Small extracellular vesicles (sEVs) play essential roles in intercellular signaling both in normal and pathophysiological conditions. Comprehensive studies of dsDNA associated with sEVs are hampered by a lack of methods, allowing efficient separation of sEVs from free-circulating DNA and apoptotic bodies. In this work, using controlled culture conditions, we enriched the reproducible separation of sEVs from free-circulated components by combining tangential flow filtration, size-exclusion chromatography, and ultrafiltration (TSU). EV-enriched fractions (F2 and F3) obtained using TSU also contained more dsDNA derived from the host genome and mitochondria, predominantly localized inside the vesicles. Three-dimensional reconstruction of high-resolution imaging showed that the recipient cell membrane barrier restricts a portion of EV-DNA. Simultaneously, the remaining EV-DNA overcomes it and enters the cytoplasm and nucleus. In the cytoplasm, EV-DNA associates with dsDNA-inflammatory sensors (cGAS/STING) and endosomal proteins (Rab5/Rab7). Relevant to cancer, we found that EV-DNA isolated from leukemia cell lines communicates with mesenchymal stromal cells (MSCs), a critical component in the BM microenvironment. Furthermore, we illustrated the arrangement of sEVs and EV-DNA at a single vesicle level using super-resolution microscopy. Altogether, employing TSU isolation, we demonstrated EV-DNA distribution and a tool to evaluate the exact EV-DNA role of cell–cell communication in cancer.

The understanding of medulloblastoma (MB) progression is limited by the lack of minimally invasive monitoring methods. Extracellular vesicles (EVs) carrying disease-specific signatures are promising for liquid biopsies, but clinical translation is hindered by inconsistent isolation techniques. This study compares small EVs (sEVs) and their proteomes from blood plasma (BP) and cerebrospinal fluid (CSF) in MB. Using ultrafiltration and size exclusion chromatography (UF-SEC), we isolated sEVs from pediatric patient samples. sEV proteins from matched CSF-BP samples from MB patients (MBCSF/MBBP), healthy BP controls (HCBP), and MB cell lines (MBCL) were analyzed by liquid chromatography-tandem mass spectrometry, subjected to Gene Ontology and Cytoscape analyses, and compared to published MB, CSF, and EV datasets. By optimizing UF-SEC for small volumes, we found that CSF-sEVs are smaller and elute in later SEC fractions. Proteins linked to the extracellular matrix (ECM) were enriched in MBCSF and MBCL, while integrin binding showed inconsistent patterns between MBCSF and MBBP. MBBP and HCBP showed no significant differences. Fourteen proteins from MB datasets were identified in our analysis and primarily enriched in CSF. These findings support CSF-sEVs as more informative than BP-sEVs for MB diagnosis and monitoring, emphasize the need for fluid-specific sEV isolation, and suggest that ECM components and integrins may mediate MB progression.