Diversity Of Cellular Communication In Glioblastoma

Abstract BACKGROUND Owing to recent advances in understanding of the active functional states exhibited within glioblastoma (GBM), intra-tumoral cellular signaling has moved into focus of neuro-oncological research. In our study, we aim to explore the diversity of transcellular signaling and investigate correlations to transcriptional dynamics and cellular behavior. MATERIAL AND METHODS Electrophysiological mapping of primary GBM cultures was performed by planar microelectrodes, in conjunction with calcium imaging in a human neocortical section based GBM model. Exposure to conditions that are physiologically present within the tumor was carried out to identify specific signaling cells of interest and signaling diversity presented as response to specific environmental conditions. Transcriptional dynamics and plasticity were examined by means of scRNA-sequencing with CRISPR based perturbation, spatial transcriptomics and deep long-read RNA-sequencing. RESULTS Electrophysiological profiles of primary GBM cell lines revealed highly variable network activity. Despite these different characteristics, all profiled primary cell-lines exhibited characteristics of scale-free networks, confirmed in a human neocortical GBM model. When the GBM was allowed to grow in “in-vivo” like environment, basal activity was significantly increased, owing to interactions with elements within the neural environment. Cellular signaling was directly correlated to changes in the environment, like hypoxia or glutamatergic activation, and total inhibition of electrical signaling was achieved only with a combination of both gap junction and synaptic inhibitors. Using single-cell sequencing and proteomics, we identified several genes related to synaptogenesis that plays a crucial role in network formation and consequently transcellular signaling. CRISPR based perturbation of these genes resulted in alterations in cellular morphology and decreased cellular connectivity, with electrical signaling being significantly attenuated. Single-cell sequencing of perturbed tumor cells in the GBM model revealed a loss of developmental lineages and significant reduction of cellular stress response state. CONCLUSION Our findings highlight the role of electrical signaling in glioblastoma. Cellular stressors induce intercellular signaling, leading to transcriptional adaptation suggesting that there exists a highly complex and powerful mechanism for dynamic transcriptional state adaptation.