Tumor Treating Fields Enhance Temozolomide‐Associated Growth Suppression in Glioblastoma Cells and Patient‐Derived Organoids Through Cell‐Cycle Blockade, Apoptosis, and SMAD‐Associated Signaling

Abstract

ABSTRACT Aims Glioblastoma (GBM) remains difficult to treat despite multimodal therapy. Tumor Treating Fields (TTF) disrupts mitosis and provides clinical benefit in GBM, but the molecular responses associated with TTF and the effects of combining TTF with temozolomide (TMZ) remain incompletely defined. Methods TMZ, TTF, and TMZ + TTF were evaluated in U251 and SHG140 GBM cells and patient‐derived organoids. TTF was applied at 200 kHz and 1.75 V/cm RMS. Viability, colony formation, migration‐related phenotypes, apoptosis, cell‐cycle distribution, Western blotting, qRT‐PCR, RNA‐seq, and organoid morphology/immunofluorescence analyses were performed. SB525334 was used to assess SMAD‐associated signaling. Results Combined TMZ + TTF treatment more strongly suppressed cell viability, clonogenic growth, and migration‐related phenotypes than either monotherapy, produced greater disruption of organoid structural integrity, and was accompanied by increased cleaved caspase‐3 activation and apoptotic populations. RNA‐seq identified TTF‐associated transcriptomic changes involving chromatin‐associated processes, DNA replication, and cell‐cycle pathways. TTF was associated with selected SMAD‐related signaling changes, G2/M accumulation, altered checkpoint signaling, and enhanced proapoptotic signaling. SB525334 partially attenuated TTF‐associated SMAD phosphorylation, growth suppression, and cleaved caspase‐3 induction. Conclusion TTF enhances TMZ‐associated antitumor responses in GBM cell lines and organoids, accompanied by cell‐cycle blockade, apoptosis‐associated changes, and selected SMAD‐related signaling responses.