Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis

Doyle, Laura M.; Wang, Michael Zhuo

doi:10.3390/cells8070727

Cited by 2,039 publications

(2,059 citation statements)

References 153 publications

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“…Thus, we used the alternative method that works with small sample volumes for higher recovery of EVs. An important factor that should be considered for EV characterization isolated using the kit is lipoprotein, which might affect EV purification 60 . In our study, lipoprotein was detected in serum‐derived EVs that could interfere the analysis of EV small RNAs.…”

Section: Discussionmentioning

confidence: 77%

“…Finally, we used the commercially available isolation kit for EV isolation in our study. Ultracentrifugation is commonly used for EV isolation as the gold standard method in research setting 60 ; however, we could not obtain sufficient blood samples for ultracentrifugation. Thus, we used the alternative method that works with small sample volumes for higher recovery of EVs.…”

Section: Discussionmentioning

confidence: 90%

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Predicting the presence of breast cancer using circulating small RNAs, including those in the extracellular vesicles

et al. 2020

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Emerging evidence indicates that small RNAs, including microRNAs (miRNAs) and their isoforms (isomiRs), and transfer RNA fragments (tRFs), are differently expressed in breast cancer (BC) and can be detected in blood circulation. Circulating small RNAs and small RNAs in extracellular vesicles (EVs) have emerged as ideal markers in small RNA‐based applications for cancer detection. In this study, we first undertook small RNA sequencing to assess the expression of circulating small RNAs in the serum of BC patients and cancer‐free individuals (controls). Expression of 3 small RNAs, namely isomiR of miR‐21‐5p (3′ addition C), miR‐23a‐3p and tRF‐Lys (TTT), was significantly higher in BC samples and was validated by small RNA sequencing in an independent cohort. Our constructed model using 3 small RNAs showed high diagnostic accuracy with an area under the receiver operating characteristic curve of 0.92 and discriminated early‐stage BCs at stage 0 from control. To test the possibility that these small RNAs are released from cancer cells, we next examined EVs from the serum of BC patients and controls. Two of the 3 candidate small RNAs were identified, and shown to be abundant in EVs of BC patients. Interestingly, these 2 small RNAs are also more abundantly detected in culture media of breast cancer cell lines (MCF‐7 and MDA‐MB‐231). The same tendency in selective elevation seen in total serum, serum EV, and EV derived from cell culture media could indicate the efficiency of this model using total serum of patients. These findings indicate that small RNAs serve as significant biomarkers for BC detection.

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Section: Discussionmentioning

confidence: 77%

Section: Discussionmentioning

confidence: 90%

Predicting the presence of breast cancer using circulating small RNAs, including those in the extracellular vesicles

et al. 2020

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“…Of note, the use of a caspase-inhibitor to induce necroptosis might interfere with meaningful proteolytic events during this process. In addition, post-translational modifications (PTM), such as phosphorylation, might be missed by the bottom-up MS sample processing approach 97 . Thus, future research should aim to use a necroptotic stimulus that does not include pharmacological caspase inhibition, and also to apply alternative proteomic approaches, e.g., PTM proteomics or semi-tryptic digest of proteins.…”

