Methotrexate

All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center (protocol 2011-0118). Unless otherwise stated, 100 freshly dissociated melanoma cells were injected subcutaneously into the right flanks of NSG mice. When tumours became palpable, in some experiments mice were injected subcutaneously with N-acetyl-cysteine (NAC) (Sigma, 200 mg/kg/day in 200 μl PBS, pH 7.4) or PBS as a control. Mice were injected with their last NAC dose 10 minutes before being sacrificed for end point analysis. In experiments where mice received NAC via the drinking water, NAC was dissolved in PBS at 1mg/ml and the water was changed every 2 days. In other experiments methotrexate (Tocris, 1.25 mg/kg/day in 100 μl PBS) was injected intraperitoneally 5 days per week. Mice that received methotrexate were simultaneously administered Thymidine (Sigma, 3 mg/mouse/day in 100 μl PBS) and Hypoxanthine (Sigma, 750 μg/mouse/day in 100 μl PBS) to prevent suppression of nucleotide biosynthesis. Tumour growth was monitored weekly with a caliper. Experiments were terminated when any tumour in the cohort reached 2.5 cm in size. At the end of experiments, blood was collected by cardiac puncture. Organs were analyzed for micrometastases and macrometastases by bioluminescence imaging and visual inspection.