Elucidating transcriptomic and functional heterogeneity of microglia in low-grade glioma and glioma-associated epilepsy.

PROJECT SUMMARY/ABSTRACT Low-grade glioma (LGG) is a disease that, despite the modern standard of care, frequently recurs or progresses to high- grade glioma, such as glioblastoma (GBM). Most LGG patients suffer from seizures on presentation and with recurrence. A growing body of work investigating how epileptiform activity affects the glioma microenvironment has suggested that this glioma-associated epilepsy (GAE) drives increased glioma proliferation and invasion by way of direct neuron-glioma signaling. However, other mechanisms by which GAE may drive glioma remain unclear. In contrast to prior work in the field, this proposal focuses on investigating the correlation between GAE and transcriptomic and functional changes in microglia, a cell type known to be critical in driving GBM progression. Microglia are well known to exhibit transcriptomic changes and phagocytic overactivity targeting synapses in temporal lobe epilepsy (TLE). However, despite the well- validated roles this cell type plays in both GBM and TLE, they have remained incompletely explored in both LGG and GAE. Intriguingly, it has been shown that there is dendritic loss in human epileptic peritumoral cortex, and dendritic loss has been shown to be microglia-mediated in many diseases, including epilepsy. In turn, it has also been shown that phagocytosis of neuronal elements drives anti-inflammatory signaling, inducing a set of cytokines that parallel microglial signatures shown to predict poorer prognosis in GBM. This proposal will test the hypothesis that GAE is associated with enrichment of microglial populations exhibiting overactive phagocytosis of synapses. Moreover, it will determine whether phagocytosis of synapses correlates with upregulation of microglial signatures suggestive of pro-tumorigenic function. In aim 1, I will use single-nucleus sequencing and immunohistochemistry or RNAscope to identify microglial subtypes differentially enriched in GAE and LGG and validate my findings in situ. I will then examine correlation of GAE and LGG-associated microglial signatures with clinical outcomes in the TCGA dataset. In aim 2, I will use Single Cell Optical Phenotyping and Expression sequencing (SCOPE-seq) to pair imaging-based measurements of single microglial phagocytic capacity with single-cell RNA-sequencing data from the same cell. I will examine whether GAE is associated with aberrantly elevated microglial synaptic phagocytosis, will identify microglial subtypes that exhibit greater synaptic phagocytic capacity and study whether phagocytosis of synapses drives upregulation of microglial signatures that suggest pro-tumorigenic function or parallel those found to predict worse clinical outcomes in GBM. This project will elucidate how microglial phagocytosis and the microglial transcriptome are differentially perturbed in LGG and GAE and may reveal novel microglial therapeutic targets that may be modulated to slow LGG progression.