Sham treatment or ABT-888 was administered 30 minutes prior to irradiation. Anesthetized mice were imaged with BLI and subsequently transported to the small animal radiation research platform (SARRP). Using the guidance software utility of the SARRP, bioluminescent images were co-registered by manual fusion with CBCT images and the isocenter of the tumor was identified and Epacadostat aligned with the central axis of the beam, as previously described 20. Mice were irradiated with the SARRP using 225 kVp x-ray beams at a dose rate of 2.5 Gy/minute using varying collimator widths adapted to the optical image of the tumor (gross tumor volume) plus a 5 mm radial
margin for set up error (planning target volume). Mice underwent BLI twice per week until day 9 and weekly thereafter to assess tumor response and were humanely euthanized when moribund, if they experienced weight gain or loss in excess of 20% of pre-treatment weight, or if tumor burden increased
more than 10-fold as determined by BLI. Two-tailed Student’s t test was utilized to assess statistically significant differences between groups (P < .05). Kaplan-Meier curve was constructed for survival analysis with log-rank test. The effects of increasing doses of radiation and ABT-888, individually and concurrently, on cell viability were assessed to determine levels of radiation dose-enhancement (Figure 1). Significant reductions in cell viability were seen with single-fraction learn more radiation doses exceeding 2 Gy at 2, 4, 6 and 8 days post-treatment. The IC10, IC20 and IC50 of radiation were calculated
to be 0.5 Gy, 2 Gy and 5 Gy, respectively (Figure 1A, 6 days post-treatment). Increasing doses of ABT-888 had little effect on cell viability until doses exceeding 5 μmol/l were used. The IC10 for treatment with ABT-888 alone was calculated to be 10 μmol/l and this dose was utilized for subsequent in vitro studies ( Figure 1B). Significant radiosensitization was noted when ABT-888 was added to cells irradiated with vehicle alone. Co-treatment with 1 μmol/l, 10 μmol/l and 100 μmol/l of ABT-888 led to radiation dose enhancement factors of 1.29, 1.41 and 2.36 (P < .05), MYO10 respectively ( Figure 1C). Minimal intrinsic cytotoxicity was noted when cells were treated with ABT-888 alone at these same doses. Radiation-induced DNA damage results in relatively immediate activation of PARP and accumulation of ribosylated protein products, such as PAR, primarily through single-strand breaks and BER. Therefore, PARP and PAR protein levels were measured as a function of time to assess the impact of treatment with radiation. An immediate and significant increase was noted in PAR levels following treatment with 10 Gy consistent with single-strand DNA damage, which persisted through the 30 minute time point before returning to control levels (Figure 2A).