Glioblastomas (GB) can intenerate into neural networks which drives their progression, however, the extent to which GBs integrate is closely related to epigenetic signatures (neural-low and neural-high). The microenvironmental mechanisms facilitating this remain unclear. Here, we dissect the ecosystem of tumor-integrated neurons leveraging a human cortical slice model and multi-modal spatial analysis.

Patient-derived cell lines from neural-low and neural-high GBs (n=6) were injected into human cortical slices (n=3). The signaling activity was evaluated within tissue slices using advanced calcium imaging. Functional connectivity with neurons was visualized using EnvA-pseudotyped rabies virus retrograde tracing and was overlaid with MERFISH spatial transcriptomics, which allowed for direct comparison between the distinct microenvironments of connected and unconnected neurons. We validated our findings using immunohistochemistry and confocal microscopy.

Automated analysis of calcium imaging revealed higher activity in the neural-high tumor cell lines with structurally different signaling patterns. Retrograde tracing showed significantly higher connectivity between malignant cells and neurons in the neural high celllines. The overlay of retrograde tracing with spatial transcriptomics underlined increased functional interactions between neurons and malignant cells in neural-high tumors, which host distinct ecosystems enriched for synaptogenesis-inducing inflammatory myeloid cells. Tumor-connected neurons specifically reside within this inflammatory niche, contrasting sharply with the environment of unconnected neurons. This unique "synaptic ecosystem" drives the increased synaptic density, coupling, and invasiveness characteristic of the neural- high phenotype.

Epigenetically defined neural-high GB establishes a specialized inflammatory microenvironment that orchestrates neuronal connectivity, contributing to accelerated progression, which highlights the neuron-tumor niche as a crucial target for phenotype-specific therapies based on epigenetic profiling.