Establishment and characterization of an in vitro 3D ovarian cancer model for drug screening assays.

ObjectiveThree-dimensional (3D) culture systems offer a more physiologically relevant environment than conventional two-dimensional (2D) cultures, particularly for studying tumor biology. Here, we systematically compared two widely used 3D platforms—poly(2-hydroxyethyl methacrylate) (Poly-HEMA, PH)-coating and ultra-low attachment (ULA) plates—to evaluate their impact on pancreatic cancer (PCa) cell behavior. We assessed spheroid morphology, chemotherapeutic response, invasion potential, and adhesion molecule expression in two PCa cell lines (PANC-1 and SU.86.86).ResultsSpheroid morphology differed markedly between PH and ULA platforms, with ULA generally promoting larger and more cohesive spheroids. Gemcitabine resistance was the highest in SU.86.86 spheroids on ULA plates. Matrigel invasion assays revealed enhanced single-cell migration in SU.86.86 spheroids grown on PH, whereas ULA spheroids exhibited broader matrix degradation and collective invasion. Moreover, gene and protein expression levels of key adhesion molecules, including E-Cadherin, N-Cadherin, and integrins, varied between platforms in both cells. These findings demonstrate that 3D culture systems distinctly influence PCa cell characteristics and highlight the necessity of selecting appropriate culture environment for studying tumor biology and drug response. Further mechanistic studies are warranted to uncover how different 3D environments shape tumor cell behavior and therapy resistance.

Objective: Cancer, characterized by uncontrolled cell proliferation and invasion into surrounding tissues, is a leading cause of global mortality. Traditional two-dimensional (2D) cell culture systems fail to adequately replicate the tumor microenvironment (TME). In contrast, three-dimensional (3D) culture systems, which better simulate cell–cell and cell–extracellular matrix (ECM) interactions, have become powerful tools in biomedical research. This study aims to compare the spheroid formation capacity of A549 lung cancer cells using three different 3D culture methods: ultra-low attachment (ULA) plates, agarose hydrogel, and the hanging drop technique. The primary objective is to identify the most effective spheroid formation method for A549 cells and to provide findings that can guide future biomedical research, particularly in cancer modeling, drug screening studies, and investigations of the tumor microenvironment.Materials and Methods: A549 cells were cultured using three different 3D culture methods: ultra-low attachment plates, agarose hydrogel, and the hanging drop method. In the ultra-low attachment method, spheroid formation was observed at cell densities of 5,000, 10,000, and 30,000 cells/ml. In the agarose hydrogel method, agarose concentrations of 1%, 1.5%, and 2% were used to evaluate cell aggregation and spheroid stability. In the hanging drop method, cells aggregated under the influence of gravity. Spheroid diameter and area were analyzed using ImageJ software.Results: In this study, the spheroid formation capacity of A549 lung cancer cells was evaluated using three different three-dimensional (3D) culture methods. The ultra-low attachment (ULA) plate method allowed cell aggregation; however, the resulting structures were not large or compact enough to be classified as spheroids. The hanging drop method showed that cells formed small clusters by day 3 but failed to develop a compact and stable spheroid structure by day 7. The agarose hydrogel method, particularly at a 2% agarose concentration, demonstrated the highest spheroid formation capacity compared to the other methods. In this method, spheroid formation began at 72 hours depending on cell density, with significant growth observed at a density of 30,000 cells/ml (p < 0.0001). Trypan Blue staining results indicated that 2% agarose and cell densities of 10,000–30,000 cells/ml provided the highest cell viability. Specifically, 4,800 viable cells were counted at a density of 30,000 cells/ml, while 3,600 viable cells were observed at 10,000 cells/ml. These findings suggest that the agarose hydrogel method, especially at 2% agarose concentration and higher cell densities, offers optimal spheroid formation and cell viability for A549 lung cancer cells.Conclusion: This study demonstrated that the agarose hydrogel method effectively promoted stable and organized spheroid formation in A549 lung cancer cells. Notably, the 2% agarose concentration was identified as the most effective condition for maintaining cell viability and optimizing spheroid size. In contrast, the ultra-low attachment (ULA) plate and hanging drop methods exhibited limited spheroid formation capacity, resulting in less compact and disorganized structures. These findings emphasize the critical role of three-dimensional (3D) cell culture methods in biomedical research, particularly for experimental tumor modeling and drug screening studies. In this context, the agarose hydrogel method, with its high spheroid formation capacity and ability to support cell viability, emerges as a promising 3D culture model that warrants further exploration in cancer research.

