Molecular profiling of ex vivo prostate cancer CAF models captures stromal heterogeneity and drug vulnerabilities.

Cancer-associated fibroblasts (CAFs) are central architects of the prostate cancer (PCa) microenvironment, yet their phenotypic diversity and druggable vulnerabilities remain largely uncharted. Here, we present an integrative multi-omics characterization of primary ex vivo CAFs from seven treatment-naïve PCa patients. Using single-cell RNA sequencing (scRNA-seq), we uncover substantial transcriptional heterogeneity among CAFs, with distinct gene expression programs related to extracellular matrix remodeling, inflammation, immune modulation, and metabolic reprogramming. This phenotypic diversity was further supported by variable expression of canonical stromal markers, including FAP, SULF1, VIM, CAV1, and αSMA. Transcription factor network analysis revealed SOX, FOX, and STAT3 family members as key regulators of pro-tumorigenic CAF states. To probe therapeutic vulnerabilities, we performed high-throughput drug sensitivity and resistance testing (DSRT) across 396 oncology compounds. CAFs exhibited broad sensitivity to multikinase inhibitors, with dasatinib, midostaurin, and FGFR inhibitors (AZD4547, erdafitinib) emerging as top stromal-directed candidates. These findings underscore the plasticity of prostate CAFs and reveal actionable vulnerabilities, supporting the development of targeted stromal therapies to disrupt tumor-stroma interactions in PCa.