Optimizing Size Exclusion Chromatography for Extracellular Vesicle Enrichment and Proteomic Analysis from Clinically Relevant Samples

The field of extracellular vesicle (EV) research has rapidly expanded in recent years, with particular interest in their potential as circulating biomarkers. Proteomic analysis of EVs from clinical samples is complicated by the low abundance of EV proteins relative to highly abundant circulating proteins such as albumin and apolipoproteins. To overcome this, size exclusion chromatography (SEC) has been proposed as a method to enrich EVs whilst depleting protein contaminants; however, the optimal SEC parameters for EV proteomics have not been thoroughly investigated. Here, quantitative evaluation and optimization of SEC are reported for separating EVs from contaminating proteins. Using a synthetic model system followed by cell line‐derived EVs, it is found that a 10 mL Sepharose 4B column in PBS produces optimal resolution of EVs from background protein. By spiking‐in cancer cell‐derived EVs to healthy plasma, it is shown that some cancer EV‐associated proteins are detectable by nano‐LC‐MS/MS when as little as 1% of the total plasma EV number are derived from a cancer cell line. These results suggest that an optimized SEC and nanoLC‐MS/MS workflow may be sufficiently sensitive for disease EV protein biomarker discovery from patient‐derived clinical samples.     

Combination of size-exclusion chromatography and ion exchange adsorption for improving the proteomic analysis of plasma-derived extracellular vesicles

Extracellular vesicles (EVs) are lipid membrane vesicles released by live cells that carry a variety of biomolecules, including nucleic acids, lipids, and proteins. Recently, proteins in plasma-derived EVs have emerged as novel biomarkers with essential functions in the diagnosis and prognosis of human diseases. However, the current methods of isolating EVs from plasma often lead to coisolated impurities in biological fluids. Therefore, before performing any research protocol, the process of extracting EVs from plasma for proteomic analysis must be optimized. In this study, two EV isolation strategies, size exclusion chromatography (SEC) and SEC combined with ion exchange adsorption (SEC + IEA), were compared in terms of the purity and quantity of protein in EVs. Our results demonstrated that, compared to single-step SEC, SEC combined with IEA could produce plasma-derived EVs with a higher purity by decreasing the abundance of lipoprotein. Additionally, with MS analysis, we demonstrated that the combination approach maintained the stability and improved the purity of EVs in many plasma samples. Furthermore, by combining SEC with IEA, more cancer-associated proteins were detected in the plasma of various cancer samples.     

The protein interaction network of extracellular vesicles derived from human colorectal cancer cells

Various mammalian cells including tumor cells secrete extracellular vesicles (EVs), otherwise known as exosomes and microvesicles. EVs are nanosized bilayered proteolipids and play multiple roles in intercellular communication. Although many vesicular proteins have been identified, their functional interrelationships and the mechanisms of EV biogenesis remain unknown. By interrogating proteomic data using systems approaches, we have created a protein interaction network of human colorectal cancer cell-derived EVs which comprises 1491 interactions between 957 vesicular proteins. We discovered that EVs have well-connected clusters with several hub proteins similar to other subcellular networks. We also experimentally validated that direct protein interactions between cellular proteins may be involved in protein sorting during EV formation. Moreover, physically and functionally interconnected protein complexes form functional modules involved in EV biogenesis and functions. Specifically, we discovered that SRC signaling plays a major role in EV biogenesis, and confirmed that inhibition of SRC kinase decreased the intracellular biogenesis and cell surface release of EVs. Our study provides global insights into the cargo-sorting, biogenesis, and pathophysiological roles of these complex extracellular organelles.     

