LAUSR

ABSTRACT Glioblastoma (GBM), the most aggressive primary brain tumour in adults, presents significant challenges due to its universal recurrence and limited survival rates. A key driver of GBM progression is the subpopulation of brain tumour‐initiating cells (BTICs), which contribute to therapy resistance and interact with the tumour microenvironment, particularly the cellular components in the subventricular zone (SVZ). Extracellular vesicles (EVs) are critical mediators of intercellular communication, carrying bioactive molecules, such as proteins and RNAs, that can modulate the behaviour of recipient cells. This study investigates the role of EVs in GBM's communication with non‐cancer cells. We utilised the proximity‐labelling system TurboID to achieve global and unbiased labelling of proteins within GBM‐derived EVs. By inducing TurboID expression in primary‐cultured human BTICs from GBM patients, we achieved efficient biotinylation of EV proteins without compromising vesicle integrity, and performed proteomic analysis of biotinylated proteins, which revealed a diverse cargo within BTIC‐EVs. Our method marks the first implementation of TurboID for unbiased global labelling of EV protein cargo in primary cells. This approach facilitates the investigation of EV‐mediated communication and potential therapeutic targets, contributing to the understanding of GBM's complex interactions with the brain's microenvironment and identification of biomarkers for improved diagnosis and treatment response.