MULTI-MODAL PROFILING OF PEDIATRIC HIGH-GRADE GLIOMA SINGLE CELLS USING PATCH-SEQ

Abstract Pediatric and adult high-grade gliomas are characterized by extensive intra-tumoral transcriptional heterogeneity. When measured by single cell RNA sequencing, gliomas reveal themselves as continuums of stemness and differentiation programs with important implications for therapy, but to date this transcriptional information has not been directly linked to physiological behaviors of cells. Recent work from our group establishes the electrical integration of glioma cells into neural circuitry. One subpopulation of glioma cells participates in glutamatergic synaptic communication with neurons, and a distinct subpopulation of cells sense and respond to extracellular potassium flux of neuronal networks by an entirely distinct mechanism. Our data support a model in which both modes of electrical communication are critical to glioma growth, but current associations between the electrophysiological properties of a cell, its transcriptional profile and developmental state are correlational in nature. Patch-seq is needed to clarify the relationship between transcriptional profiles of quiescent/cycling stem-like cells and the observed electrophysiological behaviors. Put more simply, patch-seq will clarify where the synaptically-connected glioma cells exist along a developmental hierarchy. METHODS Here, we adapt a recently described technique called patch-seq to record the electrophysiological profiles of individual pediatric high-grade glioma cells by whole cell patch-clamp and subsequently isolate their mRNA for single cell sequencing by smart-seq2 and analysis using Seurat. In this way, we couple electrophysiological and transcriptomic profiles to unambiguously assign functional identities to cells with transcriptional profiles along a developmental hierarchy. RESULTS We report the successful adaptation of patch-seq for use with patient-derived diffuse intrinsic pontine glioma (DIPG) xenografts in acute brain slice preparations, enabling evaluation of single glioma cells integrated in intact neural circuitry. CONCLUSIONS Data synthesizing the electrophysiological and transcriptomic profiles of single glioma cells in the context of the developmental hierarchy will be presented.