Background: Glioblastoma malignancy is strongly driven by the formation of tumor microtubes (TMs), which promote intercellular connectivity and calcium (Ca²⁺) wave propagation. The mechanisms by which Tumor Treating Fields (TTFields) influence these TM-mediated processes remain incompletely understood. This study aims to elucidate the effects of TTFields on the structural and functional organization of tumor cell networks and to assess potential frequency-dependent modulation of signaling pathways, including NF-κB. Methods: A comprehensive set of biological model systems is being utilized, including 2D glioblastoma cell monolayers, 3D brain organoids, and in vivo , awake, head-fixed mouse models with chronic cranial windows for longitudinal imaging. Live-cell imaging with confocal and multiphoton microscopy enables real-time observation of morphological and functional tumor dynamics. Quantification of Ca²⁺ signaling is being performed using Cellpose-based segmentation and custom Python analysis pipelines. Immunohistochemistry and spatial transcriptomics (Visium HD) are currently employed to dissect molecular mechanisms; COMET-based immunofluorescence and RNAscope FISH are planned to enable spatially resolved multi-omics. Results: TTFields induced a marked disruption of glioblastoma network architecture and function. Specifically, treatment resulted in >50% reduction in global GCaMP8s-mediated Ca²⁺ activity, a decrease in pacemaker-like cell populations, and significant reductions in synchronization and Ca²⁺ co-activity within S24 glioblastoma cells in both 2D and 3D models. Conclusion & Outlook: TTFields disrupt glioblastoma network integrity and Ca²⁺ signaling, potentially reducing tumor aggressiveness. Preliminary data from 3D brain tumor organoids and in vivo models support our previous in vitro results regarding TTFields-induced activity changes. Parallel studies in patient-derived organoids explore frequency-dependent signaling effects - including NF-κB and MAPK pathways - via spatial transcriptomic profiling. Together, these efforts aim to further elucidate the mechanistic underpinnings of TTFields action and their impact on glioblastoma plasticity and network organization.