Conventional in vitro assays are based on cells grown on two-dimensional (2D) substrates, which are not representative of the true in vivo cell environment. In tissue environments, cells interact with neighboring cells and the extracellular matrix (ECM). Three-dimensional (3D) cell culture methods allow cells to grow in structures more resembling the in vivo environment. Cells can develop cell-cell and cell-ECM interactions in 3D. The RAFT™ 3D Culture System uses a collagen matrix at physiologically relevant concentrations. Cells and neutralized collagen are mixed and subsequently incubated at 37°C to allow the formation of a cell-seeded hydrogel. Specialized RAFT™ Absorbers are placed on top of the hydrogels. The RAFT™ Absorbers gently remove abundant medium, thus compacting the cell/collagen hydrogel. Additional epithelial or endothelial cells may be added as overlays on top to study co-cultures or more complex cultures. Another commonly used 3D cell culture model is spheroids. Spheroids are compact aggregates of cells that are generated without the addition of exogenous ECM – e.g. in so-called ultra-low attachment (ULA) plates or by the hanging-drop method. While 3D cultures more accurately mimic the in vivo cell environment, it might be difficult to analyze cells in 3D due to the dense, tissue-like structure of these cultures compared to 2D cell monolayers. This presentation explains how to measure cell viability in both RAFT™ 3D cell cultures and in spheroid cultures with the ViaLight™ Plus Cell Proliferation and Cytotoxicity BioAssay. The ViaLight™ Assay is based on the bioluminescent detection of cellular ATP as a measure of cell viability. Only minor modifications of the standard two-step protocol that is used for 2D cell cultures are required for using the assay for 96-well and 24-well RAFT™ 3D cell cultures as well as for spheroid cultures in 96-well ULA plates. Two different cell types were used in this study: Normal Human Dermal Fibroblasts (NHDFneo) and the colon cancer cell line HCT-116. Both cell types efficiently form spheroids in ULA plates. Also in RAFT™ 3D cultures HCT-116 cells build compact aggregate-like structures, whereas NHDFneo grow in as individual cells interspersed in the collagen scaffold. By elongating the first step of the Vialight™ Assay, the lysis step, from 10 minutes to 30 minutes, a linear performance of the assay for cell numbers of up to 96,000 cells in the 96-well format and 480,000 cells in the 24-well format could be obtained for RAFT™ cultures. For spheroid cultures an extension of the lysis time to 60 minutes was required to obtain efficient lysis of spheroids with a diameter of up to 400μm, as confirmed by CalceinAM and propidium iodide staining. In summary this presentation shows that analyzing cells in 3D cultures can easily and routinely be done by slightly adjusting standard 2D cell culture assays like the ViaLight™ Assay. Citation Format: Stefanie Buesch, John Langer, Sabine Schaepermeier, Lubna Hussain, Jeffrey Bergeron, Volker Vogel, Jenny Schroeder. Analyzing cell viability in 3-D tissue models with the ViaLight™ plus cell proliferation and cytotoxicity bioassay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 607.

Scaffold-free 3D culturing enhance pluripotency, immunomodulatory factors, and differentiation potential of Wharton’s jelly-mesenchymal stem cells

