Spheroid Proliferation Research Articles

Brightfield and Fluorescence Imaging using 3D PrimeSurface® Ultra-Low Attachment Microplates

Abstract Culturing cells in three-dimensions (3D) has become a well established approach as it is more representative of the in vivo environment than traditional two-dimensional (2D) cultures. Allowing cells to interact with each other in a spheroid creates a micro-environment which mimics in vivo tissue and is a better model for examining the effect of drugs in cancer. Developing uniform spheroids becomes especially important as it forms the basis for robust and reliable assays. S-BIO PrimeSurface® cultureware are ultra low attachment (ULA) dishes and plates that promote scaffold free, self assembly spheroid formation. The plates are pre-coated with a proprietary hydrophilic polymer that enables spontaneous spheroid formation of uniform size. PrimeSurface 96 and 384 ULA plates have good optical clarity making them highly suitable for brightfield and fluorescent imaging. Imaging technologies such as the BioTek Cytation™ 5 allows researchers to study not only spheroid proliferation through brightfield imaging, but also phenotypic events such as hypoxia, apoptosis, or necrosis induction through the use of fluorescent probes and fluorescence imaging. Incorporation of z-stacking and projection techniques in the Gen5™ Microplate Reader and Imager Software create in-focus images of spheroidal cells, allowing accurate, robust, and repeatable determination of the effect of test molecules or conditions. In this app note, we present data generated with BioTek Cytation 5 using PrimeSurface ULA plates to develop simple robust spheroid assays for brightfield and fluorescence imaging.

Increased ETV4 expression correlates with estrogen-enhanced proliferation and invasiveness of cholangiocarcinoma cells

BackgroundCholangiocarcinoma (CCA) is one of the worst prognosis cancer. The survival time of CCA patients is related to serum estrogen levels and estrogen has been found to enhance the proliferation and invasiveness of CCA cells in vitro. This has led to the suggestion that estrogen may play an important role in the progression of CCA. This study tests the relevance of the previous in vitro findings in vivo using a mouse xenograft model of CCA, and investigates possible signaling mechanisms involved.MethodsKKU-213 and KKU-139 CCA cell lines were used in the experiments, xenografted to nude mice and treated with a potent estrogenic agent, 17β-estradiol (E2), and/or with tamoxifen (TAM), an estrogen antagonist.ResultsThe results demonstrated that E2 could accelerate growth of the xenograft-tumor and the effect was inhibited by TAM. PCR array screening of E2 responsive genes suggested ETV4 as a promising candidate intracellular mediator. ETV4-knockdown CCA cells were generated and these showed a diminished responsiveness to E2 in both cell and spheroid proliferation assays, and in invasion tests. These results point to ETV4 as a possible mediator of E2-activated CCA progression and as a potential target of TAM-mediated inhibition.ConclusionsFinally, TAM may be suggested as an adjunctive treatment of CCA to improve the conventional cytotoxic method with more patient toleration.

Self-Renewal and CSCs In Vitro Enrichment: Growth as Floating Spheres.

Cancer stem cells (CSC) are a vital component to the progression and reoccurrence of cancers, making them a primary target of study for both fundamental understanding of cancer biology and the development of effective and targeted treatments. CSCs reside in a complex 3D microenvironment, and the 3D spheroids are an indispensable tool in tumor biology due to their 3D structure and replication of the tumor microenvironment. Within this chapter the methodology for CSC isolation, suspension culture in hanging drop model, and characterization assays for CSC are described. First, the methodology for identifying and isolating CSCs from patient tumors, ascites, or cancer cell lines is described through the use of FACS analysis. Next, a detailed description of 3D hanging drop model for generating CSC spheroids is provided, followed by maintenance and monitoring techniques for extended 3D culture. Analysis methods are described for the quantification of CSC spheroid proliferation and viability tracking, throughout culture by on-plate alamarBlue fluorescence. Additional viability assays are described utilizing confocal microscopy with Live/Dead Viability/Cytotoxicity Kit. The characterization of CSCs populations within spheroids is described through FACS analysis. Further, an immunohistochemistry procedure is described for cell-cell and cell-matrix interaction assessment. Finally, several notes and tips for successful experiments with 3D CSC spheroids on the hanging drop model are provided. These methods are not only applicable to CSCs within a variety of tumor cell types, for not only understanding the fundamental tumor biology, but also for drug screening and development of preclinical chemotherapeutic strategies.

