Abstract 156: An ascitic fluid-derived organoid platform for Hispanic ovarian cancer patients to capture heterogeneity and disparity
Authors: Hassan Md Mynul , Wahab Riajul , Sanchez Sheralyn , Toy Eugene P. , Reddy Sireesha Y. , Roy Sourav , Al-Hilal Taslim A. 
Published: April 2023

Abstract
Introduction: Ovarian cancer (OC) is a fatal gynecological and heterogeneous disease usually diagnosed at stages after peritoneal seeding. Current medical intervention involves surgical removal of both ovaries and the uterus followed by chemotherapy. Thus, experimental models, established from primary tumor, may not mimic the real-time characteristics, heterogeneity, and disparity of OC after surgical debulking; a stage where OC survive and grow in peritoneal lavage fluid and form malignant ascites. Therefore, we present a protocol that enables efficient derivation and long-term expansion of ascites-derived organoids (AsO) culture to study their heterogeneity and disparity.
Methods: Malignant ascites, fresh tumors and whole blood were obtained at the time of surgical debulking and drainage of ascites/pleural effusion under an approved IRB protocol; patients characteristic included ethnicity: Hispanic and pathology: Mucinous Adenocarcinoma, Serous Grade 3, Carcinosarcoma grade 3, FIGO Stage IIB/IIIC (N = 3; treatment naïve). Within four hours of collection, an aliquot of tumors/ascites were used, and the cellular components were collected and resuspended in Matrigel®/complete organoid culture media (60:40) that is supplemented with B-27 supplement, N-acetyl-cysteine, R-spondin 1, Noggin, Nicotamide, EGF, FGF 10, Forksolin, A8301, hydrocorticosone, Heregulin β-1, β-Estradiol, Y27632, and N2; 30,000 cells per well were seeded and grown for 10 days before passaging. RNA sequencing was performed on AsO and tumor-derived organoids (TuO), and that of whole tissues as controls. Single-cell RNA sequencing (scRNAseq) was performed on 1 million live cells collected from malignant ascites (N = 3 patients). Tissues and organoids were fixed and sectioned for H&E, IHC, and IF staining.
Results: At the time of submission of this abstract, we have established 6 organoid lines from 3 Hispanic OC patients, representing different subtypes of OC. AsO or TuO recapitulate histological and genomic features from which they were derived, illustrating interpatient heterogeneity. Organoids were positive for OC protein biomarkers, such as PAX8 and cytokeratin. AsO showed morphological variation between patient subtypes, mostly forming dense organoid structure. The scRNAseq data have been analyzed to understand the distinctive features of individual cells and subcellular heterogeneity. RNA sequencing data confirmed that AsO and TuO recapitulated the features of their tissue of origin, but also illustrated genomic and phenotypical differences between both organoid types.
Conclusion: Our efforts to make Hispanic-centered AsO biobank faithfully recapitulates OC hallmarks and can be subjected to real-time drug screening against chemoresistant tumors, genetic manipulations and cancer disparity research by comparing the AsO of non-Hispanic Whites.
Citation Format: Md Mynul Hassan, Riajul Wahab, Sheralyn Sanchez, Eugene P. Toy, Sireesha Y. Reddy, Sourav Roy, Taslim A. Al-Hilal. An ascitic fluid-derived organoid platform for Hispanic ovarian cancer patients to capture heterogeneity and disparity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 156.