Examination of brain tumor-associated astrocytes at single-cell resolution

Abstract

Astrocytes are multifunctional cells of the central nervous system (CNS), are intimately linked into the immune modulatory processes that govern homeostasis and neuroinflammation, and display significant functional heterogeneity. The discovery of specific astrocyte subsets associated with various neurological diseases, including Multiple Sclerosis (MS), has prompted the investigation of astrocyte subsets within the brain tumor microenvironment (TME). As bulk transcriptomic analyses fail to address this question, there is need for high-resolution data to deconvolute complex astrocyte reactive programs found in the TME. To this end, we established a syngeneic model of glioblastoma (GBM) by orthotopic implantation of the GL261 cell line in transgenic C57BL6 mice expressing a fluorescent reporter under the control of the pan-astrocyte marker Aldh1l1. Aldh1l1-expressing astrocytes harvested 15 days after GL261 orthotopic tumor implantation were analyzed by droplet-based single-cell RNA sequencing (DropSeq). We identified 14 distinct astrocyte subsets based on transcriptional similarities, and at least 4 subsets that were specifically enriched in the TME. The most highly expanded astrocyte subset (cluster 0) presented a mixed phenotype with upregulation of both pro- and anti-inflammatory pathways, along with loss of homeostatic neurosupportive functions. We observed strong evidence of interferon-gamma (IFNg) signaling, leading us to hypothesize that cluster 0 astrocytes could represent an analogous subset to the regulatory IFNg-induced TRAIL+ astrocyte population observed in a model of the chronic autoinflammatory disease MS. The cluster 0 signature was similar to that of the previously reported T cell-killing TRAIL+ astrocytes [1]. In vivo, we observed increased TRAIL expression on astrocytes in the TME, along with evidence of transcriptionally activating STAT1 phosphorylation. To validate our findings in mice, we performed single-nucleus RNA sequencing from 10 GBM patients and detected increased expression of IFNg–TRAIL axis components, although cell type identification was not yet possible. By use of an independent cohort of human GBM, we validated the presence of an astrocyte subset associated with early recurrence that showed signs of IFNg signaling. With this study, we aim to shed light on astrocyte subpopulations in the GBM microenvironment and to uncover regulatory pathways, such as immunosuppressive IFNg signaling, that may represent a therapeutic target.