Fig. 2. Polyamines accumulate in TAMCs in GBM and can be efficiently depleted in vivo.

(A) Mice were implanted with 7.5 × 10⁴ CT-2A tumor cells, and after 14 days of tumor engraftment, TAMCs were isolated via Gr1 magnetic bead isolation and CD8⁺ T cells were isolated via CD8β⁺ magnetic bead isolation. (B) In vitro generated TAMCs pretreated with DFMO before being cocultured with decreasing ratios of splenic CD8⁺ T cells labeled with CellTrace Violet dye. (C) The same assay was performed using OT-1 T cells stimulated with SIINFEKL peptide. Forty-eight (C) and 72 (B) hours after coculture, T cell proliferation was analyzed via flow cytometric analysis. (D and E) Mice were implanted with 7.5 × 10⁴ CT-2A tumor cells, and after 5 to 6 days of tumor engraftment, 1% DFMO water was supplied ad libitum to experimental mice. After 7 days of DFMO water treatment, TAMCs were magnetically isolated for bulk metabolomics (D) or pulsed in vitro with ¹³C-arginine for 4 hours before metabolite isolation (E). (F) Schematic overview of polyamine metabolism. (A, D, and E) Each sample is n = 8 to 10 mice pooled per sample, three pooled samples per group. (B and C) Suppressor assays were carried out with n = 3 per each ratio tested, representative of two independent experiments. All statistics in this figure were analyzed by unpaired Student’s t tests: *P < 0.05, **P < 0.01, and ***P < 0.001; ns, not significant. All LC/MS data were normalized to total ion count (TIC). i.c., intracranial.