Abstract Due to the rarity of ovarian cancer stem cells (OvCSC; <0.2% total population), we developed a 3D hanging drop array model, in which as few as 10 OvCSCs (isolated from primary malignant ascites based on ALDH+ CD133+) can be stably incorporated into spheroids for study of drug sensitivity and tumor biology. Our platform can be utilized to: i) quantify drug sensitivity of chemotherapeutic agents in the context of OvCSCs; ii) distinguish drug responses for the same drugs between several patient samples; thereby uniquely suited for the development of personalized therapeutics including the context of OvCSCs. Three patient samples were evaluated (stage IIIcstage IV). Robust proliferation rates were observed in spheroids, ranging from 5.3 fold (progressive Pt.259++) to 6.5 fold(resistant Pt224++) to 8.4 fold(recurrent Pt152++). Examination of OvCSC populations by Day 7 indicated that ALDH+ CD133+ cells had differentiated within spheroids to form progenies of ALDH- CD133-, ALDH+ CD133-, and CD133+ ALDH- cells. Maintenance of the ALDH+ CD133+ pool was also observed (0.04-0.7%). Composition of progenies was different between the three different patient samples: In Pt259, <1% CD133+ and <5% ALDH+; Pt224 with ~25% ALDH+ progeny; and Pt152 with ~20% CD133+ progeny. 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. Progressive Pt259 samples were maximally sensitive to Cisplatin/673A, while Pt224 and Pt152 were more resistant to drug(20-40% higher viability). Combination dose of Cisplatin/Ruxolitinib targeted CD133+ populations, but also resulted in increased ALDH+ cells, indicating a potential differentiation of CD133+ cells into ALDH+ cells to escape cell-death. By isolating cells that escape 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. Lastly, OvCSC spheroids (10 cells/drop) initiated tumors in immunodeficient mice (2 spheroids – 80% success rate; 10 spheroids – 100% success rate). Conclusions: We have developed a hanging drop array model that incorporates primary ovarian cancer stem cells into a 3D spheroid, capable of initiating tumors in immuno-deficient mice. OvCSCs differentiate into heterogeneous progenies within spheroids, correlating with differing responses to conventional and stem cell targeting drug treatments, providing a platform for personalized therapeutics. We also developed a spheroid model that can mimic tumor re-emergence using cells that escape first line chemotherapy. This platform can be utilized to study spheroid and cancer stem cell biology, and model tumor re-emergence to identify new targeted therapeutics. This work was supported by the DOD OCRP Early Career Investigator Award W81XWH-13-1- 0134 (GM). Citation Format: Shreya Raghavan PhD, Maria Ward BS, Pooja Mehta MSc, Ronald J Buckanovich MD PhD, Geeta Mehta PhD. CHEMORESISTANCE, TUMOR INITIATION AND TUMOR RE–EMERGENCE TRENDS IN MALIGNANT ASCITES–DERIVED OVARIAN CANCER STEM CELL SPHEROIDS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP22.