Journal of extracellular vesicles

Primary tumours can establish long‐range communication with distant organs to transform them into fertile soil for circulating tumour cells to implant and proliferate, a process called pre‐metastatic niche (PMN) formation. Tumour‐derived extracellular vesicles (EV) are potent mediators of PMN formation due to their diverse complement of pro‐malignant molecular cargo and their propensity to target specific cell types (Costa‐Silva et al., 2015; Hoshino et al., 2015; Peinado et al., 2012; Peinado et al., 2017). While significant progress has been made to understand the mechanisms by which pro‐metastatic EVs create tumour‐favouring microenvironments at pre‐metastatic organ sites, comparatively little attention has been paid to the factors intrinsic to recipient cells that may modify the extent to which pro‐metastatic EV signalling is received and transduced. Here, we investigated the role of recipient cell cholesterol homeostasis in prostate cancer (PCa) EV‐mediated signalling and metastasis. Using a bone metastatic model of enzalutamide‐resistant PCa, we first characterized an axis of EV‐mediated communication between PCa cells and bone marrow that is marked by in vitro and in vivo PCa EV uptake by bone marrow myeloid cells, activation of NF‐κB signalling, enhanced osteoclast differentiation, and reduced myeloid thrombospondin‐1 expression. We then employed a targeted, biomimetic approach to reduce myeloid cell cholesterol in vitro and in vivo prior to conditioning with PCa EVs. Reducing myeloid cell cholesterol prevented the uptake of PCa EVs by recipient myeloid cells, abolished NF‐κB activity and osteoclast differentiation, stabilized thrombospondin‐1 expression, and reduced metastatic burden by 77%. These results demonstrate that cholesterol homeostasis in bone marrow myeloid cells regulates pro‐metastatic EV signalling and metastasis by acting as a gatekeeper for EV signal transduction.

Schistosomiasis is characterized by liver fibrosis, and studies have indicated that Schistosoma japonicum (S. japonicum) eggs can limit the progression of liver fibrosis. However, the detailed molecular mechanisms are yet unclear. Extracellular vesicles (EVs) contain a selection of miRNAs for long‐distance exchange of information and act as an important pathway for host‐parasite communication. This study aimed to explore the potential role of S. japonicum egg‐derived EVs and its key miRNA in liver fibrosis. Herein, we found that S. japonicum egg‐derived EVs can inhibit the activation of hepatic stellate cells, which is mediated via the high expression of Sja‐miR‐71a. Sja‐miR‐71a in EVs attenuates the pathological progression and liver fibrosis in S. japonicum infection. Sja‐miR‐71a inhibiting TGF‐β1/SMAD and interleukin (IL)‐13/STAT6 pathways via directly targeting semaphorin 4D (Sema4D). In addition, Sja‐miR‐71a can also suppress liver fibrosis by regulating Th1/Th2/Th17 and Treg balance. This study contributes to further understanding of the molecular mechanisms underlying Schistosoma‐host interactions, and Sema4D may be a potential target for schistosomiasis liver fibrosis treatment.

Production of extracellular vesicles (EVs) involved in intercellular communication is a common capacity of most cell types. Upon encountering opsonized microorganisms, neutrophilic granulocytes release EVs that compromise bacterial growth. We carried out a systematic investigation of the involvement of potential opsonin receptors in EV‐generation from human and murine neutrophils. Applying flow cytometric, proteomic and functional analysis as well as using genetically modified mice, we demonstrate that formation of antibacterial EVs depends upon stimulation of the multifunctional Mac‐1 integrin complex, also called as complement receptor 3 (CR3), whereas activation of immunoglobulin binding Fc receptors or pattern recognition receptors alone or in combination is ineffective. Mac‐1/CR3 stimulation and downstream tyrosine kinase signalling affect both the numbers, the cargo content and the antibacterial capacity of the produced vesicles. In contrast, Mac‐1/CR3 signalling is not required for spontaneous EV formation, clearly indicating the existence of separate molecular pathways in EV biogenesis. We propose that EVs are “tailor‐made” with different composition and functional properties depending on the environmental circumstances.