Ovarian cancer remains the deadliest of gynecologic malignancies. A major challenge in ovarian cancer treatment is that it is often diagnosed at stages when the cancer has already formed metastases throughout the peritoneum. While chemotherapy shows efficacy on the original tumor, the metastatic legions are often resistant to traditional chemotherapy regimens. Thus, the development of new therapy that targets metastatic ovarian cancer is necessary. Three-dimensional culture systems of ovarian spheroids are used to model the growth of ovarian cancer at these later stages. Thus, to target advanced ovarian cancer, we focused on identifying drugs that specifically target ovarian spheroids in the laboratory, hypothesizing that these drugs would target the metastatic legions in ovarian cancer patients. To do this, we identified genes that were modulated in ovarian cancer cell lines when grown in 3D culture, developing a 3D gene signature. Using this gene signature, we queried the Connectivity Map from the Broad Institute for drugs that induced the opposite gene expression patterns as the 3D signature we identified, hypothesizing that these drugs could potentially target signaling pathways activated in ovarian spheroids. From this approach, securinine was identified as a molecule that inhibits the 3D growth of ovarian cancer cells. To understand the mechanism of action of securinine, we analyzed genes modulated by securinine treatment. Pathway analysis identified Signal Transducer and Activator of Transcription 3 (STAT3) as being a transcription factor that is inhibited by securinine. STAT3 is a latent transcription factor that is activated upon tyrosine phosphorylation. Once activated, STAT3 regulates the expression of many genes involved in survival, growth, and metastasis. STAT3 has been found to be active in ovarian cancer, and we have demonstrated that the STAT3 signaling pathway is activated and necessary in ovarian cancer spheroids. While we have found that securinine does not inhibit STAT3 tyrosine phosphorylation, securinine inhibits the expression of a STAT3 responsive reporter and STAT3 target genes, verifying that securinine inhibits STAT3 activity. Computational analysis has found that the STAT3 gene signature is enhanced in cisplatin-resistant ovarian cancer spheroids and that genes inhibited by securinine are upregulated in these cells. This raises the possibility that targeting STAT3 with securinine will be beneficial in the treatment of cisplatin-resistant ovarian cancer. Thus, using gene expression signatures of 3D growth has identified securinine as an inhibitor that targets signaling pathways activated in ovarian cancer spheroids, identifying a potential drug for the treatment of advanced-stage ovarian cancer. Citation Format: Sarah R. Walker, Zachary T. Giaccone, David A. Frank. Gene expression network--based identification of drugs targeting advanced ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B01.

Establishing an Easy-to-Use 3D BM Niche Model for the Co-Culture of Primary Bone Marrow Mesenchymal Stromal Cells (BM-MSC) with Hematopoietic Stem and Progenitor Cells (HSPC)

Ovarian cancer is a complex disease with various subtypes and diverse molecular mechanisms of pathogenesis. Chemoresistance is a common challenge in ovarian tumors, where inherent resistance or adaptive changes in the tumor microenvironment may antagonize the effects of chemotherapy through various mechanisms such as promoting metastasis or inhibition of immune cell activation. Lipid nanoparticle (LNP) mediated co-administration of RNA with chemotherapy drugs presents an opportunity to modify protein expression within the tumor microenvironment and enhance chemosensitivity. siRNA-mediated knockdown of chemo resistance genes or intratumoral administration of mRNAs encoding immunostimulatory proteins are two such examples. LNP efficacy is contingent upon effective uptake into the target tissue and subsequent cargo release from endosomes. These two processes vary significantly depending on the nature of the cargo, LNP lipid composition and target tissue; the ability to screen different lipid formulations and cargo types against tumors in vitro may be a powerful tool in development of new therapies. In vitro cell-based screening methods are valuable tools for assessing cytotoxicity of candidate drugs, however the tumor microenvironment differs starkly from conventional two-dimensional culture. Heterogeneity of substrate availability, oxygen tension and pH are all defining characteristics of tumors and these factors may also contribute to altered responses to lipid nanoparticle formulations. In this study, we compared LNP-mediated cargo uptake & expression in three different ovarian cancer cell lines cultured in 2D monolayers or 3D spheroids to investigate whether efficacy in 2D culture is recapitulated in the spheroid model. SK-OV-3, PA-1 & CaOV-3 cells were grown in monolayers and 3D spheroids and transfected with four different LNP formulations containing the ionizable lipids SM-102, ALC-0315, C12-200 or DLin-MC3-DMA. Empty pre-formed loadable LNPs were loaded with mCherry mRNA, GFP DNA or Cy5-labelled 2’3’cGAMP and expression/uptake in each model was determined via fluorescence imaging. BODIPY-cholesterol labelled versions of each LNP exhibited similar dose-dependent uptake into monolayers & spheroids independent of ionizable lipid formulation. In contrast cargo expression varied substantially across conditions in cell, cargo and culture model-dependent manners. SM-102 exhibited superior efficacy at mRNA expression in monolayer cultures across all three cell types, while C12-200 generally led to the highest DNA expression in monolayer cultures. Notably the optimal LNP formulation for cargo expression in spheroids frequently differed from 2d culture: SK-OV-3 spheroids exhibited optimal mCherry mRNA expression with C12-200 while GFP DNA expression was highest with SM-102. These results highlight the importance of 3D models for lipid nanoparticle formulation testing and demonstrate the utility of preformed loadable LNP screens in early formulation development. Citation Format: David Taylor, Valerie Forsyth, Caleb Riddering, Aileen R. Lipid nanoparticle screening in 2D vs 3D cultures: Ovarian cancer cell lines exhibit different preference for optimal lipid formulation in monolayers vs spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB185.

