Tween 80

All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of University of California, San Diego with protocol S15195. Female 4- to 6-week-old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (SCID-NOD) mice were purchased from the UCSD in-house breeding program. Mice were injected subcutaneously in both flanks with either 2×10⁶ or 2.5×10⁶ 92.1 or OMM1.3 cells, respectively. Mice were monitored twice weekly for tumor development. Tumor growth analysis was assessed as LW²/2, where L and W represent length and width of the tumor. 5 mg/ml VS-4718 was prepared in 0.5% carboxymethyl cellulose (CMC) (Sigma-Aldrich; St. Louis, MO) and 0.1% Tween 80 (Sigma-Aldrich) in sterile water. 0.1 mg/ml trametinib was prepared in 5.2% polyethylene glycol 400 (Sigma-Aldrich) and 5.3% Tween 80 (Sigma-Aldrich) in DPBS. Mice were administered 50 mg/kg VS-4718 (Verastem Oncology; Needham, MA) twice daily by oral gavage and 1 mg/kg Trametinib once daily by intra-peritoneal injection (IP); control group was treated with each vehicle. Mice were euthanized at the indicated time points and tumors were isolated for sequencing, histologic, and immunohistochemical evaluation. Results of mice experiments were expressed as mean ± SEM of a total of tumors analyzed.

For radiation studies, mice were implanted with C17-PDX as described and treated every 7 days with a sublethal dose (4 Gy) administered using a traditional cesium-137 source. For studies involving other drugs, mice were implanted with C15-PDX as described. 5-FU (Sigma-Aldrich), olaparib (O-9201; LC Laboratories), and VK-1850 were weighed and transferred to graduated tubes, and formulation reagents were added slowly dropwise in the following order: 3.5% Tween 80 (Sigma), 5% dimethylacetamide (Sigma), 20% PEG400 (Sigma), 20% propylene glycol, 35% PBS, and 16.5% water. Formulated compounds were filter-sterilized. VK-1850 was administered intraperitoneally twice daily; olaparib (20 mg/kg) was administered intraperitoneally once daily; 5-FU (20 mg/kg) was administered intraperitoneally once weekly. Idelalisib (I-7447; LC Laboratories) was weighed, transferred to a graduated tube, and formulated with 2.5% Tween 80 (Sigma) and 97.5% of a 0.5% carboxymethyl cellulose solution and administered orally once daily at a concentration of 30 mg/kg in a dose volume of 10 ml/kg body weight. The vehicle control consisted of formulation reagents without compound. All animals receiving drugs administered intraperitoneally also received the oral vehicle formulation once daily; animals treated with idelalisib also received the intraperitoneal vehicle formulation twice daily.