Reprogramming of normal fibroblasts into ovarian cancer-associated fibroblasts via non-vesicular paracrine signaling induces an activated fibroblast phenotype

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 that conditioned media from ovarian cancer lines leads to an increase in the activated state of fibroblasts demonstrated by functional assays and up-regulation of known CAF-related genes and ECM pathways. 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. Critically, the conditioned CAFs resemble a transcriptional signature with involvement of ECM remodeling. The present study provides mechanistic and functional insights about the activation and reprogramming of CAFs in the ovarian tumor microenvironment mediated by nonvesicular paracrine signaling. Moreover, it provides a translational based approach to reprogram normal fibroblasts from both uterine and ovarian origin into CAFs using tumor-derived conditioned media. Using these resources, further development of therapeutics that possess potentiality and specificity towards CAF/ECMmediated chemoresistance in OC are further warranted.