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, researchers at the University Hospital Essen 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, the researchers found that EV-DNA isolated from leukemia cell lines communicates with mesenchymal stromal cells (MSCs), a critical component in the BM microenvironment. Furthermore, they illustrated the arrangement of sEVs and EV-DNA at a single vesicle level using super-resolution microscopy. Altogether, employing TSU isolation, these researchers demonstrated EV-DNA distribution and a tool to evaluate the exact EV-DNA role of cell-cell communication in cancer.
Utilization of sEVs for diagnostic and functional studies
(A) Immunoblot analysis of TSU and PSU sEV fractions (F2–F4) obtained from OCI−AML3 and MV4−11 with CD81, TSG101, and calnexin. (B) Comparison of EV-DNA concentration of OCI−AML3 and MV4−11 TSU and PSU sEV fractions (F2–F4). (C) GeneScan-based fragment-length analyses for detecting FLT3−ITD and NPM1 mutations both in genomic DNA and in their corresponding TSU and PSU sEVs. (D) Schematic diagram showing how sEVs obtained from HEK-CD63-GFP stable cell line can be helpful for EV functional studies. (E) Immunofluorescence staining of HeLa cells cultured with HEK293T-CD63-GFP TSU and PSU sEVs. Representative confocal images showing cell nuclei stained by DAPI (blue) and GFP fluorescence (green) indicating CD63+ sEVs (shown by red arrows). Scale bar: 10 µm. (F) Quantitative analysis of GFP fluorescence derived from CD63+ TSU and PSU sEVs (n = 3). (G) Zeta potential of HEK-CD63-GFP TSU and PSU sEVs (n = 3) measured using NTA. Data are shown as the mean ± S.E.M. **** p < 0.0001.