Many antibiotics produced by bacteria also have antineoplastic activity. Examples of such bacterial compounds include actinomycin D, doxorubicin, mitoxantrone, bleomycin, mitomycin, etoposide, and others. Recently, it was identified that Xenorhabdus budapestnesis (Xbu) produces a secondary metabolite, fabclavine, that has antibiotic property. Here, for the first time we demonstrate that fabclavine exhibits potent cytotoxic activity against ovarian cancer cells and ovarian cancer stem cells and demonstrate its mechanism of action, Fabclavine was isolated and purified from cell-free supernatants of Xbu cultures and analyzed by mass spectrometry. Proliferation assays conducted on several ovarian cancer cell lines Ovcar3, OVCA433, ID8, and breast cancer cell lines MCF7 and 4T1 demonstrate that fabclavine inhibits the cancer cell proliferation at nanomolar (250-400nM) concentration. Fabclavine is ten times more potent than cisplatin in inhibiting cell proliferation of ovarian cancer cell line OVCA33 and breast cancer cell line MCF7. We also tested the effect of fabclavine in 3D culture of ovarian cancer cell lines. The results indicate, even in 3D culture, fabclavine killed 40-50% of the cells at 300nM concentration. In addition, fabclavine was also tested against ovarian cancer stem cells isolated from five ovarian cancer patients and grown in 3D culture. Fabclavine inhibited the growth of stem cells by 70-85%. In cancer cell lines fabclavine induced apoptosis in 20-30% cells after 24-hour treatment. Caspase activity assay for caspase3, caspase8, and caspase9 shows a significant increase in caspase3 and caspse8 activity with no change in caspase9 activity, suggesting an activation of the extrinsic apoptosis pathway in the fabclavine-treated cells. This observation was supported by the increase in phospho-FADD upon exposure to fabclavine. When conjugated to agarose beads, fabclavine efficiently precipitated two death receptors, DR4 and DR5, from ovarian and breast cancer cell extracts. These experiments indicate that fabclavine is a novel DR4/5 ligand and should therefore be considered as a death receptor-targeting agent for the treatment of ovarian and other tumors. We will present data on the comparison of DR4/5 binding to fabclavine versus its natural ligand, TRAIL, and will also present data from PDex and mouse xenograft models on the ex vivo and in vivo effects of fabclavine on the proliferation of ovarian tumors and cancer stem cells. Citation Format: Arvinder Kapur, Mayur Kajla, Susan Paskewitz, Allegra Cappuccini, Pooja Mehta, Geeta Mehta, Manish Patankar. A novel death receptor ligand fabclavine inhibits cancer and cancer stem cell proliferation by extrinsic apoptosis [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A16.