A potent indolylquinoline alleviates growth of human lung cancer cell tumorspheres.

To fight cancer at its roots by targeting cancer stem cells is a promising approach for therapy. Previously, an indolylquinoline derivative, 3-((7-ethyl-1H-indol-3-yl)-methyl)-2-methylquinoline (EMMQ), was reported effectively inhibiting the growth of lung cancer cells through impairment of cellular mitochondria functions. To address more on drug efficiency, the study further exploited if EMMQ can impede the propagation of tumorspheres stemmed from non-small cell lung cancer cells. EMMQ inhibited proliferation of spheroids in culture. In animal models, administration of the drug attenuated the spheroid tumorigenicity. The activated apoptosis alleviated growth of xenograft tumors in immune-deficient mice as established by the enriched tumorspheres. More evidence suggested that the reduced stemness of the spheroid tumors is attributed to apoptotic death. The findings supported that EMMQ is an eligible approach to eradicate the minor but tumorigenic lung cancer tumorspheres.

Abstract 3473: Functional analysis of the KIF22 in human prostate cancer cells

Abstract In the previous study, we identified genes that were differentially expressed between monolayers and spheroids in prostate cancer cell lines. The KIF22 gene was extracted as one of candidate genes related with spheroid formation and multicellular resistance. KIF22 is one of the kinesin superfamily proteins that are microtubule-dependent molecular motor proteins with DNA-binding capacity. KIF22 plays an important role in eukaryotic cell mitosis and macromolecule transportation. Alteration of KIF22 expression and function may lead to cancer development and progression. In this study, we explored the function of the KIF22 in prostate cancer cell lines, DU145 and LNCaP using the KIF22 siRNA. The KIF22 mRNA levels in LNCaP and DU-145 monolayers were detected, and time-dependently increased in both spheroids. The inhibition of KIF22 mRNA using siRNA affected cell proliferation in DU145 and LNCaP spheroids as well as monolayers. In addition, this suppression caused G2/M arrest and apoptosis in both cell lines. In clinical samples, its mRNA expression was significantly higher in tumor portions than in non-cancerous portions. However, the inhibition of KIF22 mRNA did not affect cell migration and invasion in DU145 and LNCaP cells. These findings suggest that KIF22 may play an important role in prostate cancer proliferation, especially spheroid formation, and have the potential to be targeted for prostate cancer treatments such as combination therapy with docetaxel. Citation Format: Rina Sakamaki, Shungo Saito, Tatsuya Kitano, Tadashi Nittami, Jieun Seo, Akimitsu Takagi, Hitoshi Ishiguro, Hiroji Uemura, Masatoshi Watanabe. Functional analysis of the KIF22 in human prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3473. doi:10.1158/1538-7445.AM2017-3473

Chemosensitization and inhibition of pancreatic cancer stem cell proliferation by overexpression of microRNA-205

Treatment of pancreatic cancer with gemcitabine (GEM) is limited due to its rapid plasma metabolism and development of chemoresistance. MicroRNA (miRNA) regulates cancer stem cell (CSC) maintenance and induces chemoresistance in cancer cells. In this study, we observed differential downregulation of miR-205 (miR-205-5p) in human pancreatic cancer tissues and cells. Compared to GEM-sensitive MIA PaCa-2 cells, miR-205 was highly downregulated in GEM-resistant MIA PaCa-2R cells. Lentivirus-mediated overexpression of miR-205 inhibits MIA PaCa-2R cell proliferation after GEM-treatment. Further investigation confirmed that miR-205 alone significantly reduces the proliferation of CSCs and tumor growth in mouse models. However, miR-205 in combination with GEM was more efficient in reducing the proliferation of CSCs and 3D spheroids. Moreover, miR-205 overexpressing MIA PaCa-2R cells induced orthotopic tumor growth was significantly inhibited after intravenous administration of GEM-conjugated methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate)-graft-gemcitabine-graft-dodecanol (mPEG-b-PCC-g-GEM-g-DC) (mPEG-b-PCC-g-GEM-g-DC) polymeric micelles. Also, a reduction in CSCs, EMT and chemoresistance markers was observed in miR-205 overexpressing MIA PaCa-2R cells. Immunohistochemical analysis of orthotopic tumors showed a decrease in drug resistance protein caveolin-1 and cell proliferation marker Ki-67 in combination treatment. Overall, our findings suggest that miR-205 resensitizes GEM-resistant pancreatic cancer cells to GEM and acts as a tumor suppressor miRNA.

