Fibroblast Isolation from Mammary Gland Tissue and Syngeneic Murine Breast Cancer Models

Cancer-associated fibroblasts (CAFs) are vital within the tumor ecosystem, regulating tumor growth, dissemination, and response to therapy through crosstalk with tumor cells, infiltrating immune and vascular cells, as well as components of the extracellular matrix (ECM). CAFs have thus emerged as potential therapeutic targets to complement cancer cell-targeted therapies. To study CAF-tumor cell crosstalk ex vivo, robust isolation methods of primary CAFs are required. Here, we present protocols to isolate, expand, and culture two types of fibroblasts: (1) healthy murine mammary gland fibroblasts, a key source of the CAF population in breast tumor models and (2) CAFs derived from syngeneic murine breast tumors. Isolated mammary fibroblasts and CAFs are suitable for use in a variety of downstream cellular and molecular experiments. We expect these methods to be useful to scientists studying the properties of fibroblasts and CAFs and the interaction between CAFs and the various components of the tumor microenvironment (TME).