Isolation and Culture of Tumor-associated IMCs

Tumour-associated IMCs were freshly isolated from Braf^{+/LSL−V600E};CreER^+/0 mice and cultured in serum-free Dulbecco's modified Eagle medium (DMEM) (Invitrogen) as previously described (Kamata et al., 2015 (link), 2020 (link)). Atorvastatin (3.3 µM, Generon), lonafarnib (1-5 µM, Tocris Bioscience), GGTI-298 (8 µM, Tocris Bioscience), ML141 (10 µM, Merck) and/or EHT1864 (1-10 µM, Tocris Bioscience) were added to the serum-free IMC culture for 72 h. IMCs cultured for 96 h in serum-free DMEM were treated with the CCR1 inhibitor J113863 (5 µM, Tocris Bioscience) for 1-24 h as indicated. For membrane protein purification and detergent-insoluble protein analysis, primary IMCs were cultured for 48-72 h in DMEM containing 1% foetal bovine serum (Invitrogen) supplemented with Atorvastatin (3.3 µM), epoxomicin (0.05 µM, Sigma-Aldrich) and/or MG132 (3.3 µM, Sigma-Aldrich).