Synergistically Controlled Stemness and Multilineage Differentiation Capacity of Stem Cells on Multifunctional Biointerfaces

Demonstrations of defined and controlled stem cell culture or differentiation advance fundamental life science research and the possibility of curing diseases. Here, the multifunctional concept of using defined matrix/interface modifications to support cell culture is demonstrated to provide synergistic coupling of concurrently immobilized fibroblast growth factor 2 and chitosan, and the resulting cell culture matrix/interface enables the proliferation of spheroids of adipose stem cells with enhanced stemness and the capacity to (trans‐)differentiate into multiple cell lineages in the mesoderm, endoderm, and ectoderm. The modification and the coimmobilization are performed using a straightforward one‐step vapor‐based coating technology applicable to a wide range of cell culture materials. The coating technology raises the important prospect of designing a material interface that incorporates effective and sustainable factors to determine stem cell fate through the use of a facile multicomponent modification approach on culture substrates.

Autophagy Induction Results in Enhanced Anoikis Resistance in Models of Peritoneal Disease.

Development of BNIP3/BNIP3L-targeting agents or autophagy-targeting agents may reduce morbidity and mortality associated with peritoneal carcinomatosis and sarcomatosis. Mol Cancer Res; 15(1); 26-34. ©2016 AACR.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Establishment and Characterization of an In Vitro Model of Ovarian Cancer Stem-like Cells with an Enhanced Proliferative Capacity.

The establishment of cancer stem-like cell (CSC) culture systems may be instrumental in devising strategies to fight refractory cancers. Inhibition of the Rho kinase ROCK has been shown to favorably affect CSC spheroid cultures. In this study, we show how ROCK inhibition in human serous ovarian cancer (SOC) cells can help establish a CSC system, which illuminates cancer pathophysiology and its treatment in this setting. In the presence of a ROCK kinase inhibitor, spheroid cultures of SOC cells expressed characteristic CSC markers including ALDH1A1, CD133, and SOX2, along with differentiation and tumorigenic capabilities in mouse xenograft models of human SOC. High expression levels of ALDH, but not CD133, correlated with spheroid formation CSC marker expression and tumor forming capability. In clinical specimens of SOC, high levels of ALDH1A1 correlated with advanced stage and poor prognosis. Pharmacologic or genetic blockade of ALDH blocked cell proliferation and reduced expression of SOX2, the genetic ablation of which abolished spheroid formation, whereas SOX2 overexpression inhibited ALDH1A1 expression and blocked spheroid proliferation. Taken together, our findings illustrated a new method to culture human ovarian CSC, and they defined a reciprocal regulatory relationship between ALDH1A1 and SOX2, which impacts ovarian CSC proliferation and malignant progression.

PO17A NOVEL IMMUNOHISTOCHEMICAL METHODOLOGY FOR THE INVESTIGATION OF STEM CELL-LIKE SUBPOPULATIONS IN GLIOMA SPHEROIDS AND MIGRATORY CELLS

INTRODUCTION: Targeting tumour invasion may represent a complementary treatment when added to standard care in highly infiltrating tumours such as glioblastomas. Invasiveness is believed to be related to stem cell-like qualities of distinct subpopulations of glioma cells. The characterisation of phenotypic differences between invading and non-invading cells will aid the development of specific anti-migratory drugs. A novel immunohistochemical approach was taken to phenotypically characterise invasive migratory glioma cells after drug treatment in a 3D spheroid invasion assay. METHOD: Spheroids generated from U87 and U251 cells were embedded in a collagen matrix and treated over 72 hours with the anti-migratory drugs LiCl and Bio-Indirubin. A protocol was established to prepare the collagen embedded spheroids and migratory cells for immunohistochemistry (IHC). Markers of proliferation, apoptosis and stemness were optimised for IHC. RESULTS: In both U87 and U251, protein expression profiles in cells maintained in the original core were distinct from profiles in migratory cells. The most striking change of expression was observed for SOX-2 in U251, where drug treatment led to a reduction of SOX-2 levels in core-maintained cells whereas migrating cells exhibited higher levels of SOX-2. We could also quantify drug-induced changes in proliferation and apoptotic events in spheroids generated from both cell lines using this method. CONCLUSION: A novel IHC protocol allowed the investigation of the invasive behaviour of cells in a 3D experimental model. Protein expression profiles of markers of stemness indicate that subpopulations of cells exist within glioma spheroids that may respond differently to anti-migratory drugs.

