OS08.2.A Three layers of neuronal-like mechanisms driving brain tumor invasion

Abstract

Abstract Background Glioblastoma are characterized by their infiltration into the whole brain. Membrane protrusions of glioma cells called tumor microtubes are subcellular structures contributing to glioma cell invasion. They are the anatomical building block to build a functional and therapy-resistant tumor cell network interconnected by tumor microtubes (TMs) that characterizes a glioblastoma cell subpopulation, while other subpopulations appear unconnected to other glioma cells. The biological role of glioblastoma cells lacking connections with each other remains unclear. Furthermore, how neurogliomal synapses influence this subpopulation is unclear. Material and Methods Time-lapse in vivo two-photon microscopy and single-cell RNA-sequencing are combined to characterize functional different subpopulations of glioblastoma cells. Intravital, augmented microscopy, three-dimensional calcium imaging, electrophysiology and volume electron microscopy are used to investigate the role of neuronal invasion mechanisms and the role of neurogliomal synapses for tumor microtube generation and dynamics. Results In-vivo imaging revealed that glioblastoma cells lacking connections to other tumor cells and astrocytes were the main subpopulation driving glioblastoma invasion invasion. These glioblastoma cells were characterised with single-cell RNA-sequencing. This revealed that this subpopulation is enriched for neuronal, neural progenitor-like, and non-mesenchymal-like cell states as previously described. Sparse regions enriched with such tumor-cell and astrocyte-unconnected, invasive glioblastoma cells evolve over time into regions with tumor-cell and astrocyte-connected, glioblastoma cell networks reflected by molecular cell state changes that are reflected in cell state changes in different regions of human glioblastoma. In addition, mechanisms of glioblastoma cell invasion resembled neuronal and neural progenitor patterns during brain development. Lastly, neuronal activity stimulated neurogliomal synapses and subsequently increased glioblastoma cell invasivieness by stimulating generation of new tumor microtubes and enhanced tumor microtube dynamics. Conclusion This study uncovers three novel layers of neuronal features driving glioblastoma cell invasion. We are able to connect molecular, cellular heterogeneity and functional glioblastoma cell states interlinking heterogeneity and dissemination of glioblastoma, two important hallmarks of this disease. Lastly, this study delineates a potential roadmap to clinical translation with the multidimensional characterisation of human glioblastoma.