Mouse tumor studies

All mouse studies were approved by IACUC protocol #1111 at RPCCC. All mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Human metastatic melanoma cells were implanted in the flanks subcutaneously of severe combined immunodeficient (SCID) mice as xenograft experiments. Cells from 1205LuP and 1205LuC were injected subcutaneously into the flanks of the mice at 1 × 10⁶ cells per flank with two injections per mouse. Once the tumors reached 150 cubed millimeters (mm³), treatments with the combination therapy were delivered every two to three days for a total of five treatments. NTP treatment was given as described above with two 30-second treatments given at each application to the mouse skin surface. TPZ was injected at a concentration of 20 μM with a volume of 100 microliter (μL) per injection. In experiments with NTP alone (Figure 1A), NTP treatment was applied to the skin surface every two days up to day 10 (Figure 1A). In another experiment, (Supplementary Figure 3), we compared saline injected tumors to untreated tumors, showing no statistical significance. In a third experimental series (Figure 3C and ​and3D),3D), the tumors were either untreated, or treated with NTP, TPZ, or NTP+TPZ. In all three experiments noted above, the tumors were measured every two days with calipers until day 10. Then, from day 10 to day 16, the mice were untreated. At day 16, the tumors were measured, then excised and either fixed in 10% formalin for histological analysis or frozen at –80°C for protein and RNA analysis. In these experiments, five mice were used per condition with two tumors per mouse for a total of 10 tumors analyzed per condition. The experiment was repeated for three independent experiments. Statistical analysis was performed using the unpaired student t-test with alpha set at 0.05.