Effect of Heat-Inactivated Clostridium sporogenes and Its Conditioned Media on 3-Dimensional Colorectal Cancer Cell Models

Traditional cancer treatments, such as chemotherapy and radiation therapy continue to have limited efficacy due to tumor hypoxia. While bacterial cancer therapy has the potential to overcome this problem, it comes with the risk of toxicity and infection. To circumvent these issues, this paper investigates the anti-tumor effects of non-viable bacterial derivatives of Clostridium sporogenes. These non-viable derivatives are heat-inactivated C. sporogenes bacteria (IB) and the secreted bacterial proteins in culture media, known as conditioned media (CM). In this project, the effects of IB and CM on CT26 and HCT116 colorectal cancer cells were examined on a 2-Dimensional (2D) and 3-Dimensional (3D) platform. IB significantly inhibited cell proliferation of CT26 to 6.3% of the control in 72 hours for the 2D monolayer culture. In the 3D spheroid culture, cell proliferation of HCT116 spheroids notably dropped to 26.2%. Similarly the CM also remarkably reduced the cell-proliferation of the CT26 cells to 2.4% and 20% in the 2D and 3D models, respectively. Interestingly the effect of boiled conditioned media (BCM) on the cells in the 3D model was less inhibitory than that of CM. Thus, the inhibitive effect of inactivated C. sporogenes and its conditioned media on colorectal cancer cells is established.

Bioengineered Scaffolds for 3D Analysis of Glioblastoma Proliferation and Invasion.

The invasion of malignant glioblastoma (GBM) cells into healthy brain is a primary cause of tumor recurrence and associated morbidity. Here, we describe a high-throughput method for quantitative measurement of GBM proliferation and invasion in three-dimensional (3D) culture. Optically clear hydrogels composed of thiolated hyaluronic acid and gelatin were chemically crosslinked with thiol-reactive poly(ethylene glycol) polymers to form an artificial 3D tumor microenvironment. Characterization of the viscoelasticity and aqueous stability indicated the hydrogels were mechanically tunable with stiffness ranging from 18 Pa to 18.2 kPa and were resistant to hydrolysis for at least 30 days. The proliferation, dissemination and subsequent invasion of U118 and U87R GBM spheroids cultured on the hydrogels were tracked in situ with repeated fluorescence confocal microscopy. Using custom automated image processing, cells were identified and quantified through 500 µm of gel over 14 days. Proliferative and invasive behaviors were observed to be contingent on cell type, gel stiffness, and hepatocyte growth factor availability. These measurements highlight the utility of this platform for performing quantitative, fluorescence imaging analysis of the behavior of malignant cells within an artificial, 3D tumor microenvironment.

Compressive Stress Inhibits Proliferation in Tumor Spheroids through a Volume Limitation

In most instances, the growth of solid tumors occurs in constrained environments and requires a competition for space. A mechanical crosstalk can arise from this competition. In this article, we dissect the biomechanical sequence caused by a controlled compressive stress on multicellular spheroids (MCSs) used as a tumor model system. On timescales of minutes, we show that a compressive stress causes a reduction of the MCS volume, linked to a reduction of the cell volume in the core of the MCS. On timescales of hours, we observe a reversible induction of the proliferation inhibitor, p27Kip1, from the center to the periphery of the spheroid. On timescales of days, we observe that cells are blocked in the cell cycle at the late G1 checkpoint, the restriction point. We show that the effect of pressure on the proliferation can be antagonized by silencing p27Kip1. Finally, we quantify a clear correlation between the pressure-induced volume change and the growth rate of the spheroid. The compression-induced proliferation arrest that we studied is conserved for five cell lines, and is completely reversible. It demonstrates a generic crosstalk between mechanical stresses and the key players of cell cycle regulation. Our results suggest a role of volume change in the sensitivity to pressure, and that p27Kip1 is strongly influenced by this change.

