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

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 b...

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Bibliographic Details
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
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Summary: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.
ISSN:2072-6694
2072-6694
DOI:10.3390/cancers14092068