Early Cellular Responses to Tumor Treating Fields in Glioblastoma

Abstract Background Tumor Treating Fields (TTFields) are a clinically validated therapy that has contributed to improved survival outcomes in glioblastoma patients. A deeper understanding of the underlying cellular mechanisms of TTFields could further advance therapeutic strategies. Here we explore the early impact of TTFields on cell cycle dynamics to inform potential synergistic adjunctive approaches. Methods We analyzed TTFields-induced alterations in cell cycle distribution in synchronized glioblastoma cells via flow cytometry, and in asynchronous cells through live-cell imaging of PIP-FUCCI-transduced cells. Replication stress was assessed via the DNA fiber assay. To investigate potential therapeutic synergies, we evaluated the effects of TTFields together with cell cycle–targeting agents (Chk1, ATR, and Wee1 inhibitors) on clonogenic survival. Finally, we validated the impact of TTFields in tumor-bearing mice using the inovivo™ system. Results TTFields induced a pronounced G2-phase arrest in synchronized glioblastoma cells. Importantly, live-cell imaging revealed that cell cycle progression delays occurred very early after TTFields exposure in S, G2 and G1 cell cycle phases. TTFields triggered replication stress, and pharmacological inhibition of Wee1, ATR, or Chk1 significantly increased the sensitivity of glioblastoma cells to TTFields. Notably, these early alterations in cell-cycle progression and replication dynamics occurred in the absence of overt DNA damage following TTFields exposure in both in vitro and in vivo glioblastoma models. Conclusions TTFields induce early alterations in cell-cycle progression and replication dynamics without evidence of overt DNA damage at the investigated time points in glioblastoma models. These findings identify cell-cycle checkpoint pathways as a potential therapeutic vulnerability and support further evaluation of cell-cycle–targeting agents concurrently with TTFields.