Syngeneic Orthotopic Mammary Tumor Metastasis

Balb/c and FVB/N female mice were purchased from Charles River Laboratory. The generation and characterization of Ddr2^−/− mice has been previously described (Corsa et al., 2016 (link)). The Ddr2^−/− allele has been backcrossed onto both FVB/N and Balb/c genetic backgrounds (>10 generations). MMTV-PyMT mice were purchased from Jackson Laboratories and maintained on FVB/N background. For syngeneic orthotopic transplants, 1×10⁶ 4T1 cells (Balb/c) were injected into the fourth mammary fat pad of 8-week-old Balb/c females and allowed to reach end stage (2 cm primary tumor). Primary tumors and lungs were collected at end stage and processed for immunohistochemistry as previously described (Brenot et al., 2018 (link)). Lungs were processed in three step-sections of 50 µm steps with two serial sections of each step. Metastatic tumor nodules were counted and averaged per lobe of lung (five lobes) per mouse. Syngeneic lung colonization experiments were conducted by injecting 7.5×10⁵ PyMT-derived FVB/N tumor cells with or without 1×10⁵ CAFs (from FVB/N mice) of indicated genotypes into the tail vein of 8-week-old FVB/N female mice. After 25 days the lungs were harvested and processed as above. All animal experiments were performed according to Washington University Institutional Animal Care and Use Committee under protocol #2019-1042.