Early diagnosis of colon cancer (CC) is clinically important, as it can significantly improve patients' survival rate and quality of life. Although the potential role for small extracellular vesicles (sEVs) in early detection of many diseases has been repeatedly mentioned, systematic screening of plasma sEVs derived early CC specific biomarkers has not yet been reported. In this work, plasma sEVs enriched fractions were derived from 15 early‐stage (TisN0M0) CC patients and 10 normal controls (NC). RNA sequencing identified a total number of 95 sEVs enriched fraction derived miRNAs with differential expression between CC and NC, most of which (60/95) was in well accordance with tissue results in the Cancer Genome Atlas (TCGA) dataset. Among those miRNAs, we selected let‐7b‐3p, miR‐139‐3p, miR‐145‐3p, and miR‐150‐3p for further validation in an independent cohort consisting of 134 participants (58 CC and 76 NC). In the validation cohort, the AUC of 4 individual miRNAs ranged from 0.680 to 0.792. A logistic model combining two miRNAs (i.e. let‐7b‐3p and miR‐145‐3p) achieved an AUC of 0.901. Adding the 3rd miRNA into this model can further increase the AUC to 0.927. Side by side comparison revealed that sEVs miRNA profile outperformed cell‐free plasma miRNA in the diagnosis of early CC. In conclusion, we suggested that circulating sEVs enriched fractions have a distinct miRNA profile in CC patients, and sEVs derived miRNA could be used as a promising biomarker to detect CC at an early stage.

Proliferation of vascular smooth muscle cells (VSMCs) plays crucial roles in vascular remodelling and stiffening in hypertension. Vascular adventitial fibroblasts are a key regulator of vascular wall function and structure. This study is designed to investigate the roles of adventitial fibroblasts‐derived extracellular vesicles (EVs) in VSMC proliferation and vascular remodelling in normotensive Wistar‐Kyoto rat (WKY) and spontaneously hypertensive rat (SHR), an animal model of human essential hypertension. EVs were isolated from aortic adventitial fibroblasts of WKY (WKY‐EVs) and SHR (SHR‐EVs). Compared with WKY‐EVs, miR155‐5p content was reduced, while angiotensin‐converting enzyme (ACE) content was increased in SHR‐EVs. WKY‐EVs inhibited VSMC proliferation of SHR, which was prevented by miR155‐5p inhibitor. SHR‐EVs promoted VSMC proliferation of both strains, which was enhanced by miR155‐5p inhibitor, but abolished by captopril or losartan. Dual luciferase reporter assay showed that ACE was a target gene of miR155‐5p. MiR155‐5p mimic or overexpression inhibited VSMC proliferation and ACE upregulation of SHR. WKY‐EVs reduced ACE mRNA and protein expressions while SHR‐EVs only increased ACE protein level in VSMCs of both strains. However, the SHR‐EVs‐derived from the ACE knockdown‐treated adventitial fibroblasts lost the roles in promoting VSMC proliferation and ACE upregulation. Systemic miR155‐5p overexpression reduced vascular ACE, angiotensin II and proliferating cell nuclear antigen levels, and attenuated hypertension and vascular remodelling in SHR. Repetitive intravenous injection of SHR‐EVs increased blood pressure and vascular ACE contents, and promoted vascular remodelling in both strains, while WKY‐EVs reduced vascular ACE contents and attenuated hypertension and vascular remodelling in SHR. We concluded that WKY‐EVs‐mediated miR155‐5p transfer attenuates VSMC proliferation and vascular remodelling in SHR via suppressing ACE expression, while SHR‐EVs‐mediated ACE transfer promotes VSMC proliferation and vascular remodelling.

Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory diseases, and cancer. Stromal cells play a crucial role in regulating angiogenesis through the release of soluble factors or direct contact with endothelial cells. Here, we analysed the properties of the extracellular vesicles (EVs) released by bone marrow mesenchymal stromal cells (MSCs) and explored the possibility of using them to therapeutically target angiogenesis. We demonstrated that in response to pro‐inflammatory cytokines, MSCs produce EVs that are enriched in TIMP‐1, CD39 and CD73 and inhibit angiogenesis targeting both extracellular matrix remodelling and endothelial cell migration. We identified a novel anti‐angiogenic mechanism based on adenosine production, triggering of A_2B adenosine receptors, and induction of NOX2‐dependent oxidative stress within endothelial cells. Finally, in pilot experiments, we exploited the anti‐angiogenic EVs to inhibit tumour progression in vivo. Our results identify novel pathways involved in the crosstalk between endothelial and stromal cell and suggest new therapeutic strategies to target pathological angiogenesis.

