Simple SummaryMedulloblastoma is the most prevalent malignant paediatric brain tumour, where metastasis and recurrence account for 95% of medulloblastoma-associated deaths. Secretion of extracellular vesicles (EVs) has emerged as a pivotal mediator for communication in the tumour microenvironment during metastasis. We investigated whether sEVs and exosomes mediate communication between medulloblastoma cells and their surroundings to drive metastasis. Metastatic exosomes were shown to potentiate medulloblastoma migration via the active protease, matrix metalloproteinase-2 (MMP-2), on their surface, resulting in degradation of the extracellular matrix (ECM) and creating routes for medulloblastoma cells to invade into the surrounding environment. Knockdown of MMP-2 and its activator extracellular matrix metalloproteinase inducer (EMMPRIN) reduced this invasive potential. Our observations also highlight the potential of MMP-2 as a biomarker for metastatic medulloblastoma. Together, our findings reveal unique insights into the pathogenesis of medulloblastoma and highlight the need to explore alternative therapeutic approaches to impair MMP-driven mechanisms of tumour invasion and migration.AbstractExtracellular vesicles (EVs) have emerged as pivotal mediators of communication in the tumour microenvironment. More specifically, nanosized extracellular vesicles termed exosomes have been shown to contribute to the establishment of a premetastatic niche. Here, we sought to determine what role exosomes play in medulloblastoma (MB) progression and elucidate the underlying mechanisms. Metastatic MB cells (D458 and CHLA-01R) were found to secrete markedly more exosomes compared to their nonmetastatic, primary counterparts (D425 and CHLA-01). In addition, metastatic cell-derived exosomes significantly enhanced the migration and invasiveness of primary MB cells in transwell migration assays. Protease microarray analysis identified that matrix metalloproteinase-2 (MMP-2) was enriched in metastatic cells, and zymography and flow cytometry assays of metastatic exosomes demonstrated higher levels of functionally active MMP-2 on their external surface. Stable genetic knockdown of MMP-2 or extracellular matrix metalloproteinase inducer (EMMPRIN) in metastatic MB cells resulted in the loss of this promigratory effect. Analysis of serial patient cerebrospinal fluid (CSF) samples showed an increase in MMP-2 activity in three out of four patients as the tumour progressed. This study demonstrates the importance of EMMPRIN and MMP-2-associated exosomes in creating a favourable environment to drive medulloblastoma metastasis via extracellular matrix signalling.