Glioblastoma-associated fibroblasts enhance vascular stiffness and confer to poor prognosis

Background: Glioblastoma (GBM) lacks normal fibroblasts but contains cancer-associated fibroblasts (CAFs) that shape the tumor microenvironment (TME). However, their specific functions remain unclear. We identified a distinct subset of CAFs marked by COL6A3, termed glioblastoma-associated fibroblasts (GAFs), which drive vascular dysfunction and hypoxia. correlating with higher tumor grade and poor patient prognosis. We hypothesized that COL6A3-mediated endothelial stiffness exacerbates GBM progression and investigated its therapeutic potential. Methods and results: Using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomic analysis, we characterized the GBM vascular landscape and identified a distinct COL6A3 + GAF subset. COL6A3 + GAFs were enriched in perivascular hypoxic niches and associated with a higher tumor grade and poor prognosis. These GAFs impair vascular function via COL6A3-ITGA1 signaling, leading to increased endothelial stiffness, vascular permeability, and hypoxia. We identified myeloid-derived cells (MDCs) as a potential source of GAFs. CRISPR-Cas9-mediated COL6A3 knockout were used to assess the role and functional significance of COL6A3 in vitro and in vivo. Orthotopic GBM mouse models and functional assays further validated GAF-mediated vascular dysregulation and promoted tumor proliferation. Conclusions: Our study defines COL6A3 + GAFs as a key feature of GBM, driving vascular dysfunction and hypoxi, and suggests COL6A3 as a promising therapeutic target for disrupting fibrosis-angiogenesis crosstalk in GBM.