Tumor treating fields (TTFields) delay DNA damage repair following radiation treatment of glioma cells

Abstract

Background: Tumor Treating Fields (TTFields) are an anti-neoplastic treatment modality delivered via application of alternating electric fields using insulated transducer arrays placed directly on the skin in the region surrounding the tumor. A Phase 3 clinical trial has demonstrated the effectiveness of continuous TTFields application in patients with glioblastoma during maintenance treatment with Temozolomide. The goal of this study was to evaluate the efficacy of combining TTFields with radiation treatment (RT) in glioma cells. We also examined the effect of TTFields transducer arrays on RT distribution in a phantom model and the impact on rat skin toxicity.

Methods: The efficacy of TTFields application after induction of DNA damage by RT or bleomycin was tested in U-118 MG and LN-18 glioma cells. The alkaline comet assay was used to measure repair of DNA lesions. Repair of DNA double strand breaks (DSBs) were assessed by analyzing γH2AX or Rad51 foci. DNA damage and repair signaled by the activation pattern of phospho-ATM (pS1981) and phospho-DNA-PKcs (pS2056) was evaluated by immunoblotting. The absorption of the RT energy by transducer arrays was measured by applying RT through arrays placed on a solid-state phantom. Skin toxicities were tested in rats irradiated daily through the arrays with 2Gy (total dose of 20Gy).

Results: TTFields synergistically enhanced the efficacy of RT in glioma cells. Application of TTFields to irradiated cells impaired repair of irradiation- or chemically-induced DNA damage, possibly by blocking homologous recombination repair. Transducer arrays presence caused a minor reduction in RT intensity at 20 mm and 60 mm below the arrays, but led to a significant increase in RT dosage at the phantom surface jeopardizing the "skin sparing effect". Nevertheless, transducer arrays placed on the rat skin during RT did not lead to additional skin reactions.

Conclusions: Administration of TTFields after RT increases glioma cells treatment efficacy possibly by inhibition of DNA damage repair. These preclinical results support the application of TTFields therapy immediately after RT as a viable regimen to enhance RT outcome. Phantom measurements and animal models imply that it may be possible to leave the transducer arrays in place during RT without increasing skin toxicities.

Keywords: DNA damage repair; Glioma; Radiation treatment; Radiosensitization; TTFields.

Fig. 1

Surviving fraction of U-118 MG cells treated with 200 kHz TTFields (1.7 V/cm RMS) for 72 h (a). The efficacy of the combined treatment of TTFields and irradiation with 4 Gy was tested when 72 h TTFields treatment was applied immediately after RT or 1 h, 4 h, and 24 h after RT in U-118 MG cells (b). The efficacy of the combined treatment (c) of TTFields and irradiation with 4 Gy in U-118 MG cells RT (red column) and TTFields (pale blue column) treatments alone were compared with untreated cells (white column) – The combination treatment (dark blue column) was compared with RT alone (red column). Surviving fraction of U-118 MG cells treated with bleomycin alone or in combination with 200 kHz TTFields (1.7 V/cm RMS) for 72 h (d)

Fig. 2

TTFields Delay Irradiation-Induced DNA Damage Repair in glioma cells. U-118 MG cells were irradiated with 4 Gy and immediately treated with TTFields applied for 1 h, 2 h or 24 h (a-b) or treated with bleomycin followed by TTFields application (c-d). Effect on DNA repair was measured as tail moment in the comet assay

Fig. 3

TTFields Treatment after RT causes the Retention of γH2AX Foci formation. a. U-118 MG cells were irradiated with 4 Gy RT and immediately treated with TTFields for 1 h, 2 h, or 24 h. a pATM (pS1981) or total ATM expression was examined by immunoblotting with α-tubulin used to confirm equal loading. b-c Effect of RT (4 Gy), TTFields or their combination on formation and resolution of γH2AX foci was analyzed by immunofluorescence with DAPI used for counterstaining of cell nuclei. Scale bar - 5 μm. In (c) the average γH2AX foci in cells with more than 5 foci were quantified

Fig. 4

TTFields Influence DNA Damage Repair by Homologous Recombination in Glioma Cells. a pDNA-PKcs (pS2056) and total DNA-PK were compared between U-118 MG cells either untreated or treated with RT or TTFields alone or their combination at indicated time points post RT (4 Gy). Lamin B was used as loading control. b U-118 MG cells were transfected with an intact pGL2-Luc vector or vector that was linearized with either HindIII or EcoRI. Luc activity was measured in cells prior and post 24 h TTFields treatment. c-d U-118 MG cells were irradiated with 4 Gy and immediately treated with TTFields for 1 h, 2 h, or 24 h. c Rad 51 foci formation was analyzed by immunofluorescence at 24 h post treatment. Rad 51 foci (Red) and DAPI (blue) stained nuclei are shown. Scale bar - 5 μm. d The average Rad51 foci in cells with more than 5 foci are shown

Fig. 5

Irradiation Absorbance by TTFields Ceramic Transducer Arrays. Insulated ceramic arrays with hydrogel were placed on a solid-state phantom (a). Dosimetry was measured at the depths of 0 mm (b), 20 mm (c), and 60 mm (d) – dimensions in the figure are not to scale. Dosimetry of RT in phantom with or without ceramic TTFields arrays at the phantom surface (e), and at 20 mm (f) and 60 mm (g) are given

Fig. 6

Effect of low dose RT through TTFields ceramic transducer arrays on the rat skin. Ceramic transducer arrays placed on the rat’s dorsal skin (6a-b). The five treatment groups are shown in 6c, Groups 4 and 5 received RT. Effect of TTFields and RT on the weight of non-tumor bearing rats (d). The group average body weight (g) over 2 weeks are shown. Figures e through j show the gross histological assessment of erythema (e) and microscopic histology assessment of inflammation (f), edema (g), hemorrhages (h) and fibrosis (i). Mild (Score 1) to moderate (Score 2) necrosis was observed in the epidermis of all RT groups (j). For scoring criteria please see Table 1