7. The grenz zone in dermatofibroma - An inconsistent truth. A histopathological study of 86 cases

<h3>Background</h3> Dermatofibroma is a benign dermal fibrohistocytic lesion with a number of typical diagnostic features, including the presence of an uninvolved zone of the papillary dermis, referred to as a grenz zone. We identified a number of cases in consultation practice in which the absence of a grenz zone led the pathologist to question the diagnosis of dermatofibroma in otherwise typical cases. <h3>Aim</h3> To examine the frequency of the presence of a grenz zone in dermatofibroma and to explore the extent to which the presence of this feature can be explained by ‘off-centre' sectioning of a spheroid proliferation. <h3>Methods</h3> We performed a retrospective study of 86 routine cases of dermatofibroma from the files of PathWest Laboratory Medicine at Sir Charles Gairdner Hospital. Haematoxylin and eosin stained sections for each case were concurrently reviewed by two derma-topathologists to confirm the diagnosis of dermatofibroma and to assess for the presence or absence of a grenz zone. <h3>Results</h3> Between 1 and 4 sections were examined for each case of dermatofibroma. A grenz zone was identified in 67% of cases. However, only 49% of cases of dermatofibroma showed a grenz zone in all examined sections. <h3>Conclusions</h3> While a grenz zone is frequently seen in dermatofibroma, its presence is not essential for the diagnosis. Furthermore, in approximately 50% of cases at least one section can be identified in which this feature is absent. We suggest that the identification of a grenz zone in many cases is in fact an artefact of ‘off-centre' sectioning of a spheroid lesion apposed to the horizontal under-surface of the epidermis and that the presence or absence of a grenz zone may not be of significant discriminatory value in the diagnosis of dermatofibroma.

Abstract A73: A bioengineered ovarian cancer microenvironment to assess protease-associated functions

Abstract Cancer-associated proteases promote peritoneal dissemination and chemoresistance in malignant progression. In this study, kallikrein-related peptidases 4, 5, 6, and 7 (KLK4-7)-cotransfected ovarian cancer cells were embedded in a bioengineered three-dimensional (3D) microenvironment that contains RGD motifs for integrin engagement to analyse their spheroid growth and survival after chemotreatment. KLK4-7-cotransfected cells formed larger spheroids and proliferated more than controls in 3D, particularly within RGD-functionalized matrices, which was reduced upon integrin inhibition. In contrast, KLK4-7-expressing cell monolayers proliferated less than controls, emphasising the relevance of the 3D microenvironment and integrin engagement. In a spheroid-based animal model, KLK4-7-overexpression induced tumor growth after 4 weeks and intraperitoneal spread after 8 weeks. Upon paclitaxel administration, KLK4-7-expressing tumors declined in size and showed less metastatic outgrowth. KLK4-7-expressing spheroids showed 53% survival upon paclitaxel treatment, accompanied by enhanced chemoresistance-related factors; their survival was further reduced by combination treatment of paclitaxel with KLK (22%) or MAPK (6%) inhibition. The concomitant presence of KLK4-7 in ovarian cancer cells together with integrin activation drives spheroid formation and proliferation. Combinatorial approaches of paclitaxel and KLK/MAPK inhibition may be more efficient for late-stage disease than chemotherapeutics alone as these inhibitory regimens reduced cancer spheroid growth to a greater extent than paclitaxel alone. Citation Format: Daniela Loessner, Simone C. Rizzi, Kathryn S. Stok, Viktor Magdolen, Dietmar W. Hutmacher, Judith A. Clements. A bioengineered ovarian cancer microenvironment to assess protease-associated functions. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A73.

Abstract 5062: The role of fibroblast MMP2 in breast to lung metastasis.

