Response of breast cancer carcinoma spheroids to combination therapy with radiation and DNA-PK inhibitor: Growth arrest without a change in α/β ratio.

PURPOSE Agents that increase tumor radiosensitivity are of interest in improving outcomes in radiotherapy (XRT). DNA-PK inhibitors lead to radiosensitization and also to alterations in cell adhesion proteins. We investigated the combination of radiation and a DNA-PK inhibitor in monolayer vs spheroid culture cells. Materials and Methods: Using a HER2 positive mammary carcinoma cell line, NT2.5, we investigated the impact of NU7441, a potent and selective DNA-PK inhibitor together with external beam irradiation in both 2D monolayer and 3D spheroid cell culture systems. Colony formation assays were performed for cell from monolayer culture cells and spheroids after different dose of external beam irradiation or incubated with NU7441 (5 µM) for 24 h after irradiation. Results: In monolayer culture cells, α/β increased from 3.0 ± 0.2 Gy (XRT alone) to 6.9 ± 0.2 Gy (XRT + NU7441). Corresponding α/β values for cells obtained by disaggregating treated spheroids were 3.6 ± 0.7 Gy (XRT alone) and 3.5 ± 0.2 Gy (XRT + NU7441). In contrast to the minimal change in α/β, spheroids survival was highly sensitive to NU7441 incubation. 21 days after 0, 2, 4, 6, 8, or 10 Gy XRT alone 100, 83, 75, 63, 56, and 31%, respectively, of the irradiated spheroids remained intact; when NU7441 treatment was involved, 56% remained intact at 2 Gy and 13% at 4 Gy, no spheroids survived to 3 weeks at 6 Gy or more. We also found a progressive increase in fragmentation for spheroids exposed only to NU7441. The discrepancy between the minimal change in α/β from cells derived from spheroids and the spheroid growth response was not related to poor penetration of NU7441 since the colony formation assay results for different sizes of spheroids (180 to 400 µm) treated with 4 Gy with or without NU7441were not significantly different. Conclusions: DNA-PK inhibitor NU7441 radiosensitized 2D cultured NT2.5 monolayer cells but not cells obtained from 3D cultured spheroids. The combination, NU7441 and radiation led to an increase in spheroid fragmentation compared to spheroids treated only with radiation.