Glioblastoma hijacks neuronal mechanisms for brain invasion.

Glioblastomas are incurable tumors infiltrating the brain. A subpopulation of glioblastoma cells forms a functional and therapy-resistant tumor cell network interconnected by tumor microtubes (TMs). Other subpopulations appear unconnected, and their biological role remains unclear. Here, we demonstrate that whole-brain colonization is fueled by glioblastoma cells that lack connections with other tumor cells and astrocytes yet receive synaptic input from neurons. This subpopulation corresponds to neuronal and neural-progenitor-like tumor cell states, as defined by single-cell transcriptomics, both in mouse models and in the human disease. Tumor cell invasion resembled neuronal migration mechanisms and adopted a Lévy-like movement pattern of probing the environment. Neuronal activity induced complex calcium signals in glioblastoma cells followed by the de novo formation of TMs and increased invasion speed. Collectively, superimposing molecular and functional single-cell data revealed that neuronal mechanisms govern glioblastoma cell invasion on multiple levels. This explains how glioblastoma's dissemination and cellular heterogeneity are closely interlinked.

Autonomous rhythmic activity in glioma networks drives brain tumour growth.

Diffuse gliomas, particularly glioblastomas, are incurable brain tumours1. They are characterized by networks of interconnected brain tumour cells that communicate via Ca2+ transients2-6. However, the networks' architecture and communication strategy and how these influence tumour biology remain unknown. Here we describe how glioblastoma cell networks include a small, plastic population of highly active glioblastoma cells that display rhythmic Ca2+ oscillations and are particularly connected to others. Their autonomous periodic Ca2+ transients preceded Ca2+ transients of other network-connected cells, activating the frequency-dependent MAPK and NF-κB pathways. Mathematical network analysis revealed that glioblastoma network topology follows scale-free and small-world properties, with periodic tumour cells frequently located in network hubs. This network design enabled resistance against random damage but was vulnerable to losing its key hubs. Targeting of autonomous rhythmic activity by selective physical ablation of periodic tumour cells or by genetic or pharmacological interference with the potassium channel KCa3.1 (also known as IK1, SK4 or KCNN4) strongly compromised global network communication. This led to a marked reduction of tumour cell viability within the entire network, reduced tumour growth in mice and extended animal survival. The dependency of glioblastoma networks on periodic Ca2+ activity generates a vulnerability7 that can be exploited for the development of novel therapies, such as with KCa3.1-inhibiting drugs.

Deep intravital brain tumor imaging enabled by tailored three-photon microscopy and analysis.

Intravital 2P-microscopy enables the longitudinal study of brain tumor biology in superficial mouse cortex layers. Intravital microscopy of the white matter, an important route of glioblastoma invasion and recurrence, has not been feasible, due to low signal-to-noise ratios and insufficient spatiotemporal resolution. Here, we present an intravital microscopy and artificial intelligence-based analysis workflow (Deep3P) that enables longitudinal deep imaging of glioblastoma up to a depth of 1.2 mm. We find that perivascular invasion is the preferred invasion route into the corpus callosum and uncover two vascular mechanisms of glioblastoma migration in the white matter. Furthermore, we observe morphological changes after white matter infiltration, a potential basis of an imaging biomarker during early glioblastoma colonization. Taken together, Deep3P allows for a non-invasive intravital investigation of brain tumor biology and its tumor microenvironment at subcortical depths explored, opening up opportunities for studying the neuroscience of brain tumors and other model systems.