Fig. 1. TAMCs up-regulate the arginine-polyamine axis in brain tumors.

(A to C) Mice were implanted with 7.5 × 10⁴ CT-2A tumor cells, and after 14 days of tumor engraftment, myeloid cells from the brain (TAMCs) and spleen were isolated via Gr1 magnetic bead isolation and analyzed via LC-MS/MS for metabolomics (A) (table S1) and RNA-seq (B). (A) Normalized peak areas of samples were graphed in MetaboAnalyst (74). RNA-seq data were visualized using GSEA heatmap analysis for arginine metabolic genes (B). (C) Left: Fpkm values comparing hallmark arginine metabolite genes. Right: Metabolite levels in TAMCs versus peripheral myeloid cells. (D) Flow cytometric expression of arginase-1 in matched peripheral versus tumor myeloid cells of both humans and mice. (E) Schematic of the arginine metabolic pathway. (F) In vitro generated CD8⁺ T cells and TAMCs were cultured in ¹³C-arginine SILAC medium for 2 hours before LC-MS/MS. (G) TAMC and CD8⁺ T cells were isolated from tumor-bearing mice and pulsed ex vivo with ¹³C-arginine for 2 hours [4 hours in (H)] and analyzed via LC-MS/MS. (A and B) Each replicate is n = 10 mice pooled per sample, three independent experiments (a total of n = 30 mice). (C) Five matched human and four matched mouse samples were analyzed for arginase-1 expression. (D) n = 4 matched human samples were analyzed for bulk metabolite analysis. Significance was calculated by Student’s t test: **P < 0.01 and ***P < 0.001.