Figure 3. Glutamine antagonism reduces MDSCs by increasing cell death and inhibiting tumor CSF3 secretion.

(A) MDSCs from 4T1 tumor–bearing mice were treated with DON (1 μM) for 24 hours, and the active caspase-3 level was analyzed by immunoblot. Actin was used as loading control. (B–D) 4T1 tumor–bearing mice were treated with JHU083 (1 mg/kg) starting on day 14 after tumor inoculation. (B) After 7 hours of the first treatment and following every daily treatment, active caspase-3 on PMN-MDSCs and Mo-MDSCs from blood was analyzed by flow cytometry at the indicated time points (n = 5/group). (C) Cell numbers and percentages of MDSCs from tumor-infiltrating leukocytes (TIL) were counted and analyzed by flow cytometry (n = 5/group). (D) Serum (n = 16/group) and tumors (n = 4/group) were collected from 4T1 tumor–bearing mice and CSF3 was measured by ELISA (top). After 6 hours of treatment with or without DON (1 μM), Csf3 mRNA levels were measured in 4T1 cells (bottom left) (n = 3 technical replicates). Csf3 mRNA levels were measured from in vivo 4T1 tumor lysates by q-PCR (n = 5 mice) (bottom right). (E) Percentages of PMN-MDSCs and Mo-MDSCs from empty vector (EV) 4T1 or CSF3-overexpressing (OE) 4T1 tumor–bearing mice were analyzed by flow cytometry. (F) C/EBPβ levels were measured by immunoblotting of GFP⁺ sorted tumor cells from 4T1 tumor–bearing mice (left) and 4T1 tumor cells with or without DON (1 μM) treated for 24 hours (right). (G) PMN-MDSCs and Mo-MDSCs from EV 4T1 or C/EBPβ-OE 4T1 tumor–bearing mice were analyzed by flow cytometry. Data are representative of at least 2 (E–G) or 3 (A–D) independent experiments and are presented as the mean ± SD. NS, not significant. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001 by 2-way ANOVA with post hoc multiple t tests (B, C, E, and G) or unpaired t test (D).