Abstract Breast cancer is the second most common fatal malignancy of women in the United States. Matrix metalloproteinases (MMPs) have been implicated in virtually all stages of breast cancer. MMP2 was identified as part of a subset of genes upregulated in breast to lung metastases (Minn). These studies focused on epithelial derived MMP2, however, a host of other sources have emphasized the importance of stromal cell derived MMP2 in breast cancer growth. We have directed our studies to investigate the role of MMP2 from stromal cells in tumor outgrowth at the secondary site. We hypothesize that stromal MMP2 at the secondary site contributes to metastatic progression. Spontaneous metastasis models have shown that MMP2−/− mice demonstrate reduced lung metastatic lesions compared to wildtype (WT) animals. In an experimental metastasis model using luciferase labeled tumor cells, we have found that MMP2−/− mice have a significant reduction in lung tumor outgrowth compared to WT animals as measured by bioluminescent imaging. The reduction in growth only manifests approximately seven days post inoculation, suggesting that decreased tumor growth is due to defects in tumor outgrowth and not seeding ability or survival. Immunohistochemical staining of Ki67 revealed that MMP2 selectively facilitates the proliferation of tumor cells in the lung. MMP2 in our model is primarily localized to stromal cells surrounding lung tumor nodules. We determined by dual labeling for MMP2 and vimentin or αSMA positive areas that fibroblasts were the major cellular source of MMP2. Hematopoietic cells (CD45 positive areas) and endothelial cells (CD31 positive areas) showed minimal expression of MMP2 and were ruled out as a significant source. To determine if there is a direct role of MMP2 in tumor cell proliferation, we took an in vitro approach and analyzed tumor cell proliferation after treatment with recombinant protein or fibroblast conditioned media. There was no difference in two-dimensional (2D) proliferation after either treatment, suggesting that MMP2 plays an indirect role in the proliferation of tumor cells. In our present studies, we investigate the role of MMP2 in fibroblasts and the effect this has on the outgrowth of tumor cells in the lung. A knock down approach was used to assess the impact of MMP2 on 2D fibroblast proliferation by cell counting. Our results demonstrated no difference in 2D proliferation between control and MMP2 knockdown fibroblasts. We have developed a 3D co-culture system to evaluate the role of MMP2 in fibroblast promotion of tumor spheroid proliferation. Our present data suggests that MMP2 mediated recruitment and functionality of tumor associated fibroblasts promotes tumor outgrowth via proliferation. Results from these experiments will lead to new insights on the role of MMP2 in fibroblasts and how these cells then enable a permissive environment for tumor outgrowth at secondary sites. Minn, AJ et al. Nature. 2005 Jul 28;436(7050):518-24 Citation Format: Andreia Bates, Barbara Fingleton. The role of fibroblast MMP2 in breast to lung metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5062. doi:10.1158/1538-7445.AM2013-5062

Isotropic stress reduces cell proliferation in tumor spheroids

In most instances, tumors have to push their surroundings in order to grow. Thus, during their development, tumors must be able to both exert and sustain mechanical stresses. Using a novel experimental procedure, we study quantitatively the effect of an applied mechanical stress on the long-term growth of a spherical cell aggregate. Our results indicate the possibility to modulate tumor growth depending on the applied pressure. Moreover, we demonstrate quantitatively that the cells located in the core of the spheroid display a different response to stress than those in the periphery. We compare the results to a simple numerical model developed for describing the role of mechanics in cancer progression.

Abstract 5270: Evaluation of Cell-able spheroid culture system for culturing patient derived primary tumor cells

Abstract Tumor cell microenvironment has significant impact on growth kinetics, cell signaling and response to drug treatments. 3D models are more biologically relevant models compared to 2D models and have gained preference among researchers and drug developers. It is well recognized that primary tumor cell cultures grown in 2D monolayer quickly lose the ability to proliferate while 3D culture conditions on extracellular matrix allows for spheroid formation and proliferation. Cell-able, a novel micropatterned plate has cell adhesion areas in the well bottom where the size and shape of these areas are tightly controlled. Micro-fabrication of the well bottom enables rapid cell attachment and spheroid formation. Cell-able plates are potentially an alternative to culturing cells in 3D without extracellular matrix. We have evaluated spheroid formation, morphology, duration of viability, histopathology and expression of markers in patient-derived primary tumor cells from different tumor types when cultured on Cell-able plates compared to Cultrex extracellular matrix (ECM) coated plates. In addition we have utilized Cell-able plates to grow patient-derived tumor cells and evaluate the anti-proliferative responses to cytotoxic and targeted agents using high content imaging coupled with immunofluorescence characterization enabling subpopulation analysis within the spheroids. Our results indicate that patient-derived tumor cells from multiple indications as well as tumor cell lines can be cultured on Cell-able plates as spheroids that resemble the micro architecture of tumors and are therefore a suitable 3D model. The results of high content imaging analysis suggest that Cell-able plates could be used as a valuable tool in in vitro predictive assay models in oncology drug development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5270. doi:1538-7445.AM2012-5270