Unbiased proteomic profiling of host cell extracellular vesicle composition and dynamics upon HIV‐1 infection

Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surrounding cells. Specific markers to distinguish different EVs (e.g. exosomes, ectosomes, enveloped viruses like HIV) are still lacking. We have developed a proteomic profiling approach for characterizing EV subtype composition and applied it to human Jurkat T cells. We generated an interactive database to define groups of proteins with similar profiles, suggesting release in similar EVs. Biochemical validation confirmed the presence of preferred partners of commonly used exosome markers in EVs: CD81/ADAM10/ITGB1, and CD63/syntenin. We then compared EVs from control and HIV‐1‐infected cells. HIV infection altered EV profiles of several cellular proteins, including MOV10 and SPN, which became incorporated into HIV virions, and SERINC3, which was re‐routed to non‐viral EVs in a Nef‐dependent manner. Furthermore, we found that SERINC3 controls the surface composition of EVs. Our workflow provides an unbiased approach for identifying candidate markers and potential regulators of EV subtypes. It can be widely applied to in vitro experimental systems for investigating physiological or pathological modifications of EV release.     

Toward Standardization of Mesenchymal Stromal Cell‐Derived Extracellular Vesicles for Therapeutic Use: A Call for Action

Extracellular vesicles (EVs), which include a variety of nano‐sized membrane‐encapsulated particles, are released to the extracellular microenvironment by the vast majority of cells and carry lipids, proteins, mRNA, and miRNA or non‐coding RNA. Increasing evidence suggests the great versatility and potential of EV‐based applications in humans. In this issue, van Balkom et al. explore and compare the reported proteomic signature of mesenchymal stromal cell (MSC)‐derived small EVs. In particular, their paper offers a valuable approach and point of view on MSC‐EV manufacturing and therapeutic potential. Briefly, van Balkom et al. aimed to identify a common protein signature that may be useful in ensuring the homogeneity of therapeutic MSC‐EVs. In addition to excessive variability in EV‐producing cell sources and culture conditions, the harvesting time for the EV‐containing conditioned medium, and EV isolation procedure, the authors found a specific protein signature from the publicly available MSC‐EVs proteome. In light of their findings and those from the plentiful studies published in this continuously growing area of research, potential focus areas and issues are outlined for the more rational design and optimization of MSC‐EV production and potency for therapeutics.     

Quantitative Proteomic Analysis of Seminal Plasma,Sperm Membrane Proteins,and Seminal Extracellular Vesicles Suggests Vesicular Mechanisms Aid in the Removal and Addition of Proteins to the Ram Sperm Membrane

Quantitative proteomic studies are contributing greatly to the understanding of the spermatozoon through the provision of detailed information on the proteins spermatozoa acquire and shed in the acquisition of fertility. Extracellular vesicles (EVs) are thought to aid in the delivery of proteins to spermatozoa in the male reproductive tract. The aim of this study is to isolate, identify and quantify EV proteins isolated from ram seminal plasma. Ram sperm plasma membrane proteins are also isolated using nitrogen cavitation and identified to better understand the interplay of proteins between the sperm membrane and extracellular environment. The categorization of proteins enriched in the EV population according to their function revealed three main groupings: vesicle biogenesis, metabolism, and membrane adhesion and remodeling. The latter group contains many reproduction‐specific proteins that show demonstrable links to sperm fertility. Many of these membrane‐bound proteins show testicular expression and are shed from the sperm surface during epididymal maturation (e.g., testis expressed 101; TEX101 and lymphocyte Antigen 6 Family Member K; LY6K). Their association with seminal EVs suggests that EVs may not only deliver protein cargo to spermatozoa but also assist in the removal of proteins from the sperm membrane.     