Section: Discussionmentioning

confidence: 99%

Proteomic analysis of necroptotic extracellular vesicles

Shlomovitz

Arad

Erlich

et al. 2020

Preprint

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Necroptosis is a regulated and inflammatory form of cell death. We, and others, have previously reported that necroptotic cells release extracellular vesicles (EVs). We have found that necroptotic EVs are loaded with proteins, including the phosphorylated form of the key necroptosis-executing factor, mixed lineage kinase domain-like kinase (MLKL). However, neither the exact protein composition, nor the impact, of necroptotic EVs have been delineated. To characterize their content, EVs from necroptotic and untreated U937 cells were isolated and analyzed by mass spectrometry-based proteomics. A total of 3337 proteins were identified, sharing a high degree of similarity with exosome proteome databases, and clearly distinguishing necroptotic and control EVs. A total of 352 proteins were significantly upregulated in the necroptotic EVs. Among these were MLKL and caspase-8, as validated by immunoblot. Components of the ESCRTIII machinery and inflammatory signaling were also upregulated in the necroptotic EVs, as well as currently unreported components of vesicle formation and transport, and necroptotic signaling pathways. Moreover, we found that necroptotic EVs can be phagocytosed by macrophages to modulate cytokine and chemokine secretion. Finally, we uncovered that necroptotic EVs contain tumor neoantigens, and are enriched with components of antigen processing and presentation. In summary, our study reveals a new layer of regulation during the early stage of necroptosis, mediated by the secretion of specific EVs that influences the microenvironment and may instigate innate and adaptive immune responses. This study sheds light on new potential players in necroptotic signaling and its related EVs, and uncovers the functional tasks accomplished by the cargo of these necroptotic EVs.Word count: 14969 of apoptotic bodies 61-63 . A key feature of apoptosis is the exposure of phosphatidylserine (PS) on the outer plasma membrane, which functions as an "eat me" signal, resulting in phagocytosis and clearance of apoptotic cells and bodies. We 64 and others 65,66 have previously revealed that necroptotic cells also expose PS to the outer plasma membrane prior to its rupture. We discovered that PS-exposing necroptotic cells release extracellular vesicles (EVs), which are smaller and contain a higher protein content than apoptotic bodies 64 . Endosomal sorting complexes required for transport (ESCRT) machinery is a family of proteins that plays a role in endosomal protein transport, multivesicular endosome (MVE) formation, and budding 67 . By using a dimerizable RIPK3 or MLKL system, Gong et al. revealed ESCRTIII-dependent shedding of PS-exposing plasma membrane bubbles during necroptosis. ESCRTIII-mediated membrane shedding during necroptosis delayed cell death and enabled longer inflammatory signaling 65,68 . These results underline a major knowledge gap, as the exact protein content, as well as the biogenesis and impact, of necroptotic EVs, have not yet been elucidated. EVs are cell-derived membranous structures, divided into...

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“…Due to their role in cell-to-cell communication, exosomes have in recent years witnessed a growing interest in many fields of research, including oncology. They are 30-150nm-sized vesicles originating from the intraluminal vesicles (ILVs) within the multivesicular bodies (MVBs) as part of the endocytic machinery known as late endosomes [3,39,40]. During this process, proteins, lipids, DNA, messenger RNAs, and non-coding RNAs (ncRNAs), including miRNAs, are selectively sorted and loaded into exosomes [41][42][43].…”

Section: Characteristics Of Extracellular Vesiclesmentioning

confidence: 99%

The Crosstalk between Tumor Cells and the Microenvironment in Hepatocellular Carcinoma: The Role of Exosomal microRNAs and Their Clinical Implications

Pascut

Pratama

Võ

et al. 2020

Cancers

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The communication between hepatocellular carcinoma (HCC) cells and their microenvironment is an essential mechanism supporting or preventing tumor development and progression. Recent evidence has identified extracellular vesicles (EVs) as one of the mechanisms mediating paracrine signaling between cells. Exosomes, the most described class of EVs, deliver proteins, mRNAs, noncoding RNAs, DNA, and lipids to recipient cells, also at remote distances. MicroRNAs (miRNAs), as part of the non-coding RNA exosomal cargo, have an important role in regulating cellular pathways in targeted cells, regulating several processes related to tumor progression invasion and metastasis, such as angiogenesis, immune-escape, epithelial-to-mesenchymal transition, invasion, and multi-drug resistance. Accumulating evidence suggests exosomal miRNAs as relevant players in the dynamic crosstalk among cancerous, immune, and stromal cells in establishing the tumorigenic microenvironment. In addition, they sustain the metastasic niche formation at distant sites. In this review, we summarized the recent findings on the role of the exosome-derived miRNAs in the cross-communication between tumor cells and different hepatic resident cells, with a focus on the molecular mechanisms responsible for the cell re-programming. In addition, we describe the clinical implication derived from the exosomal miRNA-driven immunomodulation to the current immunotherapy strategies and the molecular aspects influencing the resistance to therapeutic agents.

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Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis

Cited by 2,039 publications

References 153 publications

Predicting the presence of breast cancer using circulating small RNAs, including those in the extracellular vesicles

Predicting the presence of breast cancer using circulating small RNAs, including those in the extracellular vesicles

Proteomic analysis of necroptotic extracellular vesicles

The Crosstalk between Tumor Cells and the Microenvironment in Hepatocellular Carcinoma: The Role of Exosomal microRNAs and Their Clinical Implications

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