Extracellular Vesicles In Circulation Research Articles

Extracellular vesicles: A potential new way to assess cholestasis.

Extracellular vesicles (EVs) are small, nonreplicating, lipid-encapsulated nanoparticles that carry protein and nucleic acid cargo derived from their tissue of origin. Due to their capacity to provide comparable insights to solid organ biopsy through a minimally invasive collection procedure, EVs provide an attractive biomarker source. This review will provide an insight, how EVs in circulation may provide a novel way to assess cholestasis and will address the possibility of getting a better understanding of the mechanisms of cholestasis of pregnancy through the use of serial hepatic-specific EVs as a window.

Engineering of dendritic cell bispecific extracellular vesicles for tumor-targeting immunotherapy

Advances in the development of therapeutic extracellular vesicles (EVs) for cancer immunotherapy have allowed them to emerge as an alternative to cell therapy. In this proof-of-concept work, we develop bispecific EVs (BsEVs) by genetically engineering EV-producing dendritic cells (DCs) with aCD19 scFv and PD1 for targeting tumor antigens and blocking immune checkpoint proteins simultaneously. We find that these bispecific EVs (EVs-PD1-aCD19) have an impressive ability to accumulate in huCD19-expressing solid tumors following intravenous injection. In addition, EVs-PD1-aCD19 can remarkably reverse the immune landscape of the solid tumor by blocking PD-L1. Furthermore, EVs-PD1-aCD19 can also target tumor-derived EVs in circulation, which prevents the formation of a premetastatic niche in other tissues. Our technology is a demonstration of bispecific EV-based cancer immunotherapy, which may inspire treatments against various types of tumors with different surface antigens and even a patient-tailored therapy.

CNS endothelial derived extracellular vesicles are biomarkers of active disease in multiple sclerosis

BackgroundMultiple sclerosis (MS) is a complex, heterogenous disease characterized by inflammation, demyelination, and blood–brain barrier (BBB) permeability. Currently, active disease is determined by physician confirmed relapse or detection of contrast enhancing lesions via MRI indicative of BBB permeability. However, clinical confirmation of active disease can be cumbersome. As such, disease monitoring in MS could benefit from identification of an easily accessible biomarker of active disease. We believe extracellular vesicles (EV) isolated from plasma are excellent candidates to fulfill this need. Because of the critical role BBB permeability plays in MS pathogenesis and identification of active disease, we sought to identify EV originating from central nervous system (CNS) endothelial as biomarkers of active MS. Because endothelial cells secrete more EV when stimulated or injured, we hypothesized that circulating concentrations of CNS endothelial derived EV will be increased in MS patients with active disease.MethodsTo test this, we developed a novel method to identify EV originating from CNS endothelial cells isolated from patient plasma using flow cytometry. Endothelial derived EV were identified by the absence of lymphocyte or platelet markers CD3 and CD41, respectively, and positive expression of pan-endothelial markers CD31, CD105, or CD144. To determine if endothelial derived EV originated from CNS endothelial cells, EV expressing CD31, CD105, or CD144 were evaluated for expression of the myelin and lymphocyte protein MAL, a protein specifically expressed by CNS endothelial cells compared to endothelial cells of peripheral organs.ResultsQuality control experiments indicate that EV detected using our flow cytometry method are 0.2 to 1 micron in size. Flow cytometry analysis of EV isolated from 20 healthy controls, 16 relapsing–remitting MS (RRMS) patients with active disease not receiving disease modifying therapy, 14 RRMS patients with stable disease not receiving disease modifying therapy, 17 relapsing-RRMS patients with stable disease receiving natalizumab, and 14 RRMS patients with stable disease receiving ocrelizumab revealed a significant increase in the plasma concentration of CNS endothelial derived EV in patients with active disease compared to all other groups (p = 0.001). Conclusions: For the first time, we have identified a method to identify CNS endothelial derived EV in circulation from human blood samples. Results from our pilot study indicate that increased levels of CNS endothelial derived EV may be a biomarker of BBB permeability and active disease in MS.

Characterization and Therapeutic Use of Extracellular Vesicles Derived from Platelets.