Proteomic and phosphoproteomic landscape of salivary extracellular vesicles to assess OSCC therapeutical outcomes

Circulating extracellular vesicles (EVs) have emerged as an appealing source for surrogates to evaluate the disease status. Herein, we present a novel proteomic strategy to identify proteins and phosphoproteins from salivary EVs to distinguish oral squamous cell carcinoma (OSCC) patients from healthy individuals and explore the feasibility to evaluate therapeutical outcomes. Bi-functionalized magnetic beads (BiMBs) with Ti (IV) ions and a lipid analog, 1,2-Distearoyl-3-sn-glycerophosphoethanolamine (DSPE) are developed to efficiently isolate EVs from small volume of saliva. In the discovery stage, label-free proteomics and phosphoproteomics quantification showed 315 upregulated proteins and 132 upregulated phosphoproteins in OSCC patients among more than 2500 EV proteins and 1000 EV phosphoproteins, respectively. We further applied targeted proteomics by coupling parallel reaction monitoring with parallel accumulation-serial fragmentation (prm-PASEF) to measure panels of proteins and phosphoproteins from salivary EVs collected before and after surgical resection. A panel of three total proteins and three phosphoproteins, most of which have previously been associated with OSCC and other cancer types, show sensitive response to the therapy in individual patients. Our study presents a novel strategy to the discovery of effective biomarkers for non-invasive assessment of OSCC surgical outcomes with small amount of saliva.     

Cerebrospinal Fluid Extracellular Vesicles Undergo Age Dependent Declines and Contain Known and Novel Non-coding RNAs

Brain development requires precise orchestration of cellular events through the coordinate exchange of information between distally located cells. One mechanism by which intercellular communication is achieved is through the transfer of extracellular vesicles (EVs). Exosomes are EVs that carry lipids, nucleic acids, and proteins and are detectable in most biological fluids including cerebrospinal fluid (CSF). Here we report that CSF EV concentrations undergo age dependent fluctuations. We characterized EV RNA content by next generation small RNA sequencing and miRNA microarray analysis and identified a temporal shift in CSF EV content. CSF EVs encapsulated miRNAs that contain a conserved hnRNPA2/B1 recognition sequence. We found that hnRNPA2/B1-containing EVs were produced by choroid plexus epithelial cells and that hnRNPA2/B1 containing EVs decreased with age. These results provide insight into EV exchange of miRNAs within the central nervous system and a framework to understand how changes in EVs may have an important impact on brain development.     

Imaging flow cytometry challenges the usefulness of classically used extracellular vesicle labeling dyes and qualifies the novel dye Exoria for the labeling of mesenchymal stromal cell–extracellular vesicle preparations

Background aimsExtracellular vesicles (EVs) are involved in mediating intercellular communication processes. An important goal within the EV field is the study of the biodistribution of EVs and the identification of their target cells. Considering that EV uptake is assumed to be important for EVs in mediating intercellular communication processes, labeling with fluorescent dyes has emerged as a broadly distributed strategy for the identification of EV target cells and tissues. However, the accuracy and specificity of commonly utilized labeling dyes have not been sufficiently analyzed.MethodsBy combining recent advances in imaging flow cytometry for the phenotypic analysis of single EVs and aiming to identify target cells for EVs within therapeutically relevant mesenchymal stromal cell (MSC)-EV preparations, the authors explored the EV labeling efficacy of various fluorescent dyes, specifically carboxyfluorescein diacetate succinimidyl ester, calcein AM, PKH67, BODIPY TR ceramide (Thermo Fisher Scientific, Darmstadt, Germany) and a novel lipid dye called Exoria (Exopharm Limited, Melbourne, Australia).ResultsThe authors’ analyses qualified Exoria as the only dye that specifically labeled EVs within the MSC-EV preparations. Furthermore, the authors demonstrated that Exoria labeling did not interfere with the immunomodulatory properties of the MSC-EV preparations as tested in a multi-donor mixed lymphocyte reaction assay. Within this assay, labeled EVs were differentially taken up by different immune cell types.ConclusionsOverall, the results qualify Exoria as an appropriate dye for the labeling of EVs derived from the authors’ MSC-EV preparations. This study also demonstrates the need for the development of next-generation EV characterization tools that are able to localize and confirm the specificity of EV labeling.     

