Targeting the cell-cycle effects of tumor treating fields (ttfields) to increase its efficacy

Abstract Tumor Treating Fields (TTFields) are a novel, non-invasive FDA-approved treatment modality for glioblastoma (GBM) that utilises alternating electric fields. Our research aims to elucidate the effect of TTFields on the cell cycle to advance the understanding of TTFields and find novel targets for increasing its efficacy. We studied the cell cycle progression using live-cell imaging (inovitro Live™) of PIP-FUCCI-transduced GBM cells, EdU incorporation, RPA foci and fiber assay (in U251-MG, SNB-19, A172). These tools allow us to track individual cells evaluate the time spent in each cell cycle phase, and study the ultimate cell fate in control and treatment conditions concerning the cell cycle and replication fork dynamics. Statistical testing was performed by 2-way ANOVA, Tukey-corrected for multiple comparisons, and Kruskal-Wallis test. TTFields causes an immediate delay in S and G2 cell cycle phase, with a subsequent G1 arrest. Wee1 inhibition by AZD1775 forces cells through S-phase and abrogates the subsequent TTFields-induced G2-checkpoint, preventing DNA repair. Combined treatment with TTFields and AZD1775 led to decreased incorporation of EdU and an increase in S-phase-specific phospho-RPA foci. TTFields reduced significantly the DNA fiber length after 12, 24, 48 and 72 hrs of exposure (10.2, 8.3, 7.1 and 5.6 vs 13 um), with also an increase in replication fork asymmetry at these time points (0.56, 0.45, 0.49, and 0.47 vs 0.9 ratio) respectively. CombiningTTFields with either Wee1, ATR, or Chk1 inhibition synergistically decreased clonogenic survival. TTFields induced cell cycle effects with the induction of replication stress, which is aggravated by inhibition of Wee1. Additionally, Wee1, ATR, and Chk1 inhibition dramatically boost the efficacy of TTFields in vitro. This combination strategy has promising clinical potential and will be tested in vivo in different GBM xenografts. Cell signaling and signaling pathways