Autologous blood products, such as platelet-rich plasma (PRP), are gaining increasing interest in different fields of regenerative medicine. Although growth factors, the main components of PRP, are thought to stimulate reparation processes, the exact mechanism of action and main effectors of PRP are not fully understood. Plasma contains a high amount of extracellular vesicles (EVs) produced by different cells, including anucleated platelets. Platelet-derived EVs (PL-EVs) are the most abundant type of EVs in circulation. Numerous advantages of PL-EVs, including their ability to be released locally, their ease of travel through the body, their low immunogenicity and tumourigenicity, the modulation of signal transduction as well as the ease with which they can be obtained, has attracted increased attention n. This review focuses briefly on the biological characteristics and isolation methods of PL-EVs, including exosomes derived from platelets (PL-EXOs), and their involvement in the pathology of diseases. Evidence that shows how PL-EVs can be used as a novel tool in medicine, particularly in therapeutic and regenerative medicine, is also discussed in this review.

In Situ Membrane Biotinylation Enables the Direct Labeling and Accurate Kinetic Analysis of Small Extracellular Vesicles in Circulation.

Circulating small extracellular vesicles (sEVs) are naturally occurring nanosized membrane vesicles that convey bioactive molecules between cells. Conventionally, to evaluate their behaviors in vivo, circulating sEVs have to be isolated from the bloodstream, then labeled with imaging materials in vitro, and finally injected back into the circulation of animals for subsequent detection. The tedious isolation-labeling-reinfusion procedures might have an undesirable influence on the natural properties of circulating sEVs, thereby changing their behaviors and the detected kinetics in vivo. Herein, we proposed an in situ biotinylation strategy to directly label circulating sEVs with intravenously injected DSPE-PEG-Biotin, aiming to evaluate the in vivo kinetics of circulating sEVs more biofriendly and accurately. Such an analysis strategy is free of isolation-labeling-reinfusion procedures and has no unfavorable influence on the natural behaviors of sEVs. The results showed that the lifetime of generic circulating sEVs in mice was around 3 days. Furthermore, we, for the first time, revealed the distinct in vivo kinetics of circulating sEV subpopulations with different cell sources, among which erythrocyte-derived sEVs showed the longest lifespan. Moreover, compared with circulating sEVs in situ or used as autograft, circulating sEVs used as allograft had the shortest lifetime. In addition, the in situ biotinylation strategy also provides a way for the enrichment of biotinylated circulating sEVs. In summary, this study provides a novel strategy for in situ labeling of circulating sEVs, which would facilitate the accurate characterization of their kinetics in vivo, thereby accelerating their future application as biomarkers and theranositic vectors.

Effects of an acute bout of exercise on circulating extracellular vesicles: tissue-, sex-, and BMI-related differences.

Exercise is recognized to evoke multisystemic adaptations that, particularly in obese subjects, reduce body weight, improve glucometabolic control, counteract sarcopenia, and lower the risk of cardiometabolic diseases. Understanding the molecular and cellular mechanisms of exercise-induced benefits is of great interest due to the therapeutic implications against obesity. The aim of the present study was to evaluate time-related changes in size distribution and cell origin of extracellular vesicles (EVs) in obese and normal-weight subjects who underwent a moderate-intensity exercise on a treadmill (at 60% of their VO2max). Blood samples were drawn before, immediately at the end of the exercise and during the postexercise recovery period (3 and 24 h). Circulating EVs were analyzed by a nanoparticle tracking analysis and flow cytometry after labeling with the following cell-specific markers: CD14 (monocyte/macrophage), CD61 (platelet), CD62E (activated endothelium), CD105 (total endothelium), SCGA (skeletal muscle), and FABP (adipose tissue). In all subjects, acute exercise reduced the release of total (i.e., 30-700 nm) EVs in circulation, predominantly EVs in the microvesicle size range (i.e., 130-700 nm EVs). The postexercise release of microvesicles was higher in normal-weight than obese subjects; after exercise, circulating levels of exosomes (i.e., 30-130 nm EVs) and microvesicles were, respectively, lower and higher in females than males. In all experimental subgroups (males vs. females and obese vs. normal-weight subjects), acute exercise reduced and increased, respectively, CD61 + and SCGA + EVs, being the effect on CD61 + EVs prolonged up to 24 h after the end of the test with subjects in resting conditions. Total EVs, exosomes, and CD61 + EVs were associated with HOMA-IR. Though preliminary, the results of the present study show that a single bout of acute exercise modulates the release of EVs in circulation, which are tissue-, sex-, and BMI specific, suggesting that the exercise-related benefits might depend upon a complex interaction of tissue, endocrine, and metabolic factors.

