P1754

C57BL/6J (000664), BALB/cJ (000651), Rag1 KO (002216) and OTI transgenic (003831) mice were obtained from the Jackson Laboratory. All mouse procedures were performed under Institutional Animal Care and Use Committee (IACUC)-approved protocols from Vanderbilt University Medical Center and conformed to all relevant regulatory standards. Mice were housed in ventilated cages with at most 5 animals per cage and provided ad libitum food and water. Mice were on 12 hour light/dark cycles which coincided with daylight in Nashville, TN. The mouse housing facility was maintained at 68–76°F and 30–70% humidity. For injectable tumor models, 8–20 week old male and female mice were used. Mice were euthanized if humane endpoint was reached (2cm dimension, ulceration, weight loss >10%). V9302 treatments were administered intraperitoneally twice daily for five days at 25mg/kg for FDG uptake or once at 75mg/kg 3hr prior to ¹⁸F-Gln injection. Rapamycin treatments were administered intraperitoneally daily for four days at 2mg/kg dissolved in 2% DMSO 30% Polyethylene Glycol 300 (Sigma Aldrich 202371), and 5% Tween 80 (sigma Aldrich P1754). Mice were randomized at first day of treatment to control or drug in an unblinded manner, with mice from the same cage receiving different treatments. Sample sizes were chosen based on prior experiments.

Female NOD.Cg-Prkdcscid Il2rgtm1wjl/SzJ mice (commonly known as NOD scid gamma, Jackson Laboratory, Maine), 6 to 8 weeks of age and weighing 18 to 20g, were used in the study of UM cells, housed in appropriate sterile filter-capped cages, and provided food and water ad libitum. All procedures were essentially as previously described (Feng et al., 2014b (link); Schrage et al., 2015 (link); Vaque et al., 2013 (link)). Briefly, exponentially growing cultures were harvested, washed, resuspended in RPMI 1640, and 2 × 10⁶ viable cells were transplanted subcutaneously into the flanks of mice. For tumor growth analysis, tumor volume was assessed as [(LW²/2); where L and W represent the length and the width of the tumor]. The animals were monitored twice weekly for tumor development. Results of animal experiments were expressed as mean ± SEM of a total of tumors analyzed. To administer VS-4718 (Verastem Oncology; Needham, MA) to mice, 10mg/ml VS-4718 was prepared in 0.5% carboxymethyl cellulose (CMC) (C5678, Sigma-Aldrich; St . Louis, MO) 0.1% Tween 80 (P1754, Sigma Aldrich; St. Louis, MO) in sterile water, 100 mg/kg administered via oral gavage twice daily, control group was treated with vehicle.