Non-Enzymatic Isolation of Cancer-Associated Fibroblasts From Human Prostate Tumor Explants

Prostate carcinoma (PCa) progression is strongly influenced by the surrounding tumor microenvironment, where cancer-associated fibroblasts (CAFs) represent the most abundant and functionally relevant stromal population. Despite their importance, the lack of stable cell lines representing CAF phenotypes limits the study of stromal-tumor interactions. To address this limitation, we provide an optimized protocol for isolating CAFs from fresh human PCa biopsies based on a mechanical procedure exploiting the specific CAF ability to migrate out from the tumor explants. This approach preserves tissue architecture and maintains CAF viability and phenotype. The resulting ex vivo CAF cultures provide a suitable model to investigate CAF biology within the tumor microenvironment. Key features • The protocol provides an optimized workflow for isolating CAFs from prostate tumor explants by exploiting their selective outgrowth. • It is an optimized, enzymatic-free procedure that minimizes fibroblast cell stress while preserving cell phenotypic features.