Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Methodological Approaches to Study Extracellular Vesicle miRNAs in Epstein–Barr Virus-Associated Cancers

Epstein Barr-virus (EBV) was the first virus identified to be associated with human cancer in 1964 and is found ubiquitously throughout the world’s population. It is now established that EBV contributes to the development and progression of multiple human cancers of both lymphoid and epithelial cell origins. EBV encoded miRNAs play an important role in tumor proliferation, angiogenesis, immune escape, tissue invasion, and metastasis. Recently, EBV miRNAs have been found to be released from infected cancer cells in extracellular vesicles (EVs) and regulate gene expression in neighboring uninfected cells present in the tumor microenvironment and possibly at distal sites. As EVs are abundant in many biological fluids, the viral and cellular miRNAs present within EBV-modified EVs may serve as noninvasion markers for cancer diagnosis and prognosis. In this review, we discuss recent advances in EV isolation and miRNA detection, and provide a complete workflow for EV purification from plasma and deep-sequencing for biomarker discovery

Multiplex protein profiling method for extracellular vesicle protein detection

Abstract Extracellular vesicles (EVs) are small nanometer-sized membrane sacs secreted into biological fluids by all cells. EVs encapsulate proteins, RNAs and metabolites from its origin cell and play important roles in intercellular communication events. Over the past decade, EVs have become a new emerging source for cancer diagnostics. One of the challenges in the study of EVs and there utility as diagnostic biomarkers is the amount of EVs needed for traditional protein analysis methods. Here, we present a new immuno-PCR method that takes advantage of commercially available TotalSeq antibodies containing DNA conjugated oligos to identify immobilized protein analysts using real-time qPCR. Using this method, we demonstrate that multiple EV surface proteins can be profiled simultaneously with high sensitivity and specificity. This approach was also successfully applied to similar protocol using cell and serum samples. We further described the development of a micro-size exclusion chromatography method, where we were able to detect EV surface proteins with as little as 10 μL of human serum when combined with immuno-PCR. Overall, these results show that the immuno-PCR method results in rapid detection of multiple EV markers from small sample volumes in a single tube

Extracellular Vesicle Integrins Distinguish Unique Cancers

The proteomic profile of extracellular vesicles (EVs) has been of increasing interest, particularly in understanding cancer growth, drug resistance, and metastatic behavior. Emerging data suggest that cancer-derived EVs carry an array of oncogenic cargo, including certain integrin proteins that may, in turn, promote cell detachment, migration, and selection of future metastatic sites. We previously reported a large comparison of secreted vesicle protein cargo across sixty diverse human cancer cell lines. Here, we analyze the distinct integrin profiles of these cancer EVs. We further demonstrate the enrichment of integrin receptors in cancer EVs compared to vesicles secreted from benign epithelial cells. The total EV integrin levels, including the quantity of integrins α6, αv, and β1 correlate with tumor stage across a variety of epithelial cancer cells. In particular, integrin α6 also largely reflects breast and ovarian progenitor cell expression, highlighting the utility of this integrin protein as a potential circulating biomarker of certain primary tumors. This study provides preliminary evidence of the value of vesicle-associated integrin proteins in detecting the presence of cancer cells and prediction of tumor stage. Differential expression of integrins across cancer cells and selective packaging of integrins into EVs may contribute to further understanding the development and progression of tumor growth and metastasis across a variety of cancer types

Dynamic Interactions of the UL16 Tegument Protein with the Capsid of Herpes Simplex Virus▿

The UL16 tegument protein of herpes simplex virus is conserved throughout the herpesvirus family. It has been reported to be capsid associated and may be involved in budding by providing an interaction with the membrane-bound UL11 protein. UL16 has been shown to be present in all the major locations that capsids are found (i.e., the nucleus, cytoplasm, and virions), but whether it is actually capsid associated in each of these has not been reported. Therefore, capsids were purified from each compartment, and it was found that UL16 was present on cytoplasmic but not nuclear capsids. In extracellular virions, the majority of UL16 (87%) was once again not capsid associated, which suggests that the interaction is transient during egress. Because herpes simplex virus (HSV) buds into the acidic compartment of the trans-Golgi network (TGN), the effect of pH on the interaction was examined. The amount of capsid-associated UL16 dramatically increased when extracellular virions were exposed to mildly acidic medium (pH 5.0 to 5.5), and this association was fully reversible. After budding into the TGN, capsid and tegument proteins also encounter an oxidizing environment, which is conducive to disulfide bond formation. UL16 contains 20 cysteines, including five that are conserved within a putative zinc finger. Any free cysteines that are involved in the capsid interaction or release mechanism of UL16 would be expected to be modified by N-ethylmaleimide, and, consistent with this, the amount of capsid-associated UL16 dramatically increased when virions were incubated with this compound. Taken together, these data suggest a transient interaction between UL16 and capsids, possibly modified in the acidic compartment of secretory vesicles and requiring a release mechanism that involves cysteines

