Mouse studies and xenograft assays

All mouse experiments complied with all relevant ethical regulations and were performed according to protocols approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center (protocol 2016–101360). Melanoma cell suspensions were prepared for injection in staining medium (L15 medium containing bovine serum albumin (1 mg/ml), 1% penicillin/streptomycin, and 10 mM HEPES (pH 7.4) with 25% high-protein Matrigel (product 354248; BD Biosciences)). Subcutaneous injections were performed in the right flank of NOD.CB17-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice in a final volume of 50 μl. Four to 8-week-old male and female NSG mice were transplanted with 100 melanoma cells subcutaneously unless otherwise specified. Mouse cages were randomized between treatments (mice within the same cage had to be part of the same treatment). Both male and female mice were used. Subcutaneous tumor diameters were measured weekly with calipers until any tumor in the mouse cohort reached 2.5 cm in its largest diameter, in agreement with the approved animal protocol. At that point, all mice in the cohort were euthanized and spontaneous metastasis was evaluated by gross inspection of visceral organs for macrometastases and bioluminescence imaging of visceral organs to quantify metastatic disease burden (see details below).

YUMM1.7 (Braf^V600E/+; PTEN^−/−; Cdkn2^−/−), YUMM3.3 (Braf^V600E/+; Cdkn2^−/−), and YUMM5.2 (Braf^V600E/+; p53^−/−) cell lines³⁰ were obtained from and authenticated by ATCC and cell lines were confirmed to be mycoplasma free using the MycoAlert detection kit (Lonza). YUMM1.7, YUMM3.3 and YUMM5.2 were transfected with dsRed2 and luciferase (dsRed2-P2A-Luc) for bioluminescence imaging. Subcutaneous injections of 20,000–50,000 cells were performed in the right flank of 6- to 8-week-old male and female C57/BL6 mice in 50 μl.

For studies that involved treatment with the MCT1 inhibitor (AZD3965, Selleckchem), when subcutaneous tumors became palpable, the mice were administered AZD3965 by oral gavage every second day in xenografted mice and every day for mice transplanted with YUMM cells (30 mg/kg body mass in 200 μl of 0.5% promethylcellulose, 0.2% Tween80 and 5% DMSO). Tumor growth was monitored weekly with a caliper. Mice were euthanized when the primary tumor reached 2.5 cm in its largest diameter. In addition to measuring subcutaneous tumor diameters, the frequency of circulating melanoma cells in the blood (obtained by cardiac puncture) was measured by flow cytometry, and metastatic disease burden was measured by total bioluminescence levels in dissected visceral organs.