Depletion of GS in CAFs remodels TAM phenotype and restrains tumor progression. (A and B) Glutamine concentrations in the TME were quantified by LC-MS in serum and TIF from engrafted Yumm1.7 melanoma model (n = 8) (A) or inducible Braf/Pten melanoma model (n = 8) (B). (C) Distributions of glutamine synthesis pathway scores and heatmap displaying normalized gene expression in glutamine synthesis pathway in different cell types from murine melanoma scRNA-seq dataset. (D) Comparison of GS activity in TAMs and CAFs in engrafted Yumm1.7 melanoma model (n = 10). (E) Correlation of GS transcription level and pro-tumorigenic TAMs signature of melanoma patients in TCGA datasets. (F) Representative images of immunofluorescence staining on sections from human patients with colon cancer. Green: anti-CD68, white: anti-CD163, blue: anti-PDPN, and red: anti-GS. Cyan arrowheads indicate GS⁺ fibroblasts (GS⁺PDPN⁺). Yellow arrowheads indicate M2-like (CD163⁺CD68⁺) macrophages. White arrowheads indicate M1-like (CD163⁻CD68⁺) macrophages. Scale bars, 50 μm. (G) Correlation of GS⁺CAFs (GS⁺PDPN⁺) with the number of M1-like (CD163⁻CD68⁺) macrophages (red) or M2-like (CD163⁺CD68⁺) macrophages (blue) per field in the tumor sections derived from human colon cancer patients. (H) qPCR analysis of relative mRNA level of Cd206, Arg1 and Cd163 (pro-tumorigenic TAM markers) in BMDMs co-cultured with NFs or CAFs under glutamine-deficient condition for 48 h. (I) qPCR analysis of relative mRNA level of Cd206, Arg1, and Cd163 in BMDMs co-cultured with Ctrl CAFs or GS^KO CAFs under glutamine-deficient condition for 48 h. (J and K) Tumor growth (J) and tumor weight (K) of Yumm1.7 melanomas from GS f/f (n = 8) and GS f/f FSP1cre mice (n = 8). (L–N) Percentages of iTAMs (L) and mTAMs (M) in Ly6G⁻CD11b⁺ myeloid cells from tumor-bearing GS f/f and GS f/f FSP1cre mice and ratio of iTAMs to mTAMs (N). (O and P) Tumor growth (O) and tumor weight (P) of Yumm1.7 melanomas from GS f/f (n = 12) and GS f/f FSP1cre mice (n = 11) treated with PBS or with anti-CSF1R antibodies. (Q) Yumm1.7 melanoma tumor cells were co-injected with either Ctrl CAFs or GS^KO CAFs on the right or left flanks of GSf/f FSP1cre mice. (R and S) Tumor growth (R) and tumor weight (S) of Yumm1.7 melanomas co-injected with Ctrl CAFs or GS^KO CAFs on GSf/f FSP1cre mice (n = 8). (T–V) Paired percentages of iTAMs (T) and mTAMs (U) in Ly6G⁻CD11b⁺ myeloid cells derived from Yumm1.7 melanomas co-injected with Ctrl CAFs or GS^KO CAFs on GSf/f FSP1cre mice and paired ratio of iTAMs to mTAMs (V). Data are the cumulative results from at least two independent experiments (A, B, D, J–N, and R–V) or are representative of at least two independent experiments with similar results (H, I, O, P, and T–V). All data are shown as mean ± SEM and were analyzed by two-tailed, paired Student’s t test (A, B, and D) and two-tailed, unpaired Student’s t test (H–P, R, and S). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns).