Tumoroid model recreates clinically relevant phenotypes of high grade serous ovarian cancer (HGSC) cells, carcinoma associated fibroblasts, and macrophages.

Ovarian cancer, the gynecological malignancy with the lowest survival rate, is significantly influenced by the tumor microenvironment. The mesenchymal subtype of high-grade serous carcinoma (HGSC) shows poor outcomes due to high stromal and low immune response. Single-cell RNA sequencing (scRNA-seq) of HGSC metastatic ascites has identified carcinoma-associated fibroblasts (CAFs), macrophages, and carcinoma-associated mesenchymal stem cells (CA-MSCs) as crucial drivers of immune exclusion, chemotherapy resistance, metastasis, and stem-like cell propagation. To explore this complex signaling, we developed heterogeneous tri-component tumoroids, incorporating HGSC cells (OVCAR3, OVCAR4, OVCAR8), primary MSCs, and U937-derived M2-like macrophages (M2-AAM) in defined ratios, each labeled with a fluorescent protein for distinct analysis. Upon a 48-hour treatment with carboplatin and/or paclitaxel, HGSC cells in tri-component tumoroids exhibited higher chemoresistance than HGSC-only spheroids. Flow cytometry revealed significant increases in cancer stem-like cell (CSC) markers CD44 and CD90 in the tri-component tumoroids. Conditioned medium from the tri-component tumoroids significantly enhanced HGSC cell migration compared to spheroids. Invasion assays further demonstrated that tri-component tumoroids penetrated monolayer of mCherry-labeled LP-9 mesothelial cells more effectively than spheroids. Additionally, scRNA-seq of tri-component tumoroids identified a unique cancer cell cluster enriched in epithelial-mesenchymal transition (EMT) and matrisome signatures, featuring a 14-gene signature linked to poor survival. MSCs in these tri-component tumoroids displayed a myofibroblastic-CAF signature, while macrophages indicated an ECM-associated and immunosuppressive phenotype. In conclusion, our 3D heterogenous tri-component tumoroids replicate key HGSC phenotypes, such as chemoresistance, CSC enrichment, migration, invasion, and EMT. This platform is invaluable for studying HGSC microenvironment interactions and preclinical testing of targeted therapies.