Abstract
<title>Abstract</title> <p>Background Glioblastoma is the most malignant and the fastest-progressing primary brain tumour. Macrophages and microglia constitute a major component of the glioblastoma microenvironment. Microglia are rapidly reprogrammed during the earliest stages of tumour development, but the mechanisms underlying this process remain unknown. Extracellular vesicles (EVs) have emerged as key mediators of intercellular communication, but their contribution to microglial modulation during tumour initiation remains largely unexplored. Here, we investigated the role of EV-mediated communication in shaping microglial function and tumour progression during the earliest stages of glioblastoma development. Methods Using a live zebrafish model of glioblastoma initiation, we visualised EV release from preneoplastic cells and monitored their uptake by microglia. EV biogenesis and release were inhibited using GW4869, and the effects on microglial phenomics, preneoplastic cell mass, and apoptosis were quantified by confocal imaging. The relationship between microglia and EV signalling was further investigated using pharmacological microglial depletion. Results EVs released from preneoplastic cells (PNCs) were internalized by microglia during tumour initiation. Pharmacological blockade of EV biogenesis and release using GW4869 partially restored ramified microglial morphology and reduced the PNC mass during early tumour development. This was accompanied by an increase in apoptosis and enhanced microglial phagocytic activity, whereas no such effects were observed in control larvae. Combined microglial depletion and EV inhibition slightly increased apoptotic cell number further without an additional decrease in PNC mass, indicating that EV-mediated regulation of tumour development involves both microglia-dependent and microglia-independent mechanisms. Conclusion Our findings demonstrate that EV-mediated communication is established during the earliest stages of glioblastoma initiation and plays a critical role in regulating microglial phenomics and tumour progression.</p>