Mouse xenograft studies

All procedures were carried out according to the Guide for the Humane Use and Care of Laboratory Animals, and the experiments were approved by the Animal Care and Use Committee of the Children’s Hospital, Boston, MA, USA (protocol reference number: 1589).

Immunodeficient strain CB17SC-M scid (C.B-Igh-1^b/IcrTac-Prkdc^scid) mice were used to generate xenograft tumors. Mice were purchased at the age of 3–4 weeks from Taconic, Germantown, NY, USA. Mice were 6 to 8 weeks old and their weight was between 17 to 25 g when 3 x 10⁶ tumor cells of the cancer cell line SNU-398 in 100 μL medium mixed with 100 μL matrigel were injected subcutaneously in both back flank regions.

Xenograft tumor nodules were followed 3x weekly, and when they reached a minimum diameter of 4mm (14–30 days after injection), drug treatments were initiated. Mice were treated either with INK128 (Intellikine Inc.), rapamycin (LC laboratories), or ganetespib (Synta Pharmaceuticals); or vehicle; or a combination of ganetespib and INK128, or ganetespib and rapamycin. Bodyweight was measured at least 3 times a week. Tumor size was measured with an electronic digital caliper in 2 dimensions (volume = a²xb/2, with a being the greater diameter). For drug administration, INK128 powder was dissolved daily in vehicle (5% NMP, 15% polyvinylpyrrolidone K30, and 80% water); and was administered by gavage 5 days/week at a dose of 1mg/kg and a volume of 100 μL. Rapamycin was prepared as a 20 mg/mL stock solution using 100% ethanol, and was mixed daily with sterile vehicle (0.25% PEG-200, 0.25% Tween-80, and water to achieve a volume of 200 μL for injection. Rapamycin was administered by intraperitoneal injection 3 times/week in a dose of 3 mg/kg. Ganetespib powder was dissolved in DMSO to concentration 50 mg/mL, with heating to 55°C. Ganetespib stock solution was diluted 1:10 in 20% cremophor RH40 (CrRH40)/80% dextrose (D5W). Ganetespib was administered by tail vein injection once/week in a dose of 50 mg/kg. Control mice received vehicle treatment for one of these three drugs.