Single-cell RNA sequencing reveals intra-tumoral heterogeneity of glioblastoma and a pro-tumor subset of tumor-associated macrophages characterized by EZH2 overexpression

Glioblastoma (GBM) is a highly heterogeneous disease with poor clinical outcome.To comprehensively dissect molecular landscape of GBM and heterogeneous distribution and potential role of Enhancer of zeste homolog 2 (EZH2) in tumor microenvironment (TME).Single-cell RNA sequencing (scRNA-seq) analysis was performed in GBM samples from 8 patients. Deconvolution analysis, immunofluorescence (IF) microscopy, reverse-transcription quantitative polymerase chain reaction (RT-qPCR), colony formation experiments, and Cell Counting Kit-8 (CCK-8) assays were performed to confirmed the potential role of EZH2 in TME cells.Malignant cells exhibited remarkable heterogeneity in abnormal metabolic patterns. A mesenchymal-2-like (MES2-like) GBM subcluster with glial-immune dual feature was firstly discovered, which were associated with highly activated hallmark pathways, immune evasion associated transcription factor (IRF8), and poor survival. The oncogene, EZH2, was heterogeneously expressed in malignant cells and immune cells consistent with proliferative genes, cell-cycle transcription factors, and similar activated hallmark pathways. In a tumor-associated macrophages (TAMs) subset (macrophage.3), EZH2 was highly expressed with similar changes of transcriptomic dynamics with cell-cycle genes and macrophages M2-phetotype genes. In addition, the subset tightly interacted with malignant cells. Deconvolution analysis showed increased abundance of the subset in GBM compared to low-grade glioma (LGG) and significant association with worse prognosis. Functional verification experiments confirmed the pro-tumor role of TAMs with EZH2 overexpression in GBM.Our study illustrated a MES2-like GBM subcluster characterized by glial-immune dual feature and highlighted the pro-tumor role of a TAMs subset characterized by EZH2 overexpression.