Abstract A52: Serially passaging ovarian cancer spheroids as an in situ model for emergence of chemoresistance and enrichment of cancer stem cells

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.