Author efforts: J

Author efforts: J.M., A.R., and M.S.L. of = 30 mice). (C) Five matched human and four matched mouse samples were analyzed for arginase-1 expression. (D) = 4 matched human samples were analyzed for bulk metabolite analysis. Significance was calculated by Students test: **< 0.01 and ***< 0.001. Statistical analysis revealed that TAMCs exhibit a 3.27-fold increase in ornithine compared to splenic myeloid cells. There was a concomitant decrease in intracellular arginine levels, indicating strong arginine catabolism (Fig. 1C). Flow cytometric analysis confirmed the up-regulation of arginase-1 in both mouse and human TAMCs compared to peripheral myeloid cells (Fig. 1D). This is consistent with previous studies of arginase-1 expression in TAMCs in glioma (< 0.001). Similarly, spermidine levels were 3.82-fold up-regulated in TAMCs (1.02 108 6.4 106) versus spleens (2.7 107 2.5 106; < 0.001). In CD8+ T cells, there was a pattern toward a decrease of putrescine in tumors (= 0.1), with an increase in spermidine levels in tumors (0.12-fold increase; < 0.05). The role of polyamines in myeloid immunosuppression has been resolved previously, as Yu (= 8 to 10 mice pooled per sample, three pooled samples per group. (B and C) Suppressor assays were carried out with = 3 per each ratio tested, representative of two impartial experiments. All statistics in this physique were analyzed by unpaired Students assessments: *< 0.05, **< 0.01, and ***< 0.001; ns, not significant. All LC/MS data were normalized to total ion count (TIC). i.c., intracranial. To determine whether DFMO can block polyamine generation in vivo, we implanted mice with CT-2A and, after 5 days of tumor engraftment (which was sufficient time for tumor establishment as verified by neuropathological examination), administered 1% DFMO in their drinking water ad libitum. After 7 days of water treatment, TAMCs were isolated and compared to splenic myeloid cells using LC-MS/MS (Fig. 2D). While splenic myeloid cells showed no changes in polyamine content, TAMCs had significant reductions in their polyamine content. This suggests that de novo polyamine generation is required only within the TME. To understand whether this reduction is specific to the arginine-ornithine-polyamine axis, we performed a 4-hour 13C-arginine relative isotopic incorporation ex vivo (Fig. 2E). While there was no difference in the amount of 13C-labeled ornithine in DFMO-treated animals (suggesting that M+5 ornithine incorporation is at steady state), the amount GSK1292263 of labeled putrescine was almost entirely diminished in the TAMCs of DFMO-treated mice (< 0.001; Fig. 2E). There was no change in 13C-labeled putrescine in peripheral myeloid cells, supporting a tumor-specific phenomenon. To address the possibility of steady-state labeling, we performed a NEDD9 13C-arginine metabolite flux analysis over multiple time points and found that ornithine flux was reduced in DFMO-treated animals at 1 hour (< 0.001), while it remained steady GSK1292263 after 2 hours (fig. S3B). Putrescine labeling occurred beginning at 4 hours of flux in which DFMO-treated animals never had putrescine labeling (fig. S3C). These facts suggest that DFMO treatment stymies arginase activity, while it abolishes ODC1 activity. There was no change in 13C-labeled urea cycle metabolites, confirming the RNA-seq and bulk metabolomics data (fig. S4). This suggests that the urea cycle/iNOS pathway is usually inactive in TAMCs in glioma. We also analyzed the bulk metabolites that significantly changed by DFMO treatment in TAMCs (fig. S5) to determine other effects of polyamine inhibition. We found a broad array of metabolites down-regulated by DFMO treatment that was impartial of arginine metabolism, such as < 0.001), as indicated in Fig. 3A. Considering that ODC1 is usually broadly expressed in most brain tumors and inversely correlated with GSK1292263 patient survival (fig. S6), there is a possibility that inhibition of ODC1.