Characterization of plasma-derived protoporphyrin-IX-positive extracellular vesicles following 5-ALA use in patients with malignant glioma.

SummaryBackgroundMalignant gliomas are rapidly progressive brain tumors with high mortality. Fluorescence guided surgery (FGS) with 5-aminolevulinic acid (5-ALA) provides fluorescent delineation of malignant tissue, which helps achieve maximum safe resection. 5-ALA-based fluorescence is due to preferential accumulation of the fluorophore protoporphyrin-IX (PpIX) in malignant glioma tissue. Additionally, gliomas cells release extracellular vesicles (EVs) which carry biomarkers of disease. Herein, we performed animal and human studies to investigate whether 5-ALA dosed glioma cells, in vitro and in vivo, release PpIX positive EVs in circulation which can be captured and analyzed.MethodsWe used imaging flow cytometry (IFC) to characterize PpIX-positive EVs released from 5-ALA-dosed glioma cells, glioma-bearing xenograft models, as well as patients with malignant glioma undergoing FGS.FindingsWe first show that glioma cells dosed with 5-ALA release 247-fold higher PpIX positive EVs compared to mock dosed glioma cells. Second, we demonstrate that the plasma of glioma-bearing mice (n = 2) dosed with 5-ALA contain significantly higher levels of circulating PpIX-positive EVs than their pre-dosing background (p = 0.004). Lastly, we also show that the plasma of patients with avidly fluorescent tumors (n = 4) undergoing FGS contain circulating PpIX-positive EVs at levels significantly higher than their pre-dosing background (p = 0.00009) and this rise in signal correlates with enhancing tumor volumes (r 2 = 0.888).InterpretationOur findings highlight the potential of plasma-derived PpIX-positive EV-based diagnostics for malignant gliomas, offering a novel liquid biopsy platform for confirming and monitoring tumor status.

Bio-Inspired NanoVilli Chips for Enhanced Capture of Tumor-Derived Extracellular Vesicles: Toward Non-Invasive Detection of Gene Alterations in Non-Small Cell Lung Cancer.

Tumor-derived extracellular vesicles (EVs) present in bodily fluids are emerging liquid biopsy markers for non-invasive cancer diagnosis and treatment monitoring. Because the majority of EVs in circulation are not of tumor origin, it is critical to develop new platforms capable of enriching tumor-derived EVs from the blood. Herein, we introduce a biostructure-inspired NanoVilli Chip, capable of highly efficient and reproducible immunoaffinity capture of tumor-derived EVs from blood plasma samples. Anti-EpCAM-grafted silicon nanowire arrays were engineered to mimic the distinctive structures of intestinal microvilli, dramatically increasing surface area and enhancing tumor-derived EV capture. RNA in the captured EVs can be recovered for downstream molecular analyses by reverse transcription Droplet Digital PCR. We demonstrate that this assay can be applied to monitor the dynamic changes of ROS1 rearrangements and epidermal growth factor receptor T790M mutations that predict treatment responses and disease progression in non-small cell lung cancer patients.

Proteomic Characterization of Epithelial-Like Extracellular Vesicles in Advanced Endometrial Cancer.

Endometrial cancer (EC) is the most frequent gynecological cancer. Tumor dissemination affecting ∼20% of EC patients is characterized at the primary carcinoma by epithelial-to-mesenchymal transition (EMT) associated with myometrial infiltration. At distant sites, the interaction of circulating tumor cells (CTCs) with the microenvironment is crucial for metastatic colonization, with a participation of the extracellular vesicles (EVs). We comprehensively approached these primary and secondary sites to study the impact of tumor EVs on the metastatic efficiency of CTCs in EC. Tumor EVs in circulation reproduce the epithelial phenotype predominant in the primary carcinoma, whereas CTCs are characterized by an EMT phenotype. We modeled this EMT-related clinical scenario in the Hec1A endometrial cell line and characterized the epithelial-like EVs in circulation by SILAC proteome analysis. The identification of proteins involved in cell-cell and cell-matrix interaction and binding, together with in vitro evidence of an improved adhesion of CTC to a functionalized endothelium, suggests a contribution of the epithelial-like EVs in the homing of CTCs at metastatic sites. Accordingly, adhesion protein LGALS3BP was found to be significantly enriched in circulating EVs from a cohort of EC patients with a high risk of recurrence by targeted proteomics (multiple reaction monitoring), highlighting its potential in liquid biopsy in EC.

