Introduction: Epithelial ovarian cancer comprises approximately 90% of all cases of ovarian cancer and is the leading gynecologic cause of death in Western societies. Ovarian cancer patients typically respond well to first-line platinum and taxane-based chemotherapies; however, approximately 80% of these patients relapse and succumb to more chemoresistant disease. Relapse and development of chemoresistance is linked to the presence of a rare population of chemoresistant cells, termed cancer stem-like cells (CSCs), which are enriched in spheroids in the malignant ascites. Despite the high levels of chemoresistance and relapse observed in ovarian cancers, there are no in vitro models to understand the development of chemoresistance in situ. Here we implement a 3D model of the development of chemoresistance in ovarian cancer to investigate the functional and genetic changes that occur as chemoresistance develops. Using this model to gain insight into the development of chemoresistance in ovarian cancer will facilitate the development of more effective treatments. Methods: To investigate the development of chemoresistance in ovarian cancer, we utilized an in vitro model using the 3D hanging drop spheroid platform with an ovarian cancer cell line, two primary patient samples. In our model, spheroids were serially passaged every 7 days and evaluated for proliferation and response to treatment with cisplatin and a novel ALDH1A inhibitor. Concomitantly, the expression CSC markers ALDH and CD133 as well as tumor initiating capacity were analyzed. RNA-sequencing and qRT-PCR was performed on spheroids from early (P0), middle (P3), and late (P6) passages for two patient samples to establish gene signatures associated with the evolution of stemness, tumorigenicity, and chemoresistance. Lastly, a mathematical model was developed to predict the emergence of CSCs during serial passaging of ovarian cancer spheroids. Results: Our serial passage model demonstrated increased cell proliferation, enriched CSCs, and emergence of a platinum-resistant phenotype with passaging. Contrarily, serial passaged spheroids were enriched for ALDH and consequently exhibited greater sensitivity to ALDH1A inhibitor. Furthermore, in vivo tumor xenograft assays indicated that later passage spheroids were significantly more tumorigenic with higher CSC proportions, compared to early passage spheroids, validating the increased proliferative capacity predicted by our in vitro serial passage platform. RNA-sequencing revealed several gene signatures supporting the emergence of CSCs, chemoresistance, and malignant phenotypes, with links to poor clinical prognosis. Finally, our mathematical model predicted the emergence of CSC populations within serially passaged spheroids, concurring with experimental data. Conclusions: Our integrated approach illustrates the utility of the serial passage spheroid model for examining the emergence of chemoresistance in ovarian cancer in a controllable and reproducible format. Citation Format: Pooja Mehta, Micheal Bregenzer, Maria Ward Rashidi, Shreya Raghavan, Elyse Fleck, Eric Horst, Zainab Harissa, Visweswaran Ravikumar, Samuel Brady, Andrea Bild, Arvind Rao, Ronald Buckanovich, Geeta Mehta. Serially passaging ovarian cancer spheroids as an in situ model for emergence of chemoresistance and enrichment of cancer stem cells [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 A52.
Ovarian cancer is the leading cause of gynecologic malignancy, characterized by a high degree of heterogeneity and relapse leading to poor clinical outcomes. These outcomes are attributed in part to a population of cells termed cancer stem-like cells (CSCs), which are capable of repopulating tumors and are more chemoresistant and tumorigenic. These properties make CSCs a promising target for novel therapies; however, we lack a comprehensive understanding of how they are regulated by the tumor microenvironment. To address this, we have developed a tumoroid culture system wherein patient tumor cells are brought together with controlled ratios of mesenchymal stem cells (MSCs), endothelial cells (ECs), and peripheral blood mononuclear cells (PBMCs) within 384-well hanging drop arrays. This allows for comprehensive analysis of the surrounding cells in the tumor microenvironment and their influence on CSC populations and chemoresistance. The fine control of this culture system over the tumoroid cell composition coupled with the high-throughput nature of 384-well hanging drop also facilitates patient-specific high-throughput analysis of inter- and intrapatient heterogeneity and chemoresistance. Tumoroids were generated from 5 patient samples and characterized using flow cytometry detection of cell type proportions, immunofluorescence evaluation of cell localization, and progressive evaluation of cell localization with fluorescently labeled tumoroids. Patient-derived tumoroids were evaluated by single-cell RNA sequencing. Drug screening was performed on patient-derived tumoroids and control patient-derived spheroids using classic ovarian cancer therapies, carboplatin and paclitaxel, as well as three novel drugs. Additionally, tumor formation assay was performed in immunodeficient mice to compare the rate of tumor formation following injection of tumoroids versus spheroids. Finally, a mathematical model was developed to predict the evolution of cell populations within the tumoroids to facilitate further analysis. Using these methods, we successfully generated and characterized viable tumoroids with primary patient-derived tumor cells, MSCs, ECs, and PBMCs. Within these tumoroids, we observed heterogeneity both between and within patient samples reflective of clinical observations in ovarian cancers. Furthermore, we found increased CSC phenotypes in the tumoroids compared to patient-derived tumor cell-only spheroids. Patient-derived tumoroids also exhibited increased chemoresistance and tumorigenicity compared to spheroids generated with only tumor cells. Through thorough development and characterization of this patient-derived model, we present a novel 3D tumoroid model for comprehensive investigation of CSC regulation, chemoresistance, and heterogeneity in ovarian cancers, with the long-term goal of developing novel CSC targeting therapies and improving clinical outcomes. This abstract is also being presented as Poster B08. Citation Format: Micheal E. Bregenzer, Pooja Mehta, Arvind Rao, Karen McLean, Nouri Neamati, Ronald J. Buckanovich, Geeta Mehta. Patient-derived tumoroids for exploration of the ovarian cancer stem cell regulation, chemoresistance, and tumor heterogeneity [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 PR05.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.