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 cancers grow in suspension in the ascites fluid, and contain a small population of ovarian cancer stem cells (OvCSC), which are resistant to therapy. Due to the rarity of OvCSCs, we developed a 3D hanging drop platform, in which as few as one ALDH+ CD133+ cell (isolated from primary malignant ascites) can be stably incorporated into 3D spheroids. Our platform can be utilized to quantify drug sensitivity of chemotherapeutic agents in the context of OvCSCs, distinguish drug responses for the same drugs between several patient samples, and model patient-specific tumor re-emergence, making it uniquely suited for the development of personalized therapeutics. Three patient samples (Pt259, Pt224, Pt152) were evaluated and robust proliferation rates were observed in spheroids, ranging from 5.3 fold to 8.4 fold. By Day 7, ALDH+ CD133+ cells had differentiated within spheroids to form progeny of ALDH- CD133-, ALDH+ CD133-, and CD133+ ALDH- cells while maintaining an ALDH+ CD133+ population. Each patient-derived spheroid demonstrated a different composition of these progeny, which were similar to those observed in the patient samples. OvCSC spheroids had differing responses to drug treatments (cisplatin, ALDH targeting compound 673A, and JAK1/2 inhibitor ruxolitinib). Combination of cisplatin/673A targeted ALDH+ and CD133+ in all patient samples. Pt259 samples were maximally sensitive to cisplatin/673A, while Pt224 and Pt152 were more resistant (20-40% higher viability). Combination dose of cisplatin/ruxolitinib targeted CD133+ populations. By isolating cells that escaped chemotherapy, we created a spheroid model to study tumor re-emergence. ALDH+ populations re-emerged to a lower extent compared to original OvCSC spheroids, while CD133+ populations did not recover at all. Spheroids formed from the most platinum-sensitive cells (Pt259) and the most platinum-resistant cells (Pt152) following cisplatin/673A treatment were also serially passaged over 7 cycles in 7 weeks to characterize CD133+ and ALDH+ populations and evaluate their ability to reform spheroids, effectively modeling tumor re-emergence in vitro. ALDH+ OvCSC progeny reliably repopulated within these spheroids despite initial depletion following treatment. Over six serial passages, ALDH+, CD133+, and ALDH+ CD133+ populations gradually returned to original and even higher than levels seen in original patient samples. Lastly, OvCSC spheroids initiated tumors in immunodeficient mice at 100% success with only 10 spheroids injected. These tumors demonstrated a distinct response to therapy that corresponds with responses seen in spheroids, indicating that our model may be a means to screen tumors for personalized drug selection. Our patient-derived low-cell-number OvCSC spheroid platform can be utilized to study tumor biology, to model tumor re-emergence after primary chemotherapy, and to identify new targeted therapeutics from a personalized medicine standpoint. Citation Format: Shreya Raghavan, Pooja Mehta, Michael Bregenzer, Maria Ward Rashidi, Elyse Fleck, Lijun Tan, Karen McLean, Ronald Buckanovich, Geeta Mehta. Patient-specific evaluation of chemoresistance and tumor recurrence using ovarian cancer stem cell spheroids. [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 A33.
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