Tumor Treating Fields (TTFields) Sensitize Glioma Tumor Cells to Radiation Therapy by Delaying DNA Damage Repair Through Homologous Recombination

Abstract

Tumor treating fields (TTFields) are an established anti-neoplastic treatment modality in patients with Glioblastoma Multiforme brain tumors. TTFields are delivered via noninvasive application of low-intensity (1-3 V/cm), intermediate-frequency (100-300 kHz), and alternating electric fields to the region of the tumor. The combination of TTFields with chemotherapeutic agents has resulted in enhanced anti-neoplastic efficacy across a variety of tumor cell lines, as well as enhanced treatment efficacy in malignant tumors in vivo. To date, no study has examined the outcomes of combining TTFields exposure with radiation therapy (RT) and thus the therapeutic potential of such combination remains unknown. To investigate whether TTFields can improve RT treatment outcomes, we studied glioma cells treated with RT in combination with TTFields. To evaluate the efficacy of combined treatment with TTFields and RT, the surviving fractions of U-118 MG and U-87 MG glioma cells were determined. Cells were treated for 72h with TTFields (1.75 V/cm RMS, 200 kHz) applied immediately, 1, 4 or 24 h after irradiation (IR) in 4Gy or in different doses. Surviving fractions were calculated. For assessment of DNA double strand breaks, cells were stained with anti-γH2AX antibody or anti-Rad51 antibody. Immunoblot analysis was used for the assessment of activation pattern of phospho-ATM (pS1981) and phospho-DNA-PK (pS2056). Functionality of non-homologous end-joining (NHEJ) and micro-homologous DNA damage repair was assayed using pGL2-Luc vector linearized with either HindIII or EcoRI. Luciferase activity was evaluated using the dual luciferase reporter assay system. The radiomimetic agent bleomycin was used in combination with TTFields to specifically assess the effect of TTFields on double strand breaks (DSB). Alkaline comet assay was used to measure DNA lesions in cells treated with TTFields following exposure to bleomycin or RT. RT treatment efficacy was significantly enhanced when combined with TTFields therapy. The combination therapy was found to be most effective when TTFields were applied immediately after exposure to RT. On the molecular level, our results demonstrate aberrant DNA damage repair and extended retention of γH2AX and Rad51 nuclear foci in cells 24 hours after exposure to both modalities. NHEJ and microhomologous DNA damage repair were not significantly affected by TTFields. Our results suggest that in addition to exerting an anti-mitotic effect, TTFields radiosensitize glioma cells by specifically reducing IR induced DNA damage repair by homologous recombination, providing a strong rationale to investigate the use of TTFields in combination with RT in the clinical setting.