Local pO2 study in murine tumors

Before ITPP treatment, animals were randomized into two groups (ITPP or Saline). In one set of experiments (4T1 tumors, mice injected with 3 ITPP doses), animals were additionally randomized based on preliminary pO2 level in the tumor, to equalize normoxic and hypoxic tumors population. Four different protocols of ITPP treatments were studied. 4T1 tumors were subjected to either 3 (8, 12 and 16 days after tumor inoculation, 1.5 g/kg, IP) or 4 (8, 9, 15, 16 days after tumor inoculation, 1.5 g/kg, IP) doses of ITPP. B16F10 tumors were treated with 6 ITPP injections on either 5, 6, 11, 12, 17, 18 or 5, 6, 12, 13, 19, 20 days after tumor inoculation, all as an intraperitoneal dose of 2 g/kg. These treatment protocols were designed based on earlier results by Tran et al. and pilot experiments. The treatment schedules were adjusted to the tumor growth rate. Mice were randomized into two groups based on ITPP or saline (control, vehicle for ITPP) administration for each protocol: 4T1 tumors treated with 3ITPP doses: N=10 for ITPP and N=10 for control; 4 ITPP doses protocol N=10 for ITPP group and N=9 for saline-treated animals; for B16F10 tumors received six doses at day 5,6,11,12,17,18 there were N=11 for ITPP and N=7 for the control group; second 6 dose protocol (injections at day 5,6,12,13,19,20) has N=6 for ITPP group and N=5 for saline-treated animals. In vivo EPR measurements were conducted at L-band (Bruker Elexsys-II E540, Germany) CW EPR spectrometer using a surface coil (Bruker, Germany). A high-resolution ultrasound (US) imaging system designed for small experimental animals (Visual Sonics Vevo 2100, Canada) with an MS-550D transducer with a center operating frequency of 22–55 MHz was used for ultrasound measurements of 4T1 tumors. To better understand ITPP effects on tissue oxygenation, tumors were classified based on local pO2 values before treatment as normoxic (>10 mmHg) or hypoxic (<10 mmHg). Additionally, to investigate changes in tumor oxygenation during the experimental time, each local pO2 value for the selected time point was classified as normoxic (N) and hypoxic (H).