Xenograft growth delay

After approval by the Animal Care Committee (AL-15-07), xenografts were established as subcutaneous tumors in 4-to-6-week old female nude NIH III mice (Charles Rivers Laboratories, Wilmington, MA, USA) as previously described [20]. Tumor volume was measured twice weekly by digital calipers and calculated using the formula (πab²)/6 (a = largest diameter, b = smallest diameter). When the tumors reached a volume of 200–300 mm³, animals were randomly assigned to the experimental groups. Experiment endpoint was a tumor volume of 2,000 mm³. Seven mice were used in each experimental group.

Radiation was delivered using a Faxitron Cabinet X-ray System at a dose rate of 0.69 Gy/min, tube voltage of 160 KVp, current of 4 mA and filtration with 0.5 mM Al and 0.5 mM Cu. Unanesthetized animals were restrained and protected in custom-made lead jigs with only the right flank exposed and irradiated in groups of 4 in a cross-shape configuration with the flank tumors positioned to the center of the radiation field. Buparlisib (10 mg/kg), binimetinib (5 mg/kg) in 0.5% methylcellulose or their combination was given by oral gavage 4 h post radiation treatment, 5 days/week for three weeks. Sham oral gavage was 0.5% methylcellulose only. Radiation treatment (RT) consisted of 2 Gy/day, five times/week for three weeks for UT-SCC-14. For UT-SCC-15 tumors, the dose per fraction was increased to 3 Gy due to the relative radioresistance of this cell line. For each tumor there were eight treatment groups: (1) control with sham oral gavage (2) RT and sham oral gavage (3) buparlisib (4) RT followed by buparlisib (5) binimetinib (6) RT followed by binimetinib (7) buparlisib in combination with binimetinib (8) RT followed by buparlisib and binimetinib.