Disrupting glioblastoma networks with tumor treating fields (TTFields) in in vitro models

Abstract Purpose This study investigates the biological effect of Tumor Treating Fields (TTFields) on key drivers of glioblastoma’s malignancy—tumor microtube (TM) formation—and on the function and overall integrity of the tumor cell network. Method Using a two-dimensional monoculture GB cell network model (2DTM) of primary glioblastoma cell (GBC) cultures (S24, BG5 or T269), we evaluated the effects of TTFields on cell density, interconnectivity and structural integrity of the tumor network. We also analyzed calcium (Ca 2+ ) transient dynamics and network morphology, validating findings in patient-derived tumoroids and brain tumor organoids. Results In the 2DTM assay, TTFields reduced cell density by 85–88% and disrupted network interconnectivity, particularly in cells with multiple TMs. A “crooked TM” phenotype emerged in 5–6% of treated cells, rarely seen in controls. Ca 2+ transients were significantly compromised, with global Ca 2+ activity reduced by 51–83%, active and periodic cells by over 50%, and intercellular co-activity by 52% in S24, and almost completely in BG5 GBCs. The effects were more pronounced at 200 kHz compared to a 50 kHz TTFields. Similar reductions in Ca 2+ activity were observed in patient-derived tumoroids. In brain tumor organoids, TTFields significantly reduced tumor cell proliferation and infiltration. Conclusion Our comprehensive study provides new insights into the multiple effects of Inovitro-modeled TTFields on glioma progression, morphology and network dynamics in vitro. Future in vivo studies to verify our in vitro findings may provide the basis for a deeper understanding and optimization of TTFields as a therapeutic modality in the treatment of GB.