Small and Large Glioblastoma Extracellular Vesicles Differentially Regulate Endothelial and Mitochondrial Function in Blood–Brain Barrier and Peripheral Vasculature Models

Purpose. Extracellular vesicles (EVs) released by glioblastoma (GBM) cells circulate systemically and modulate tumour-host interactions, influencing vascular function both within and beyond the tumour microenvironment. The EVs span a size range from tens to hundreds of nanometres; however, the specific effects of distinct GBM-derived EV size subpopulations on endothelial function across different endothelial cell types remain poorly understood. Methods. In this study, we investigated the effects of GBM human cell line HROG36-derived small EVs (sEV) and large EVs (lEV) on barrier integrity (transendothelial electrical resistance (TEER) and tight junction (TJ) protein analysis), migration (wound healing assay), angiogenic capacity (tube formation on extracellular matrix), and mitochondrial respiration (MitoStress assay) in human brain microvascular endothelial cells hCMEC/D3 and peripheral endothelial cells HUVECs. Results. We show that sEV significantly impaired endothelial barrier integrity in both cell types, as evidenced by reduced TEER and disrupted TJ protein organisation. In contrast, lEV enhanced endothelial cell migration while suppressing angiogenic network formation, resulting in disorganized, shortened capillary-like structures. These functional alterations were accompanied by cell-type- and EV-size-specific changes in mitochondrial respiration, with sEV predominantly reducing bioenergetic capacity, whereas lEV primarily affected mitochondrial function in brain endothelial cells. Conclusion. Our data indicate that size-defined GBM-derived EV subpopulations exert distinct vascular effects, providing a rationale for EV size-based biomarkers and personalised therapeutic approaches in GBM.