Gene expression profiling of tumor-associated macrophages and in-vitro-polarized macrophages

The aim of this study was to decipher gene expression of tumor-associated macrophage in the 4T1 mouse model of breast cancer and of in-vitro-polarized macrophages. For in-vitro-polarized macrophages, Ly6C+ monocytes were isolated from splenocytes of 4T1-bearing mice using Miltenyi magnetic beads, seeded in a 6-wells plate at 1 million cells per wells in RPMI 10% FCS, penicillin/streptomycin, 50ng/ml M-CSF and cultured for 72h. M0 basal macrophages were cultured with no extra-cytokine, while we added LPS+IFN for M1, IL4 for M2 and IL6 for M6 (all at 50ng/mL). F4/80+ TAMs were isolated from enzyme-digested 4T1 tumors using Miltenyi magnetic beads, and lysed directly in Ambion's PureLink RNA Mini Kit lysis buffer.