date: 2022-04-22T04:33:17Z
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pdf:docinfo:title: Efficient Small Extracellular Vesicles (EV) Isolation Method and Evaluation of EV-Associated DNA Role in Cell?Cell Communication in Cancer
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Keywords: small extracellular vesicles; pure EVs; EV isolation; EV characterization; exosomes; EV-DNA; cell-free DNA; extracellular dsDNA; EV communication; EV in cancer
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subject: 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.
dc:creator: Venkatesh Kumar Chetty, Jamal Ghanam, Srishti Anchan, Katarina Reinhardt, Alexandra Brenzel, Márton Gelléri, Christoph Cremer, Elena Grueso-Navarro, Markus Schneider, Nils von Neuhoff, Dirk Reinhardt, Jadwiga Jablonska, Irina Nazarenko and Basant Kumar Thakur
dcterms:created: 2022-04-22T02:56:26Z
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title: Efficient Small Extracellular Vesicles (EV) Isolation Method and Evaluation of EV-Associated DNA Role in Cell?Cell Communication in Cancer
Last-Save-Date: 2022-04-22T04:33:17Z
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pdf:docinfo:keywords: small extracellular vesicles; pure EVs; EV isolation; EV characterization; exosomes; EV-DNA; cell-free DNA; extracellular dsDNA; EV communication; EV in cancer
pdf:docinfo:modified: 2022-04-22T04:33:17Z
meta:save-date: 2022-04-22T04:33:17Z
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dc:title: Efficient Small Extracellular Vesicles (EV) Isolation Method and Evaluation of EV-Associated DNA Role in Cell?Cell Communication in Cancer
modified: 2022-04-22T04:33:17Z
cp:subject: 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.
pdf:docinfo:subject: 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.
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pdf:docinfo:creator: Venkatesh Kumar Chetty, Jamal Ghanam, Srishti Anchan, Katarina Reinhardt, Alexandra Brenzel, Márton Gelléri, Christoph Cremer, Elena Grueso-Navarro, Markus Schneider, Nils von Neuhoff, Dirk Reinhardt, Jadwiga Jablonska, Irina Nazarenko and Basant Kumar Thakur
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creator: Venkatesh Kumar Chetty, Jamal Ghanam, Srishti Anchan, Katarina Reinhardt, Alexandra Brenzel, Márton Gelléri, Christoph Cremer, Elena Grueso-Navarro, Markus Schneider, Nils von Neuhoff, Dirk Reinhardt, Jadwiga Jablonska, Irina Nazarenko and Basant Kumar Thakur
meta:author: Venkatesh Kumar Chetty, Jamal Ghanam, Srishti Anchan, Katarina Reinhardt, Alexandra Brenzel, Márton Gelléri, Christoph Cremer, Elena Grueso-Navarro, Markus Schneider, Nils von Neuhoff, Dirk Reinhardt, Jadwiga Jablonska, Irina Nazarenko and Basant Kumar Thakur
dc:subject: small extracellular vesicles; pure EVs; EV isolation; EV characterization; exosomes; EV-DNA; cell-free DNA; extracellular dsDNA; EV communication; EV in cancer
meta:creation-date: 2022-04-22T02:56:26Z
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meta:keyword: small extracellular vesicles; pure EVs; EV isolation; EV characterization; exosomes; EV-DNA; cell-free DNA; extracellular dsDNA; EV communication; EV in cancer
Author: Venkatesh Kumar Chetty, Jamal Ghanam, Srishti Anchan, Katarina Reinhardt, Alexandra Brenzel, Márton Gelléri, Christoph Cremer, Elena Grueso-Navarro, Markus Schneider, Nils von Neuhoff, Dirk Reinhardt, Jadwiga Jablonska, Irina Nazarenko and Basant Kumar Thakur
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