Title : Single cell RNA sequencing reveals vascular heterogeneity and immune crosstalk in the glioma blood tumor barrier
Abstract:
Background: Glioblastoma (GBM) remains the most devastating primary brain malignancy, with a dismal 5-year survival rate of only 7.2%. The Blood-Brain Barrier (BBB), a specialized neurovascular unit composed of Endothelial Cells (ECs), Peri Cytes (PCs), and astrocytic end-feet, undergoes pathological remodeling into a dysfunctional Blood-Tumor Barrier (BTB) during glioma progression. However, the cellular heterogeneity and dynamic molecular changes underlying this BBB-to-BTB transition remain incompletely characterized.
Methods: We performed a comprehensive secondary analysis of a public single-cell RNA sequencing dataset (GEO: GSE242044), comprising 100,939 cells from normal brain samples and 83,041 cells from glioma samples. Unsupervised clustering, cell type annotation, pseudotime trajectory analysis, and cell-cell communication network inference were conducted using Seurat, Monocle2, and Cell chat pipelines.
Results: Comparative analysis revealed significant compositional shifts in the glioma microenvironment, with endothelial cell proportions increasing from 61.8% to 73.1% (p<0.01) and pericytes decreasing 3.4-fold from 15.9% to 4.7% (p<0.001). Astrocytes exhibited an 11.5-fold increase (0.25% to 2.88%, p<0.001), reflecting reactive astrogliosis. Cell-cell communication analysis demonstrated a dramatic rewiring of interaction networks: While normal tissues were dominated by pericytes-Smooth Muscle Cells (SMCs) crosstalk critical for BBB maintenance, tumor tissues shifted toward astrocytes-ECs interactions driven by pro-angiogenic VEGFA-VEGFR2 signaling.
Pseudotime trajectory analysis identified novel tumor-specific subclusters across all three vascular cell types. Tumor pericytes exhibited distinct transcriptional programs enriched for ECM remodeling, with upregulated TUSC3, DAP, NTMT1, SNF8, and RAP1B. Tumor SMCs underwent a "synthetic phenotype shift" characterized by ribosomal protein upregulation (RPL26, RPL36, RPS23, EEF1A1, RPS2), suggesting translational hyperactivation and loss of contractile function. Notably, endothelial cells diverged into two terminal disease trajectories: A CCND1-driven proliferative pathway and an HSPH1-mediated stress-resistant pathway, with coordinated upregulation of HNRNPAB across both trajectories.
Conclusions: This study reveals previously unrecognized heterogeneity in glioma-associated vascular cells and establishes a "vascular dysfunction triad" model linking pericyte ECM remodeling, SMC synthetic phenotype switching, and EC dual oncogenic activation to BTB disruption. The identified tumor-specific markers (TUSC3, RPL26, CCND1, HSPH1) represent promising therapeutic targets for precision vascular targeting strategies aimed at normalizing the tumor vasculature, enhancing drug delivery, and overcoming therapeutic resistance in glioma.
Keywords: Glioma; Blood-Tumor Barrier; Vascular heterogeneity; Single-Cell RNA sequencing; Tumor microenvironment; Pericytes; Endothelial cells
