In vivo experimental metastasis

As previously described [28], tumor cells were harvested and prepared in HBSS. For tail vein injection assays, 10⁶ K7M2 cells or 10⁴ MG63.3 cells were intravenously injected into 5–6-week-old female BALB/c or SCID-Beige mice. Mice were either treated at Day 2 after tumor cell injection (early treatment) or at Day 9, when the micro-metastases were established in the lungs (late treatment). Mice were randomly divided into four cohorts (the experiments were repeated 2–3 times with n = 3–9), receiving daily gavage of vehicle (HPBCD, twice/day), CB-839 (200 mg/kg, twice/day), metformin (300 mg/kg, once/day), or combination of CB-839 and metformin. The experiments were terminated after 30 consecutive days of treatment. Lungs of treated mice were inflated and formalin-fixed. The whole lung fluorescent images were acquired via fluorescent stereomicroscopy (Leica MGFLIII). The percent of the lung occupied by metastases area/total lung area was calculated with ImageJ software. Lung metastases were also examined using H&E stained paraffin-embedded sections. Statistical analysis was performed with GraphPad Prism.