ELUCIDATING THE IMPACT OF ASCITES ON HIGH GRADE SEROUS OVARIAN CANCER THROUGH MULTIPLE IN VITRO PATIENT-SPECIFIC MODELS

High-grade serous ovarian cancer (HGSOC) is a highly heterogeneous and aggressive malignancy characterized by marked inter-/intra-tumor variability and a dynamic tumor microenvironment (TME). The TME, and particularly ascitic fluid (AF), plays a critical role in OC progression by influencing tumor and stromal cell behavior, promoting metastasis and driving therapy resistance. Current in vitro models often fail to replicate the complexity of the TME, being limited in their ability to fully capture HGSOC biology. Here, we explored the impact of AF supplementation on multiple patient-derived in vitro models, specifically 2D cells, 3D stem-enriched spheroids and polyclonal organoids, to develop preclinical models able to capture and faithfully reproduce the heterogeneity and complexity of this tumor and its TME. We could show that AF supplementation improves culturing conditions by increasing cell proliferation, sphere formation and propagation, while also preserving the original metabolic profiles of patient-derived samples. In parallel, 2D cells and 3D spheroids revealed a model-specific effect of ascites, with a downregulation of genes involved in the biosynthesis of cholesterol and in inflammatory response in 2D cells, and an upregulation of proliferation and glycolysis in 3D spheres. Additionally, we identified a common effect of AF on both models, showing AF-dependent genes and pathways related to cell cycle regulation and cellular proliferation. Among them, CDKN2B was notably downregulated in the presence of AF, highlighting a novel regulatory mechanism directly driven by the ascitic TME in HGSOC. Integrative analyses revealed that the in vitro models were mostly enriched for tumor stroma cellular subpopulations, warranting a perspective shift from a tumor epithelial-centric view to a systematic, and now finally experimentally tractable, appraisal of the TME, particularly cancer-associated fibroblasts (CAFs), paving the way to a refined understanding of tumor progression in light of tumor-TME cross-interaction. In this context, the study underscores the critical role of AF in shaping the interactions between tumor and non-tumor cells and the surrounding microenvironment, highlighting the importance of TME for a comprehensive understanding of HGSOC biology. The integration of AF into in vitro systems represents a significant step forward in creating more defined models for studying stroma and/or tumor interactions, allowing in the future to position these models within the broader landscape of HGSOC biology and potentially guide more refined therapeutic strategies.