Prognostic value of glypican-1 for patients with advanced pancreatic cancer following regional intra-arterial chemotherapy.

Regional intra-arterial chemotherapy (RIAC) is a potential alternative treatment for advanced pancreatic cancer (APC) with fewer adverse effects than other treatment options. However, specific biomarkers to determine the prognosis of patients with APC have thus far, been unsatisfactory. Glypican-1 (GPC1) in exosomes has been identified as an early diagnostic biomarker for pancreatic adenocarcinoma. The aim of the present study was to investigate whether the presence of GPC1 in extracellular vesicles (EVs) could serve as a predictor of RIAC outcome for patients with APC. EVs in circulation were isolated and the percentage of GPC1+ EVs was measured using flow cytometry. Compared with healthy individuals, the levels of GPC1+ EVs were significantly increased in patients with APC (P<0.01). Following RIAC treatment, the percentage of GPC1+ EVs was decreased (P=0.023). Furthermore, patients with APC exhibiting a greater decrease of GPC1+ EVs experienced improved overall survival (OS) rates. In summary, the present study provides insights into identifying GPC1 as a novel prognostic biomarker for patients with APC following RIAC treatment.

Inflammaging and Frailty Status Do Not Result in an Increased Extracellular Vesicle Concentration in Circulation

In the last decades extracellular vesicles (EVs) have emerged as key players for intercellular communication. In the case of inflammation, several studies have reported that EV levels are increased in circulation during inflammatory episodes. Based on this, we investigated whether aging results in elevated EV number, as a basal proinflammatory status termed “inflammaging” has been described in aged individuals. Moreover, we also hypothesized that frailty and dependence conditions of the elderly could affect EV concentration in plasma. Results showed that inflammaging, frailty or dependence status do not result in EV increase, at least in the total number of EVs in circulation. These results open a new perspective for investigating the role of EVs in human aging and in the inflammaging process.

Circulating adipocyte-derived extracellular vesicles are novel markers of metabolic stress.

• Extensive characterization of 3T3L1 EVs identified perilipin A in their composition. • Circulating EVs are elevated in obese mice and associated with glucose intolerance. • Circulating EVs are elevated in obese human and correlated with metabolic factors. • Perilipin A and EV levels are increased in the circulation of obese mice and human. • Circulating EV and perilipin A levels decrease with low calorie intervention.

Breast Cancer-Specific miR Signature Unique to Extracellular Vesicles Includes "microRNA-like" tRNA Fragments.

Extracellular vesicles (EV), including exosomes and shed vesicles, have been implicated in intercellular communication; however, their biomarker potential is less clear. Therefore, EVs derived from MCF7 and MCF10A cells were analyzed to identify unique miRNA (miR) profiles that distinguish their origin. One characteristic common to the miR profiles of MCF7 EVs and their parent cells is the high abundance of miR-21, let-7a, miR-100, and miR-125b, and low levels of miR-205. A second characteristic is the high abundance of "miRNA-like" tRNA fragments, which is unique to the MCF7 EVs, and is not found in comparing the cellular profiles. In addition, correlations were examined in the MCF7 cellular expression levels of these five miRs and two tRNA-derived miRNAs, miR-720 and miR-1274b, and compared with the correlations in MCF7 EV levels. Interestingly, correlations in the cellular expression of miR-125b, miR-100, and let-7a are mirrored in the EVs. In contrast, correlations in tRNA-derived miRNA levels are found only in the EVs. The findings suggest that EV miR clusters can be defined based on functional miR interactions related to correlated cellular expression levels or physical miR interactions, for example, aggregation due to comparable binding affinities to common targets. These results point to using high levels of tRNA-derived small RNA fragments in combination with known miR signatures of tumors to distinguish tumor-derived EVs in circulation from EVs derived from other cell sources. Such biomarkers would be unique to the EVs where high abundances of tRNA fragments are amplified with respect to their cellular levels.