P04.16.a Deciphering Neural-Immune Reciprocal Interactions in Glioblastoma Using 3d Singel-Cell Spatial Transcriptomics

BACKGROUND The reciprocal interaction between neural and immune components in the tumor microenvironment has emerged as a critical area of study in cancer neuroscience. Recent data has shown the increasing role of myeloid cells in enabling an immunosuppressive microenvironment in glioblastoma. However, the nature and the role of T cells and other immune components forming tumor-immune synapses remain to be fully explored. Here we aim to demonstrate how immunological stimuli and inflammatory changes affect the neural connections within and around glioblastoma cells using advanced spatial transcriptomics. MATERIAL AND METHODS The study employs human cortical slice models, inoculated with tumor cells carrying retrograde tracing viruses, specifically a rabies virus, to trace neuron-tumor connections. This was followed by the inoculation of reactive T cells. We used the MERSCOPE system, which allows for the 3D reconstruction of gene expression signatures in consecutive tissue slices, spanning multiple Z-depths by combining this cutting-edge technology with new segmentation approaches. RESULTS Our spatial transcriptomic analyses revealed different cellular ecosystems in the human cortical slice model. Enhanced inflammatory stimuli can increase local tumor growth and infiltration and neuronal connectivity. We found that inhibition of T cell activation using JAK-STAT inhibition can reverse the reactive hyperconnectivity. We further identified key transcription regulators defining immune cell dynamics and triggering immunological stimuli within tumor-immune synapses. CONCLUSION The integrative multi-omic approach using imaging-based analysis and 3D single-cell spatial transcriptomics technique is a powerful tool that allows a comprehensive understanding of how immune cells influence the neural connectivity and synaptic reactivity within the peritumoral microenvironment.