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. ABSTRACT: 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

Nanoscale distribution of TLR4 on primary human macrophages stimulated with LPS and ATI

Toll-like receptor 4 (TLR4) plays a crucial role in the recognition of invading pathogens. Upon activation by lipopolysaccharides (LPS), TLR4 is recruited into specific membrane domains and dimerizes. In addition to LPS, TLR4 can be stimulated by wheat amylase-trypsin inhibitors (ATI). ATI are proteins associated with gluten containing grains, whose ingestion promotes intestinal and extraintestinal inflammation. However, the effect of ATI vs. LPS on the membrane distribution of TLR4 at the nanoscale has not been analyzed. In this study, we investigated the effect of LPS and ATI stimulation on the membrane distribution of TLR4 in primary human macrophages using single molecule localization microscopy (SMLM). We found that in unstimulated macrophages the majority of TLR4 molecules are located in clusters, but with donor-dependent variations from ∼51% to ∼75%. Depending on pre-clustering, we found pronounced variations in the fraction of clustered molecules and density of clusters on the membrane upon LPS and ATI stimulation. Although clustering differed greatly among the human donors, we found an almost constant cluster diameter of ∼44 nm for all donors, independent of treatment. Together, our results show donor-dependent but comparable effects between ATI and LPS stimulation on the membrane distribution of TLR4. This may indicate a general mechanism of TLR4 activation in primary human macrophages. Furthermore, our methodology visualizes TLR4 receptor clustering and underlines its functional role as a signaling platform