of distal enhancer regulation. PMID:27114036

  18. Genomic analysis of organismal complexity in the multicellular green alga Volvox carteri.

    PubMed

    Prochnik, Simon E; Umen, James; Nedelcu, Aurora M; Hallmann, Armin; Miller, Stephen M; Nishii, Ichiro; Ferris, Patrick; Kuo, Alan; Mitros, Therese; Fritz-Laylin, Lillian K; Hellsten, Uffe; Chapman, Jarrod; Simakov, Oleg; Rensing, Stefan A; Terry, Astrid; Pangilinan, Jasmyn; Kapitonov, Vladimir; Jurka, Jerzy; Salamov, Asaf; Shapiro, Harris; Schmutz, Jeremy; Grimwood, Jane; Lindquist, Erika; Lucas, Susan; Grigoriev, Igor V; Schmitt, Rüdiger; Kirk, David; Rokhsar, Daniel S

    2010-07-01

    The multicellular green alga Volvox carteri and its morphologically diverse close relatives (the volvocine algae) are well suited for the investigation of the evolution of multicellularity and development. We sequenced the 138-mega-base pair genome of V. carteri and compared its approximately 14,500 predicted proteins to those of its unicellular relative Chlamydomonas reinhardtii. Despite fundamental differences in organismal complexity and life history, the two species have similar protein-coding potentials and few species-specific protein-coding gene predictions. Volvox is enriched in volvocine-algal-specific proteins, including those associated with an expanded and highly compartmentalized extracellular matrix. Our analysis shows that increases in organismal complexity can be associated with modifications of lineage-specific proteins rather than large-scale invention of protein-coding capacity. PMID:20616280