Orthotopic Breast Cancer Mouse Model for PKM2 Evaluation

All animal procedures were conducted under the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) at MD Anderson Cancer Center. Female Nu/Nu nude and BALB/c mice were used as hosts for tumor xenografts and 4T1 syngeneic model, respectively. Briefly, orthotopic breast cancer mouse model was established as previously described (33 (link)). For MDA-MB-468 PKM2^WT and PKM2^Y148F cells (1 × 10⁶ cells in 50 µL medium mixed with 50 µL Matrixgel™ (BD Biosciences) were injected into the left and right mammary fat pad, respectively. For 4T1 syngeneic model, PKM2^WT- and PKM2^Y148F-expressing mouse 4T1-luc cells (5 × 10⁴ cells in 50 µL medium mixed with 50 µL Matrixgel™) were injected to the mammary fat pad. Tumor was measured weekly with a caliper, and tumor volume was calculated by the formula: π/6 × length × width². For drug treatment, mice were treated with daily oral doses 500 mg/kg 2-DG and 10 mg/kg gefitinib for two weeks (5 days per week). Tumor infiltration lymphocyte profiles in excised tumors were analyzed as described in Supplementary Information.