Media matters! Alterations in the loading and release of Histoplasma capsulatum extracellular vesicles in response to different nutritional milieus

Histoplasma capsulatum is a dimorphic fungus that most frequently causes pneumonia, but can also disseminate and proliferate in diverse tissues. Histoplasma capsulatum has a complex secretion system that mediates the release of macromolecule‐degrading enzymes and virulence factors. The formation and release of extracellular vesicles (EVs) are an important mechanism for non‐conventional secretion in both ascomycetes and basidiomycetes. Histoplasma capsulatum EVs contain diverse proteins associated with virulence and are immunologically active. Despite the growing knowledge of EVs from H. capsulatum and other pathogenic fungi, the extent that changes in the environment impact the sorting of organic molecules in EVs has not been investigated. In this study, we cultivated H. capsulatum with distinct culture media to investigate the potential plasticity in EV loading in response to differences in nutrition. Our findings reveal that nutrition plays an important role in EV loading and formation, which may translate into differences in biological activities of these fungi in various fluids and tissues.     

Protein Composition Reflects Extracellular Vesicle Heterogeneity

Extracellular vesicles (EVs) are membrane‐enclosed particles that are released by virtually all cells from all living organisms. EVs shuttle biologically active cargo including protein, RNA, and DNA between cells. When shed by cancer cells, they function as potent intercellular messangers with important functional consequences. Cells produce a diverse spectrum of EVs, spanning from small vesicles of 40–150 nm in diameter, to large vesicles up to 10 μm in diameter. While this diversity was initially considered to be purely based on size, it is becoming evident that different classes of EVs, and different populations within one EV class may harbor distinct molecular cargo and play specific functions. Furthermore, there are considerable cell type‐dependent differences in the cargo and function of shed EVs. This review focuses on the most recent proteomic studies that have attempted to capture the EV heterogeneity by directly comparing the protein composition of different EV classes and EV populations derived from the same cell source. Recent studies comparing protein composition of the same EV class(es) derived from different cell types are also summarized. Emerging approaches to study EV heterogeneity and their important implications for future studies are also discussed.     

Oncogenic Regulation of Extracellular Vesicle Proteome and Heterogeneity

Mutational and epigenetic driver events profoundly alter intercellular communication pathways in cancer. This effect includes deregulated release, molecular composition, and biological activity of extracellular vesicles (EVs), membranous cellular fragments ranging from a few microns to less than 100 nm in diameter and filled with bioactive molecular cargo (proteins, lipids, and nucleic acids). While EVs are usually classified on the basis of their physical properties and biogenetic mechanisms, recent analyses of their proteome suggest a larger than expected molecular diversity, a notion that is also supported by multicolour nano‐flow cytometry and other emerging technology platforms designed to analyze single EVs. Both protein composition and EV diversity are markedly altered by oncogenic transformation, epithelial to mesenchymal transition, and differentiation of cancer stem cells. Interestingly, only a subset of EVs released from mutant cells may carry oncogenic proteins (e.g., EGFRvIII), hence, these EVs are often referred to as “oncosomes”. Indeed, oncogenic transformation alters the repertoire of EV‐associated proteins, increases the presence of pro‐invasive cargo, and alters the composition of distinct EV populations. Molecular profiling of single EVs may reveal a more intricate effect of transforming events on the architecture of EV populations in cancer and shed new light on their biological role and diagnostic utility.     

Extracellular vesicle-mediated long-range communication in stressed retinal pigment epithelial cell monolayers

Retinal pigment epithelium (RPE) alterations in age-related macular degeneration occur in patches, potentially involving long-distance communication between damaged and healthy areas. Communication along the epithelium might be mediated by extracellular vesicles (EVs). To test this hypothesis, EVs were collected from supernatants of polarized ARPE-19 and primary porcine RPE monolayers for functional and biochemical assays. EVs from oxidatively stressed donor cells reduced barrier function in recipient RPE monolayers when compared to control EVs. The effect on barrier function was dependent on EV uptake, which occurred rapidly with EVs from oxidatively stressed donor cells. Mass spectrometry-based proteomic analysis of EVs identified HDAC6, which is known to reduce tight junction stability. Activity assays confirmed the presence of HDAC6 in EVs, and EV transfer assays using HDAC6 inhibitors confirmed its effect in monolayers. These findings demonstrate that EVs can communicate stress messages to healthy RPE cells, potentially contributing to RPE dysfunction.     

