GM-CSF and linoleic acid activate c-Myc regulon and drive SiglecF ^hi TAN development. (A and B) Relative differential transcription factor activity within each neutrophil subset in NRAS/AKT HCC mice (A) and HCC patients (B). (C) Representative histograms and c-Myc levels within SiglecF^– and SiglecF^hi TANs. (D) c-Myc expression assessed by western blot. Proteins were extracted from TANs subsets isolated from five mice. (E–G) SiglecF⁻ and SiglecF^hi TANs were treated with or without 10058-F4 (100 μM). GSEA analysis of genes defining SiglecF^hi TANs (E). Spontaneous TGFβ production (F). RIL175 cells were s.c. injected with different TAN subsets. Tumor volume at day 8. Scale bar represents 1 cm (G). (H) Representative histograms and neutral lipid levels within SiglecF^– and SiglecF^hi TANs. (I) GC-MS analysis of HCC tumors and naïve liver. (J) Relative scores of M-CSF, G-CSF, and GM-CSF signaling within neutrophil subsets from NRAS/AKT HCC mice. (K and L) BM neutrophils from NRAS/AKT HCC mice were cultured overnight with or without the addition of GM-CSF (20 ng/ml) and the indicated fatty acids (50 μM). c-Myc expression (K) and TGFβ1 production (L). (M–O) BM neutrophils from NRAS/AKT HCC mice were cultured overnight with or without the addition of GM-CSF, linoleic acid, or 10058-F4. Heatmap of genes defining SiglecF^hi TANs (M). Schematic diagram of adoptive transfer strategy (N). Tumor volume (O). Each symbol represents one mouse (C, H, K, L, and O) or TANs isolated from two to three mice (F and G). Data are mean ± SEM (F, G, K, L, and O) and are pooled from two (C, F–H, K, and L) or four (O) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Wilcoxon matched-pairs signed rank test (C and H) and one-way ANOVA (F, G, K, L, and O). MFI, median fluorescence intensity; CM, complete media. Source data are available for this figure: SourceData F7.