Macrophage Polarization and Cancer Cell Lines

Human CRC cell lines (HCT116, HT29, LoVo, and SW48), a human monocyte cell line (THP-1), and a mouse colon cancer cell line (CT26) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Rosewell Park Memorial Institute (RPMI) 1640 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin (Gibco, NY,USA) in a 5% CO₂ incubator at 37 °C. According to previous studies^{28 (link),29 (link)}, to polarize M0 macrophages, THP-1 cells (5 × 10⁶) were plated in 100-phi dishes and stimulated with 320 nm phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO, USA), followed by incubation at 37 °C for 24 h. The culture supernatant was collected and labeled M0 macrophage-conditioned medium (M0 CM). To polarize M1 or M2 macrophages, THP-1 cells were treated with PMA for 6 h, and M1-polarizing reagents (100 ng/ml lipopolysaccharide (LPS) plus 20 ng/ml interferon gamma (IFN-γ); R&D, Waltham, MA) or M2-polarizing reagents (20 ng/ml IL-4 plus 20 ng/ml IL-13) were added, followed by incubation at 37 °C for 18 h. The culture supernatant was collected and labeled M1 macrophage CM (M1 CM) or M2 macrophage CM (M2 CM). MG132 (proteasome inhibitor) and PI3Kγ inhibitors (TG100-115 and IPI-549) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Selleckchem (Houston, TX, USA), respectively.