Isolation, Culture, and Characterization of Prostate Cancer-Associated Fibroblasts

Tumor stroma can be actively shaped by tumor cells to adopt pro-tumorigenic properties, making it a focal point of cancer research. Among the stromal components, cancer-associated fibroblasts (CAFs)-often the most abundant cell type in the tumor microenvironment (TME)-play a pivotal role in promoting cancer progression through their interactions with tumor cells and other stromal components. Elucidating the mechanisms behind these interactions requires robust methods for isolating and characterizing CAFs, which can also lay the foundation for developing novel CAF-targeted cancer therapies. This study presents a method for isolating fibroblasts from both the tumor and the adjacent normal regions of radical prostatectomy specimens obtained from prostate cancer (PCa) patients. This approach enables the concurrent isolation of CAFs and their normal counterparts (NFs) from the same individual, providing a paired experimental system. Detailed protocols are provided for the maintenance of primary CAFs and NFs for downstream in vitro analyses, as well as for their immortalization to facilitate long-term studies. We also describe functional assays designed to compare the effects of CAFs and NFs on cancer cell behaviors, including proliferation, migration, and anchorage-independent growth. Two complementary approaches are detailed: treatment of cancer cells with fibroblast-derived conditioned media (CM), and direct co-culture of fibroblasts with tumor cells. Together, these methodologies represent a comprehensive toolkit for investigating the dynamic interplay between CAFs and tumor cells, not only in PCa but potentially across a range of human cancers. Moreover, molecular characterization of these interactions may reveal key mediators of CAF-driven oncogenesis, offering promising targets for therapeutic intervention.