Figure 1. Glutamine antagonism inhibits tumor growth and lung metastasis in an immune-dependent manner.
(A–F) 4T1 cells (1 × 105) were implanted subcutaneously into the mammary fat pad of BALB/cJ female mice. On days 7, 10, 13, 17, and 24, mice were injected i.p. with 250 μg anti-PD1 and/or 100 μg anti-CTLA4 antibodies. 4T1 tumor–bearing mice were treated with vehicle (NT) or JHU083 (1 mg/kg) starting on day 7 after tumor inoculation. After 7 days of treatment, a lower dose (0.3 mg/kg) of JHU083 was used. (A) Tumor size was monitored (n = 5/group). (B) On day 17, tumor weight was measured. (C) The structure of the glutamine antagonist prodrug, JHU083. 6-Diazo-5-oxo-L-norleucine (DON) is depicted in black and its ethyl and 2-amino-4-methylpentanamido promoieties are depicted in blue and red, respectively. (D–F) Whole lungs were harvested, and spontaneous lung metastases were analyzed. (D and E) To quantify tumor nodules, on day 30, lungs were inflated with 15% India ink. (D) Representative picture of lungs. (E) Quantification of tumor nodules in lungs (n = 16–19/group, 3 experiments combined). (F) Representative histology sections stained with H&E. (G) 4T1 cells (1 × 105) were implanted subcutaneously into the mammary fat pad in WT BALB/cJ, RAG1-KO, or NSG female mice. 4T1 tumor–bearing mice were treated with JHU083 (1 mg/kg) daily starting on day 7 after tumor inoculation. After 7 days of treatment, a lower dose (0.3 mg/kg) of JHU083 was used. Tumor burden and survival were assessed (n = 5/group). Data are representative of at least 3 independent experiments and are presented as the mean ± SD. NS, not significant. **P < 0.001; ****P < 0.001 by 2-way ANOVA with Tukey’s multiple-comparisons post hoc test (A), Kruskal-Wallis test with Dunn’s multiple-comparisons post hoc test (B), Mann-Whitney test (E), or log-rank (Mantel-Cox) test (G).