Background: Three-dimensional (3D) cell culture models are more extensive to be better models than two-dimensional (2D) cell culture models due to enriched cellular signaling pathways, cell to-cell contact, more representative of tissue morphology, and predictive of drug responses in vivo. Different drugs screening and various biological responses between 2D and 3D cell culture models have been reported. However, very little information is available on cell function and/or drug susceptibility caused by a difference in methodology of three dimensional culture. Here, we compared drug responses of various cancer cells against distinctive anti-cancer drugs when grown in monolayer, scaffold 3D culture (Matrigel, NanoCuluture Plate (NCP)) or scaffold-free 3D culture models (ultra-low attachment round bottom plate), and also confirmed which method is suitable for high-throughput screening for robust three-dimensional screening model. Method: We examined drug sensitivities of different lung cancer (A549, H226, H1650), breast cancer (SKBR-3, JIMT1), and prostate cancer (DU145) cell lines under monolayer or 3D cell culture conditions against several anti-cancer agents i.e. molecular targeted agent (Gefitinib, Afatinib, Temsirolimus, Vemurafenib, Vismodegib, Sorafenib), demethylation agent (Azacytidine, Decitabine), anti-microtubule agent (Docetaxel), alkylating agent (Cisplatin, Irinotecan), antimetabolite agent (5-FU, Gemcitabine, Methotrexate). The conditions of these assays are optimized for each culture method. Cell proliferation was measured by ATP assay. Furthermore, we analyzed the biological/ morphological differences among 3D culture methods (scaffold or scaffold-free culture models). Result: Large variations in drug responses were observed among the different cell culture models. Cells grown in 3D scaffold culture model (NCP) were more drug sensitive than other cell culture models. Especially docetaxel, 5-FU, and methotrexate showed clear growth inhibition in three dimensional scaffold culture models (NCP) than scaffold-free culture models (round-bottom). Conclusion: In this study, we showed that scaffold 3D culture is appropriate for anti-cancer drug-susceptibility test. We also proposed that NanoCuluture plate which is 3D scaffold culture model, can be used as a sophisticated high-throughput three-dimensional drug screening model. Citation Format: Norio Masuda, Atsushi Mizuno, M.Mamunur Rahman, Kazuya Arai, Manabu Itoh. Effect of scaffold on drug sensitivity of multicellular spheroids: Which method is close to in vivo and suitable for HTS. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 303. doi:10.1158/1538-7445.AM2015-303

This study investigated the effects of matrix on the behaviors of 3D-cultured cells of two prostate cancer cell lines, LNCaP and DU145. Two biologically-derived matrices, Matrigel and Cultrex BME, and one synthetic matrix, the Alvetex scaffold, were used to culture the cells. The cell proliferation rate, cellular response to anti-cancer drugs, and expression levels of proteins associated with drug sensitivity/resistance were examined and compared amongst the 3D-cultured cells on the three matrices and 2D-cultured cells. The cellular responses upon treatment with two common anti-cancer drugs, Docetaxel and Rapamycin, were examined. The expressions of epidermal growth factor receptor (EGFR) and β-III tubulin in DU145 cells and p53 in LNCaP cells were examined. The results showed that the proliferation rates of cells cultured on the three matrices varied, especially between the synthetic matrix and the biologically-derived matrices. The drug responses and the expressions of drug sensitivity-associated proteins differed between cells on various matrices as well. Among the 3D cultures on the three matrices, increased expression of β-III tubulin in DU145 cells was correlated with increased resistance to Docetaxel, and decreased expression of EGFR in DU145 cells was correlated with increased sensitivity to Rapamycin. Increased expression of a p53 dimer in 3D-cultured LNCaP cells was correlated with increased resistance to Docetaxel. Collectively, the results showed that the matrix of 3D cell culture models strongly influences cellular behaviors, which highlights the imperative need to achieve standardization of 3D cell culture technology in order to be used in drug screening and cell biology studies.

The glucocorticoid receptor (GR) has been implicated in tumor progression and therapy resistance, yet its role in ovarian cancer (OC) remains controversial. In particular, how GR integrates environmental cues to control OC plasticity and therapeutic responses is poorly understood. We investigated GR function in ovarian cancer (OC) cells by integrating genetic and pharmacological approaches. By using both OC cell lines and patient-derived cells, we performed a comprehensive set of phenotypic, molecular and functional assays alongside genome-wide transcriptomic analyses. We also extended these analyses to physiologically relevant 3D systems, including tumor spheroids and organotypic cultures, to better recapitulate the in vivo tumor microenvironment. We provided unprecedented evidence that GR modulates OC behavior in a context-dependent manner. Under 2D culture conditions, GR enhanced cellular heterogeneity, epithelial–mesenchymal transition and migration, thereby increasing cisplatin resistance. Conversely, in a 3D context, GR exerted a marked yet reversible antiproliferative effect, characterized by reduced protein synthesis and adaptative stress responses. Mechanistically, GR activity converged on inhibition of glycolysis and activation of gluconeogenesis. Indeed, pharmacological inhibition of glycolysis with 2-deoxyglucose phenocopied GR-induced mesenchymalization in 2D cultures and growth inhibition in 3D models. Moreover, inhibition of gluconeogenesis with metformin prevented the GR-dependent antiproliferative effect in 3D models. Consistently, the glucocorticoid budesonide further potentiates the anti-proliferative effects of GR in OC spheroids. Transcriptomic analyses revealed that GR regulates gene programs involved in extracellular matrix organization and cell adhesion, uncovering a previously unrecognized role for GR in tumor microenvironment remodeling. Our findings reveal distinct, context-dependent effect of GR in OC cells, whereby GR activation promotes chemoresistance and migratory behavior in 2D cultures, while inducing a reversible slow proliferative state under 3D conditions. These results underscore the importance of cellular context in interpreting GR activity and suggest that selective GR modulators, including budesonide, may offer new therapeutic avenues for treating advanced-stage OC.

