Single-Cell Profiling of Pediatric High-Grade Gliomas Reveals OPC-Like Subpopulations Driving Tumorigenic Lineage Transitions.

Pediatric high-grade gliomas (pHGG) were first defined as a distinct entity in the 2021 fifth edition of the WHO classification of tumors of the central nervous system. These tumors, designated primarily as Grade 4, include the following subtypes: (1) diffuse midline glioma with H3-K27 alterations (DMG, H3-K27M), (2) diffuse hemispheric glioma with H3-G34 mutations (DHG, H3G34M), and (3) diffuse pediatric-type high-grade glioma with wild-type H3 and isocitrate dehydrogenase (pHGG, H3-WT/IDH WT). Clinically, pHGGs are known for their poor outcomes and marked tumor heterogeneity. Despite this, the characteristics of the tumor microenvironment (TME) and the processes governing tumor cell lineage transitions remain incompletely understood. In this study, we used single-cell RNA sequencing (scRNA-seq) to analyze pHGG tumor cells (excluding infant-type hemispheric gliomas). Through comprehensive bioinformatic approaches-including cell proportion analysis, Gene Ontology (GO) enrichment, metabolic activity inference via scMetabolism, proliferation gene scoring, stemness assessment by CytoTRACE2, SCENT, and pseudotime trajectory analysis with Monocle2-we thoroughly investigated the TME features and heterogeneity of these aggressive brain tumors. Our findings highlight the presence of oligodendrocyte precursor cell (OPC)-like subpopulations, with epidermal growth factor receptor (EGFR)-expressing OPC-like cells emerging as a potential tumorigenic origin in diffuse midline gliomas due to their distinct stemness properties. Notably, platelet-derived growth factor receptor alpha (PDGFRA)-positive cells exhibit high specificity in DMG, suggesting greater diagnostic and therapeutic potential than EGFR. Next-generation sequencing (NGS) and multiplex immunofluorescence analyses confirmed their distinct expression pattern, supporting PDGFRA as a key molecular marker. Moreover, OPC-like cells at different differentiation states may drive lineage transitions in DMG. Together, these findings enhance our understanding of pHGG-especially DMG-and point to new avenues for targeted therapy.