Small and medium/large glioblastoma extracellular vesicles differentially regulate endothelial and mitochondrial function in blood–brain barrier and peripheral vasculature models

Abstract

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 enriched small EVs (sEV) and medium/large EVs (m/lEV) fractions derived from human GBM cell line HROG36 on endothelial barrier integrity (assessed by transendothelial electrical resistance (TEER) and tight junction (TJ) protein analysis), migration (wound healing assay), angiogenic capacity (tube formation on extracellular matrix), and mitochondrial function (assessed by measuring the oxygen consumption rate (OCR)) in human brain microvascular endothelial cells (hCMEC/D3), representing blood–brain barrier (BBB) model, and peripheral endothelial cells (HUVECs). Results: We show that sEV-enriched fractions significantly impaired endothelial barrier integrity in both cell types, as evidenced by reduced TEER and disrupted TJ protein organisation. In contrast, m/lEV-enriched fractions enhanced endothelial cell migration while suppressing angiogenic network formation, resulting in disorganised, shortened capillary-like structures. These pro-migratory and anti-angiogenic effects of m/lEVs were accompanied by a selective suppression of mitochondrial respiration in hCMEC/D3 cells, with no such effect observed in HUVECs, indicating that the functional alterations are linked to altered bioenergetic activity specifically in BBB endothelial cells. Conclusion: Our data indicate that sEV and m/lEV-enriched subpopulations derived from GBM exert distinct vascular effects, underscoring their potential as functionally distinct EV-based biomarkers and informing the development of personalised therapeutic strategies in GBM.