Generation of Murine Cancer Cell Lines

Abstract Cancer cell lines are important tools to investigate the biology of cancer and test hypotheses to improve cancer treatments. A major challenge in establishing epithelial cancer cell lines is the removal of cancer‐associated fibroblasts (CAFs). CAFs are abundant within the tumor microenvironment. CAFs generally proliferate faster than epithelial cancer cells in culture. CAFs can be mistakenly identified as cancer cells, especially when cancer cells display spindle‐shaped morphology. Among all cancer types, pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant desmoplastic stroma. Here, we describe protocols for establishing epithelial cancer cell lines from mouse models of PDAC and verifying that they are not CAFs. The approach is cost‐effective and can be used for other types of cancer. If needed, CAF cell lines can also be established and preserved using this protocol. © 2024 Wiley Periodicals LLC. Basic Protocol 1 : Isolation of cells from tumors Basic Protocol 2 : Isolation and cryopreservation of cancer cell clones Basic Protocol 3 : Assessment of the identity of cancer cell lines and CAFs by western blotting