Histone methyltransferases (HMT) are chromatin modifiers that regulate the transcriptomic landscape in normal development as well as in diseases such as cancer.1 Enhancer of Zeste 2 (EZH2), a component of the polycomb repressive complex 2 (PRC2), trimethylates histone 3 lysine 27 (H3K27me3), resulting in a more compact chromatin structure known to repress gene activation.2 Overexpression of EZH2 has been strongly implicated in oncogenesis of many different cancers, including ovarian cancer (OC),2 and increased EZH2 activity has been linked to OC resistance to platinum-based chemotherapy, presumably by inhibiting crucial tumor suppressors and genes involved in OC metastasis such as integrins.2,3 Although HMT inhibitors (HMTI) that target EZH2 specifically or HMTs in general are promising anticancer therapeutics,1 the mechanism(s) underlying EZH2 regulation is incompletely understood, and the use of clinically relevant model systems to better understand the translational potential of this important class of epigenetic modifiers is clearly an important area of investigation. In the July 1, 2013 issue of Cell Cycle, Amatangelo and colleagues investigated the effect of a specific inhibitor of EZH2, GSK343, on human epithelial ovarian cancer.4 After GSK343 treatment of ovarian cancer (OC) cells grown on a 2D monolayer, a 90% decrease in total H3K27me3 was observed, demonstrating drug specificity to EZH2, and EZH2 protein levels remained unchanged, corroborating the mechanism of GSK343 action as an inhibitor of EZH2 HMT activity. Despite this, no effect on growth was observed on OC cells in 2D culture, although previous studies have reported that reducing the HMT activity of EZH2 markedly alters cell physiology in OC and other cancers. For example, phosphorylation of EZH2 by protein kinase B (AKT) on serine 21 (S21) reduced EZH2 enzymatic activity and as well as integrin α2 expression.5 In addition, EZH2 phosphorylation on residues threonine 350 and 487 (T350 and T487) by cyclin-dependent kinase 1 and 2 (CDK1 and CDK2), respectively, inhibited cell proliferation, migration, and anchorage-independent growth, indicating a role for EZH2 HMT activity in migration and invasion.6,7 To further investigate the effect of GSK343-mediated EZH2 inhibition on OC, Amatangelo and colleagues4 extended the scope of their study by using a 3D cell culture system composed of a matrigel extracellular matrix (ECM). It is well known that the tumor microenvironment is both heterogeneous in nature, composed stromal fibroblasts, immune cells, and vascular endothelial cells, and can significantly impact both the metastatic potential of cancer cells as well as their ability to resist chemotherapy. For these reasons, the ECM closely recapitulates the tumor microenvironment, representing a more clinically relevant model system compared with monolayer culture conditions on plastic. Taking this approach, Amatangelo and colleagues observed that GSK343 treatment inhibited OC cell growth and invasion in a 3D culture and correlated with apoptosis induction in OC cells.4 The results indicate regulation of EZH2 by the OC-ECM interaction as well as improved efficacy of GSK343 to suppress the ability of OC to remodel the ECM and metastasize (Fig. 1). Figure 1. Comparing the sensitivity of epithelial ovarian cancer cells to the histone methyltransferase inhibitor (HMTI) GSK343 in 2D (i.e., plastic, upper) and 3D (i.e., matrigel extracellular matrix, lower). No effect of GSK343 on ovarian cancer ... As epigenetic therapies for OC are in the clinical arena8 (and see SGI-110 in Combination With Carboplatin in Ovarian Identifier: NCT01696032, CancerClinicalTrials.gov), the exciting new study by the Zhang lab provides compelling evidence for using 3D culture systems to gain key insight into the biological roles of EHZ2 in OC and the sensitivity of this cancer to EZH2-specific inhibitors. Furthermore, as suggested by the Zhang lab,4 the numerous discrepancies observed between the efficacy of inhibitors in 2D culture and in vivo animal systems may be better explained by using the third dimension.

