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

Simple Summary Small extracellular vesicles (sEVs) released by all cell types function as a mediator in intercellular communication that can promote cell division and survival to remodel the tumor microenvironment to develop tumor invasion and metastasis. Even though dsDNA baggage is associated with all small EV populations, the functional role of EV-DNA in cancer remains poorly understood. This is due to a lack of methods allowing the efficient separation of small EVs (sEVs) from other non-sEV components. The main aim of our study was to develop an efficient sEV isolation method along with EV-associated DNA (EV-DNA) monitoring tool to evaluate the role of EV-DNA as a mediator of cell-cell communication in cancer. Our detailed small EV-DNA characterization confirmed that isolated sEVs using the TSU method (Tangential flow filtration + Size exclusion chromatography + Ultrafiltration) are free from contaminants such as cell-free and apoptotic bodies DNA, making TSU ideal for performing EV-DNA functional studies. Next, we revealed the exact EV-DNA distribution in the recipient cells using 3D image analysis and the association of EV-DNA with key cellular proteins, which may have an essential role in cancer. In the leukemia model, EV-DNA isolated from leukemia cell lines associated with mesenchymal stromal cells (MSCs), a crucial factor in the bone marrow (BM) microenvironment. 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.