Tumor FAK supports fibrillar collagen expression in ovarian cancer ascites.

High-grade serous ovarian cancer (HGSOC) is a lethal malignancy where matrix proteins enhance ascites-associated recurrent tumor growth. Elevated focal adhesion kinase (FAK/PTK2) and collagen protein expression support tumor survival, chemoresistance, and immune evasion. However, the sources and regulation of collagen production within HGSOC tumorspheres is unclear. Using CRISPR-mediated FAK knockout and FAK re-expression in two syngeneic HGSOC models (MOVCAR and KMF), FAK loss impaired tumorsphere growth in vitro and in vivo while reducing collagen expression, as determined by RNA sequencing, quantitative RT-PCR, and protein mass spectrometry. FAK re-expression promoted spontaneous metastatic ovarian tumor dissemination. Integrated bioinformatic analyses of MOVCAR, KMF, and HGSOC patient datasets identified 56 common mRNAs co-elevated with PTK2, including COL3A1 and COL5A2. Single-cell RNA sequencing of murine KMF ascites revealed a subset of tumor cells expressing Col3a1 and Col5a2 that exhibited epithelial-to-mesenchymal pathway enrichment that was diminished by genetic or pharmacologic FAK inhibition. In primary HGSOC ascites samples, FAK inhibitor (IN10018 or VS-4718) or FAK protein degrader (PROTAC) treatment selectively reduced COL1A1, COL3A1, and COL5A2 mRNA levels in tumor cells. Further, human OVCAR8 spheroid COL1A1, COL3A1, and COL5A2 mRNA and spheroid structure were significantly impacted by FAK inhibition and blocking anti-α1 or anti-β1 integrin antibodies. As elevated PTK2 and COL3A1 expression correlate with poor ovarian cancer patient survival, and FAK, α1, and β1 integrin inhibition reduced spheroid COL3A1 protein levels, our findings support a feed-forward linkage whereby FAK regulates fibrillar collagen acting in part via collagen binding integrins supporting HGSOC tumorsphere survival and metastatic spread.