Structural Rearrangement within an Enveloped Virus upon Binding to the Host Cell▿ †

We have made the surprising discovery that the interactions of herpes simplex virus with its initial cell attachment receptor induce a rapid and highly efficient structural change in the tegument, the region of the virion situated between the membrane and the capsid. It has been known for nearly a decade that viruses can trigger host signaling pathways when they bind to receptors on the cell surface; however, until now there has been no evidence that a signal can be sent in reverse—from the “outside in”—across a viral membrane. Evidence for this signaling event was found during studies of UL16, a tegument protein that is conserved among all the herpesviruses. Previous work has demonstrated that UL16 is bound to capsids isolated from the cytoplasm of infected cells, but this interaction is destabilized during subsequent egress steps, leading to release of the extracellular virion. Pretreatment with N-ethylmaleimide, a small, membrane-permeating compound that covalently modifies free cysteines, restabilizes the interaction, thereby permitting the capsid-UL16 complex to be isolated following disruption of virions with NP-40. In the experiments described here, we found that the natural signal for release of UL16 from capsids is sent when virions merely bind to cells at 4°C. The internal change was also observed upon binding to immobilized heparin in a manner that requires viral glycoprotein C. This represents the first example of signaling across a viral envelope following receptor binding

Analysis of the Interaction between the UL11 and UL16 Tegument Proteins of Herpes Simplex Virus▿

The UL11 and UL16 tegument proteins of herpes simplex virus are conserved throughout the herpesvirus family. Previous studies have shown that these proteins interact, perhaps to link UL16-bound nucleocapsids to UL11, which resides on the cytoplasmic face of the trans-Golgi network, where maturation budding occurs. Little is known about the interaction except that it requires the leucine-isoleucine (LI) and acidic cluster motifs in UL11 and that no other viral proteins are involved. In particular, the important question of whether these two proteins bind to each other directly has not been addressed. Accordingly, UL11 and UL16 were expressed in bacteria, and the purified proteins were found to retain the ability to interact in a manner that was dependent upon the LI and acidic cluster. In an attempt to map the UL11-binding site contained in UL16, a large number of deletion mutants were constructed. The first 40 (nonconserved) amino acids were found to be dispensable, but all the other constructs failed to bind UL11 or had poor expression in transfected cells, suggesting that UL16 is very sensitive to alterations and probably lacks a multidomain structure. As an alternative strategy for identifying residues that are important for the interaction, the cysteines of UL16 were investigated, because many of these are highly conserved. Approximately half of the 20 cysteines in UL16 have been shown to be covalently modified by N-ethylmaleimide, and this treatment was found to block the interaction with UL11. Moreover, individual serine replacements of six of the most conserved cysteine residues were made, and four of these disrupted the interaction with UL11 without affecting protein stability. However, the UL11-UL16 interaction does not involve the formation of interspecies disulfide bonds, because binding occurred even when all the cysteines in UL11 were eliminated. Thus, UL16 directly interacts with UL11 and does so in a manner that requires free cysteines

Epstein-Barr virus LMP1 manipulates the content and functions of extracellular vesicles to enhance metastatic potential of recipient cells.

Extracellular vesicles (EV) mediate intercellular communication events and alterations in normal vesicle content contribute to function and disease initiation or progression. The ability to package a variety of cargo and transmit molecular information between cells renders EVs important mediators of cell-to-cell crosstalk. Latent membrane protein 1 (LMP1) is a chief viral oncoprotein expressed in most Epstein-Barr virus (EBV)-associated cancers and is released from cells at high levels in EVs. LMP1 containing EVs have been demonstrated to promote cell growth, migration, differentiation, and regulate immune cell function. Despite these significant changes in recipient cells induced by LMP1 modified EVs, the mechanism how this viral oncogene modulates the recipient cells towards these phenotypes is not well understood. We hypothesize that LMP1 alters EV content and following uptake of the LMP1-modified EVs by the recipient cells results in the activation of cell signaling pathways and increased gene expression which modulates the biological properties of recipient cell towards a new phenotype. Our results show that LMP1 expression alters the EV protein and microRNA content packaged into EVs. The LMP1-modified EVs also enhance recipient cell adhesion, proliferation, migration, invasion concomitant with the activation of ERK, AKT, and NF-κB signaling pathways. The LMP1 containing EVs induced transcriptome reprogramming in the recipient cells by altering gene expression of different targets including cadherins, matrix metalloproteinases 9 (MMP9), MMP2 and integrin-α5 which contribute to extracellular matrix (ECM) remodeling. Altogether, our data demonstrate the mechanism in which LMP1-modified EVs reshape the tumor microenvironment by increasing gene expression of ECM interaction proteins

