CAFs promote M2 macrophage polarization under glutamine-deficient condition. (A) Gating strategy of iTAMs and mTAMs. iTAMs: single cells/live cells/CD45⁺/Ly6G⁻/CD11b⁺/F4/80⁺Ly6C^int; mTAMs: single cells/live cells/CD45⁺/Ly6G⁻/CD11b^+/F4/80⁺Ly6C⁻. (B) Representative FACS plots of gating for iTAMs and mTAMs populations in spleen and tumor from Yumm1.7 melanoma-bearing mice. (C) Representative histograms of MHC II and PD-L1 in mTAMs and iTAMs from Yumm1.7 melanoma-bearing mice. (D) Validation of gating strategy by confirming M2-like macrophage markers (CD163, Arg1, MHCII, and PDL1) expression on iTAM and mTAMs. (E) Western blot validating GS knockout in macrophages by both FSP1- and LysM-driven cre. (F and G) Tumor growth (F) and tumor weight (G) of Yumm1.7 melanomas from GS f/f (n = 6) and GS f/f LysM cre mice (n = 6). (H–J) Percentages of iTAMs (H) and mTAMs (I) in Ly6G⁻CD11b⁺ myeloid cells from tumor-bearing GS f/f and GS f/f LysM cre mice and ratio of iTAMs to mTAMs (J). (K) Representative FACS plots of macrophages in the spleen of Yumm1.7 melanoma-bearing mice treated with PBS or anti-CSF1R antibodies to validate macrophage depletion. (L) Percentages of macrophages (F4/80⁺ cells in CD45⁺ immune cells). Data are representative of at least two independent experiments with similar results (A–C, E, and K) or are the cumulative results from at least two independent experiments (D, F–J, and L). All data are shown as mean ± SEM and were analyzed by two-tailed, unpaired Student’s t test (D, F–J, and I) or two-way ANOVA with Sidak’s multiple comparison test (I). ****P < 0.0001; ***P < 0.001; P > 0.05 not significant (ns). PD-L1, programmed death-ligand 1. Source data are available for this figure: SourceData FS4.