Fig. 4

Combination therapy with CB-839 and metformin reduces OS tumor growth and inhibits metastasis in vivo. a Primary tumor growth rates with the treatment of vehicle control, CB-839 (200 mg/kg orally twice a day), metformin (300 mg/kg orally once a day), and in combination for 26 days (n ≥ 9 mice). b Primary tumor volumes at day 26 post initial treatment. A significant inhibition of primary tumor growth (***p < 0.001) was observed with the combination treatment. Data displayed as individual points and a red bar indicating mean value (ANOVA followed by multiple comparisons “vehicle vs each treatment”). *p < 0.05, **p < 0.01, ***p < 0.001. c Representative images of tumors from each treatment. d–f For the experimental metastasis model, MG63.3 cells (10⁴) were intravenously injected into SCID mice. Mice were treated as above. The treatments were started at day 2 (d, e) (early treatment) or day 9 (f) (late treatment) for total of 30 days. d Representative lung fluorescent images from each treatment revealing the significant reduction of lung metastasis from mice with the combination treatment. e, f The percent lung metastases area/total lung area from each mouse were calculated as stated in the “Methods” section. CB-839 in combination with metformin significantly inhibited MG63.3 cells lung metastasis when the treatment started as the metastatic cells were just reaching the lung (e, *p = 0.0019) or the micro-metastases already formed in the lung (f, *p = 0.0371). g For the spontaneous metastasis model, MG63.3 cells (10⁶) were injected into mice orthotopically. The treatments started at day 30 after tumor cell injections. Lung metastases areas from each mouse were calculated as stated in the “Methods” section. (Kruskal-Wallis followed by multiple comparisons “vehicle vs each treatment”.) *p < 0.05, **p < 0.01, ***p < 0.001