Nanoparticle analysis sheds budding insights into genetic drivers of extracellular vesicle biogenesis

Background: Extracellular vesicles (EVs) are important mediators of cell-to-cell communication in healthy and pathological environments. Because EVs are present in a variety of biological fluids and contain molecular signatures of their cell or tissue of origin, they have great diagnostic and prognostic value. The ability of EVs to deliver biologically active proteins, RNAs and lipids to cells has generated interest in developing novel therapeutics. Despite their potential medical use, many of the mechanisms underlying EV biogenesis and secretion remain unknown. Methods: Here, we characterized vesicle secretion across the NCI-60 panel of human cancer cells by nanoparticle tracking analysis. Using CellMiner, the quantity of EVs secreted by each cell line was compared to reference transcriptomics data to identify gene products associated with vesicle secretion. Results: Gene products positively associated with the quantity of exosomal-sized vesicles included vesicular trafficking classes of proteins with Rab GTPase function and sphingolipid metabolism. Positive correlates of larger microvesicle-sized vesicle secretion included gene products involved in cytoskeletal dynamics and exocytosis, as well as Rab GTPase activation. One of the identified targets, CD63, was further evaluated for its role in vesicle secretion. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 knockout of the CD63 gene in HEK293 cells resulted in a decrease in small vesicle secretion, suggesting the importance of CD63 in exosome biogenesis. Conclusion: These observations reveal new insights into genes involved in exosome and microvesicle formation, and may provide a means to distinguish EV sub-populations. This study offers a foundation for further exploration of targets involved in EV biogenesis and secretion

Epstein-Barr Virus LMP1 Promotes Syntenin-1- and Hrs-Induced Extracellular Vesicle Formation for Its Own Secretion To Increase Cell Proliferation and Migration

LMP1 is a notable viral protein that contributes to the modification of EV content and tumor microenvironment remodeling. LMP1-modified EVs enhance tumor proliferation, migration, and invasion potential and promote radioresistance. Currently, the mechanisms surrounding LMP1 incorporation into the host EV pathways are not well understood. This study revealed that LMP1 utilizes Hrs, Syntenin-1, and specific components of the ESCRT-III complex for release from the cell, enhancement of EV production, and metastatic properties of cancer cells. These findings begin to unravel the mechanism of LMP1 EV trafficking and may provide new targets to control EBV-associated cancers.Extracellular vesicles (EVs) are important mediators of cell-to-cell communication that are involved in both normal processes and pathological conditions. Latent membrane protein 1 (LMP1) is a major viral oncogene that is expressed in most Epstein-Barr virus (EBV)-associated cancers and secreted in EVs. LMP1-modified EVs have the ability to influence recipient cell growth, migration, and differentiation and regulate immune cell function. Despite the significance of LMP1-modified EVs in EBV malignancies, very little is understood about how this protein hijacks the host EV pathway for secretion. Using the biotin identification (BioID) method, we identified LMP1-proximal interacting proteins that are known to play roles in EV formation and protein trafficking. Analysis of the identified LMP1-interacting proteins revealed an enrichment in the ESCRT pathway and associated proteins, including CD63, Syntenin-1, Alix, TSG101, Hrs, and charged multivesicular body proteins (CHMPs). LMP1 transcriptionally upregulated and increased the protein expression of EV biogenesis and secretion genes. Nanoparticle tracking and immunoblot analysis revealed reduced levels of LMP1 EV packaging and of vesicle production following the knockdown of Syntenin-1, Alix, Hrs, and TSG101, with altered endolysosomal trafficking observed when Syntenin-1 and Hrs expression was reduced. Knockdown of specific ESCRT-III subunits (CHMP4B, -5, and -6) impaired LMP1 packaging and secretion into EVs. Finally, we demonstrate that the efficient secretion of LMP1-modified EVs promotes cell attachment, proliferation, and migration and tumor growth. Together, these results begin to shed light on how LMP1 exploits host ESCRT machinery to direct the incorporation of the viral oncoprotein into the EV pathway for secretion to alter the tumor microenvironment