Simple SummaryKnowledge of the transcriptional regulation of breast cancer tumorigenesis is largely based on studies performed in two-dimensional (2D) monolayer culture models, which lack tissue architecture and therefore fail to represent tumor heterogeneity. However, three-dimensional (3D) cell culture models are better at mimicking in vivo tumor microenvironment, which is critical in regulating cellular behavior. Hence, 3D cell culture models hold great promise for translational breast cancer research.Intratumor heterogeneity of breast cancer is driven by extrinsic factors from the tumor microenvironment (TME) as well as tumor cell–intrinsic parameters including genetic, epigenetic, and transcriptomic traits. The extracellular matrix (ECM), a major structural component of the TME, impacts every stage of tumorigenesis by providing necessary biochemical and biomechanical cues that are major regulators of cell shape/architecture, stiffness, cell proliferation, survival, invasion, and migration. Moreover, ECM and tissue architecture have a profound impact on chromatin structure, thereby altering gene expression. Considering the significant contribution of ECM to cellular behavior, a large body of work underlined that traditional two-dimensional (2D) cultures depriving cell–cell and cell–ECM interactions as well as spatial cellular distribution and organization of solid tumors fail to recapitulate in vivo properties of tumor cells residing in the complex TME. Thus, three-dimensional (3D) culture models are increasingly employed in cancer research, as these culture systems better mimic the physiological microenvironment and shape the cellular responses according to the microenvironmental cues that will regulate critical cell functions such as cell shape/architecture, survival, proliferation, differentiation, and drug response as well as gene expression. Therefore, 3D cell culture models that better resemble the patient transcriptome are critical in defining physiologically relevant transcriptional changes. This review will present the transcriptional factor (TF) repertoire of breast cancer in 3D culture models in the context of mammary tissue architecture, epithelial-to-mesenchymal transition and metastasis, cell death mechanisms, cancer therapy resistance and differential drug response, and stemness and will discuss the impact of culture dimensionality on breast cancer research.

e17041 Background: G protein-coupled receptor 30 (GPR30) is a 7-transmembrane estrogen receptor that functions alongside traditional estrogen receptors to regulate the cellular responses to estrogen. Recent studies suggest that the high expression of GPR30 is associated with a poor prognosis in breast cancer or endometrial cancer. Although the role of GPR30 in ovarian cancer was unclear, we revealed that GPR30 is associated with poor prognosis in ovarian cancer. On the other hand, Epithelial-to-Mesenchymal Transition (EMT) is involved in cancer metastasis. The purpose of this study is to reveal how GPR30 was associated with poor prognosis and whether associated with EMT in ovarian cancer. Methods: We examined whether GPR30 signaling activates the EGFR-Akt pathway in an ovarian cancer cell line (Caov-3) by a Western blotting analysis. We also examined the effect of GPR30 on EMT were evaluated in Caov-3, which were cultured both in two-dimensional (2D) culture and three-dimensional (3D) culture model. GPR30 agonist, G1, was used to confirm the regulatory effects of GPR30 on the change of phenotypic modulation and EMT markers expression. Results: The phosphorylation of the EGFR and Akt could be significantly enhanced by G1 (p < 0.05) and inhibited by a Src family kinase inhibitor. In 3D culture, the stimulation of GPR30 leads the floating and sphere formation in Caov-3. G1-induced EMT was observed with related regulation of EMT markers expression at both mRNA and protein level. G1 induced the decrease of E-cadherin level and the increase of Snail and Vimentin in RT-PCR and Western blotting. Knockdown of GPR30, using siRNA, blocked G1-induced EMT. Conclusions: GPR30 increases the phosphorylation of Akt via the EGFR in ovarian cancer cells and changes ovarian cancer cells to the EMT state.GPR30 might be an important molecule related to metastasis process in ovarian cancer.

Three-dimensional cell culture models for investigating human viruses