Normal Uterine Fibroblast Are Reprogramed into Ovarian Cancer-Associated Fibroblasts by Ovarian Tumor-derived Conditioned Media

Cancer-associated fibroblasts (CAFs) are key contributors to ovarian cancer (OC) progression and therapeutic resistance through dysregulation of the extracellular matrix (ECM). CAFs are a heterogenous population derived from different cell types through activation and reprogramming. Current studies rely on uncharacterized heterogenous primary CAFs or normal fibroblasts that fail to recapitulate CAF-like tumor behavior. Here, we present a translatable-based approach for the reprogramming of normal uterine fibroblasts into ovarian CAFs using ovarian tumor-derived conditioned media to establish two well-characterized ovarian conditioned CAF lines. Phenotypic and functional characterization demonstrated that the conditioned CAFs expressed a CAF-like phenotype, strengthened proliferation, secretory, contractility, and ECM remodeling properties when compared to resting normal fibroblasts, consistent with an activated fibroblast status. Moreover, conditioned CAFs significantly enhanced drug resistance and tumor progression and resembled a CAF-like subtype associated with worse prognosis. The present study provides a reproducible, cost-effective, and clinically relevant protocol to reprogram normal fibroblasts into CAFs using tumor-derived conditioned media. Using these resources, further development of therapeutics that possess potentiality and specificity towards CAF-mediated chemoresistance in OC are further warranted. ### Competing Interest Statement Pilar de la Puente and Kristin Calar have a patent for the 3D culture method described in this manuscript, US Patent Application #2022/0228124. Pilar de la Puente is the co-founder of Cellatrix LLC; however, there has been no contribution of the aforementioned entity to the current study. Other authors state no conflicts of interest.