Human Endometrial Extracellular Vesicles Functionally Prepare Human Trophectoderm Model for Implantation: Understanding Bidirectional Maternal‐Embryo Communication

Embryo implantation into maternal endometrium is critical for initiation and establishment of pregnancy, requiring developmental synchrony between endometrium and blastocyst. However, factors regulating human endometrial–embryo cross talk and facilitate implantation remain largely unknown. Extracellular vesicles (EVs) are emerging as important mediators of this process. Here, a trophectoderm spheroid‐based in vitro model mimicking the pre‐implantation human embryo is used to recapitulate important functional aspects of blastocyst implantation. Functionally, human endometrial EVs, derived from hormonally treated cells synchronous with implantation, are readily internalized by trophectoderm cells, regulating adhesive and invasive capacity of human trophectoderm spheroids. To gain molecular insights into mechanisms underpinning endometrial EV‐mediated enhancement of implantation, quantitative proteomics reveal critical alterations in trophectoderm cellular adhesion networks (cell adhesion molecule binding, cell–cell adhesion mediator activity, and cell adherens junctions) and metabolic and gene expression networks, and the soluble secretome from human trophectodermal spheroids. Importantly, transfer of endometrial EV cargo proteins to trophectoderm to mediate changes in trophectoderm function is demonstrated. This is highlighted by correlation among endometrial EVs, the trophectodermal proteome following EV uptake, and EV‐mediated trophectodermal cellular proteome, important for implantation. This work provides an understanding into molecular mechanisms of endometrial EV‐mediated regulation of human trophectoderm functions—fundamental in understanding human endometrium–embryo signaling during implantation.     

Proteomic profiling of extracellular vesicles secreted from Toxoplasma gondii

Toxoplasma gondii infects a wide range of hosts worldwide, including humans and domesticated animals causing toxoplasmosis disease. Recently, exosomes, small extracellular vesicles (EV) that contain nucleic acids, proteins, and lipids derived from their original cells were linked with disease protection. The effect of EVs derived from T. gondii on the immune response and its relevance in a physiological context is unknown. Here we disclose the first proteomic profiling of T. gondii EVs compared to EVs isolated from a human foreskin fibroblast infected cell line cultured in a vesicle‐free medium. Our results reveal a broad range of canonical exosomes proteins. Data are available via ProteomeXchange with the identifier PXD004895.     

Amniotic fluid-derived extracellular vesicles: characterization and therapeutic efficacy in an experimental model of bronchopulmonary dysplasia

Background aimsExtracellular vesicles (EVs) are being tested for their use as novel therapeutics. However, the optimal source of EVs is currently under investigation. Amniotic fluid (AF) is a natural source of EVs that can be easily obtained for use in regenerative medicine, yet AF-EV characterization has not been fully explored.MethodsHere the authors demonstrate AF as a rich source of EVs and identify the microRNA and proteomic cargo. Bioinformatics analysis of this cargo revealed multiple pathway targets, including immunomodulatory, anti-inflammatory and free radical scavenging networks. The authors further demonstrated the therapeutic potential of this EV product as a novel preventative agent for bronchopulmonary dysplasia (BPD).ResultsIntra-tracheal administration of AF-EVs preserved alveolar development, attenuated vascular remodeling and pulmonary hypertension, decreased lung pro-inflammatory cytokine expression and reduced macrophage infiltration in an experimental BPD model.ConclusionsThe authors’ results suggest that AF is a viable biological fluid for EV harvest and that AF-EVs have strong therapeutic potential for pulmonary diseases, such as BPD, warranting further development to transition this novel EV product into the clinic.     

