Tumor xenograft mouse model and treatment

Protocols involving mice were approved by the Institutional Animal Committee of Taipei Veterans General Hospital. NOD/SCID mice were purchased from the BioLASCO Experimental Animal Center (BioLASCO, Taiwan) and used for experiments at 6–8 weeks of age. Lung cancer cells were subcutaneously injected into mice with different doses and monitored for two months to evaluate tumor initiation. Absolute tumor-initiating frequency was calculated using the L-CalcTM Version 1.1 (Stem Cell Technologies, Vancouver, Canada). After sacrifice, tumors were resected and the expression of collagen XVII, Oct4 or HK2 was evaluated by immunohistochemistry (IHC). Anti-CK7 antibody (#M7018; 1:50, Agilent Dako, Santa Clara, CA), anti-collagen XVII antibody (#ab184996; 1:200, Abcam), anti-Oct4 polyclonal antibody (#2750; 1:50, Cell Signaling), anti-HK2 polyclonal antibody (#2867; 1:200, Cell Signaling) were used as the IHC primary antibodies.

For evaluation of lung metastasis ability, 3 × 10⁵ tumor cells were injected into the tail vein and lung tissue was harvested 12 weeks later. For tumor growth inhibition, 10⁶ tumor cells were subcutaneously injected into mice. Tumor size was measured and tumor volume was calculated as: (tumor volume = length x width²/2). Mice with tumors were randomly grouped when its size approached 50 mm³. For 2-DG treatment, mice were intraperitoneally injected with saline or 2-DG (800 mg/kg in saline, daily for 15 days). For metformin treatment, mice received metformin in drinking water (1 mg/ml) for 15 days. Tumor volume was measured every 3–4 days and tumor weight was determined after sacrifice.