Efficient Small Extracellular Vesicles (EV) Isolation Method and Evaluation of EV-Associated DNA Role in Cell-Cell Communication in Cancer

• Published in: Cancers 2022-04, Vol.14 (9), p.2068
• Main Authors: Chetty, Venkatesh Kumar, Ghanam, Jamal, Anchan, Srishti, Reinhardt, Katarina, Brenzel, Alexandra, Gelléri, Márton, Cremer, Christoph, Grueso-Navarro, Elena, Schneider, Markus, von Neuhoff, Nils, Reinhardt, Dirk, Jablonska, Jadwiga, Nazarenko, Irina, Thakur, Basant Kumar
• Format: Article
• Language: English
• Subjects: Apoptosis; Bone marrow; Cancer; Cell culture; Cell division; Cell interactions; Cell membranes; Cell survival; Chromatography; Contaminants; Cytoplasm; Deoxyribonucleic acid; DNA; Extracellular vesicles; Genomes; Image processing; Inflammation; Laboratories; Leukemia; Mesenchyme; Metastases; Microscopy; Mitochondria; Mutation; Proteins; Stromal cells; Tumor cell lines; Tumor microenvironment; Tumors; Ultrafiltration
• ISSN: 2072-6694; 2072-6694
• DOI: 10.3390/cancers14092068

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.