Extracellular vesicles (EVs) are emerging as potent non‐invasive biomarkers. However, current methodologies are time consuming and difficult to translate to clinical practice. To analyse EV‐encapsulated circulating miRNA, we searched for a quick, easy and economic method to enrich frozen human serum samples for EV. We compared the efficiency of several protocols and commercial kits to isolate EVs. Different methods based on precipitation, columns or filter systems were tested and compared with ultracentrifugation, which is the most classical protocol to isolate EVs. EV samples were assessed for purity and quantity by nanoparticle tracking analysis and western blot or cytometry against major EV protein markers. For biomarker validation, levels of a set of miRNAs were determined in EV fractions and compared with their levels in total serum. EVs isolated with precipitation‐based methods were enriched for a subgroup of miRNAs that corresponded to miRNAs described to be encapsulated into EVs (miR‐126, miR‐30c and miR‐143), while the detection of miR‐21, miR‐16‐5p and miR‐19a was very low compared with total serum. Our results point to precipitation using polyethylene glycol (PEG) as a suitable method for an easy and cheap enrichment of serum EVs for miRNA analyses. The overall performance of PEG was very similar, or better than other commercial precipitating reagents, in both protein and miRNA yield, but in comparison to them PEG is much cheaper. Other methods presented poorer results, mostly when assessing miRNA by qPCR analyses. Using PEG precipitation in a longitudinal study with human samples, we demonstrated that miRNA could be assessed in frozen samples up to 8 years of storage. We report a method based on a cut‐off value of mean of fold EV detection versus serum that provides an estimate of the degree of encapsulation of a given miRNA.

Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in order to obtain an acceptable level of data comparability. Size and concentration of EVs can be determined by nanoparticle tracking analysis (NTA). However, both the heterogeneity of EVs and the choice of instrument settings may cause an appreciable analytical variation. Intra‐assay (within‐day, n = 6) and inter‐assay (day‐to‐day, n = 6) variations (coefficient of variation, % CV) of different preparations of EVs and artificial vesicles or beads were determined using two NanoSight NS500 instruments, located at different laboratories. All analyses were performed by the same operator. The effect of applying identical software settings or instrument‐optimised settings for each sample type and instrument was also evaluated. Finally, the impact of different operators and the use of two different software versions were investigated. The intra‐assay CVs were 1–12% for both EVs and artificial samples, measured on the same instrument. The overall day‐to‐day variation was similar for both instruments, ranging from 2% to 25%. However, significantly different results were observed between the two instruments using identical software settings. The effect of applying instrument‐optimised settings reduced the mismatch between the instruments, resulting in little to no significant divergences. The impact of using different operators and software versions when analysing silica microspheres and microvesicles from monocytes using instrument‐optimised settings on the same instrument did not contribute to significant variation compared to the overall day‐to‐day variation of one operator. Performance differences between two similar NTA instruments may display significant divergences in size and concentration measurements when analysing EVs, depending on applied instrument settings and technical conditions. The importance of developing a streamlined and standardised execution of analysis, as well as monitoring longitudinal variation parameters on both biological and synthetic samples, should be highlighted.

Both mesenchymal stem cells (MSCs) and their corresponding small extracellular vesicles (sEVs, commonly referred to as exosomes) share similar immunomodulatory properties that are potentially beneficial for the treatment of acute graft versus host disease (aGvHD). We report that clinical grade Wharton's Jelly‐derived MSCs (WJMSCs) secrete sEVs enriched in programmed death‐ligand 1 (PD‐L1), an essential ligand for an inhibitory immune checkpoint. A rapid increase in circulating sEV‐associated PD‐L1 was observed in patients with aGvHD and was directly associated with the infusion time of clinical grade WJMSCs. In addition, in vitro inhibitory antibody mediated blocking of sEV‐associated PD‐L1 restored T cell activation (TCA), suggesting a functional inhibitory role of sEVs‐PD‐L1. PD‐L1‐deficient sEVs isolated from WJMSCs following CRISPR‐Cas9 gene editing fail to inhibit TCA. Furthermore, we found that PD‐L1 is essential for WJMSC‐derived sEVs to modulate T cell receptors (TCRs). Our study reveals an important mechanism by which therapeutic WJMSCs modulate TCR‐mediated TCA through sEVs or sEV‐carried immune checkpoints. In addition, our clinical data suggest that sEV‐associated PD‐L1 may be not only useful in predicting the outcomes from WJMSC clinical administration, but also in developing cell‐independent therapy for aGvHD patients.