Arrestin‐Domain Containing Protein 1 (Arrdc1) Regulates the Protein Cargo and Release of Extracellular Vesicles

Extracellular vesicles (EVs) are lipid‐bilayered vesicles that are released by multiple cell types and contain nucleic acids and proteins. Very little is known about how the cargo is packaged into EVs. Ubiquitination of proteins is a key posttranslational modification that regulates protein stability and trafficking to subcellular compartments including EVs. Recently, arrestin‐domain containing protein 1 (Arrdc1), an adaptor for the Nedd4 family of ubiquitin ligases, has been implicated in the release of ectosomes, a subtype of EV that buds from the plasma membrane. However, it is currently unknown whether Arrdc1 can regulate the release of exosomes, a class of EVs that are derived endocytically. Furthermore, it is unclear whether Arrdc1 can regulate the sorting of protein cargo into the EVs. Exosomes and ectosomes are isolated from mouse embryonic fibroblasts isolated from wild type and Arrdc1‐deficient (Arrdc1^?/?) mice. Nanoparticle tracking analysis–based EV quantitation shows that Arrdc1 regulates the release of both exosomes and ectosomes. Proteomic analysis highlights the change in protein cargo in EVs upon deletion of Arrdc1. Functional enrichment analysis reveals the enrichment of mitochondrial proteins in ectosomes, while proteins implicated in apoptotic cleavage of cell adhesion proteins and formation of cornified envelope are significantly depleted in exosomes upon knockout of Arrdc1.     

Extracellular vesicle-mediated regulation of tumor angiogenesis— implications for anti-angiogenesis therapy

Angiogenesis plays an important role in tumour progression. However, anti-angiogenesis therapy of inhibiting pro-angiogenic factors failed to meet expectations in certain types of tumour in clinical trials. Recent studies reveal that tumour-derived extracellular vesicles (EVs) are essential in tumour angiogenesis and anti-angiogenesis drug resistance. This function has most commonly been attributed to EV contents including proteins and non-coding RNAs. Here, we summarize the recent findings of tumour-derived EV contents associated with regulating angiogenesis and illustrate the underlying mechanisms. In addition, the roles of EVs in tumour microenvironmental cells are also illustrated with a focus on how EVs participate in cell-cell communication, contributing to tumour-mediated angiogenesis. It will help offer new perspectives on developing targets of anti-angiogenesis drugs and improve the efficacy of anti-angiogenesis therapies based on tumour-derived EVs.     

Multidrug resistant tumour cells shed more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart cells

BackgroundMultidrug resistance (MDR) is a serious impediment to cancer treatment, with overexpression of drug efflux pumps such as P-glycoprotein (P-gp) playing a significant role. In spite of being a major clinical challenge, to date there is no simple, minimally invasive and clinically validated method for diagnosis of the MDR phenotype using non-tumour biological samples. Recently, P-gp has been found in extracellular vesicles (EVs) shed by MDR cancer cells. This study aimed to compare the EVs shed by MDR cells and their drug-sensitive cellular counterparts, in order to identify biomarkers of MDR.MethodsTwo pairs of MDR and drug-sensitive counterpart tumour cell lines were studied as models. EVs were characterized in terms of size and molecular markers and their protein content was investigated by proteomic analysis and Western blot.ResultsWe found that MDR cells produced more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart. EVs from MDR cells contained P-gp and presented a different content of proteins known to be involved in the biogenesis of EVs, particularly in the biogenesis of exosomes.ConclusionsThe determination of the size and of this particular protein content of EVs shed by tumour cells may allow the development of a minimally-invasive simple method of detecting and predicting MDR.General significanceThis work describes for the first time that cancer multidrug resistant cells shed more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart cells, carrying a specific content of proteins involved in EV biogenesis that could be further studied as biomarkers of MDR.