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.     

Fungal Extracellular Vesicles with a Focus on Proteomic Analysis

Extracellular vesicles (EVs) perform crucial functions in cell–cell communication. The packaging of biomolecules into membrane‐enveloped vesicles prior to release into the extracellular environment provides a mechanism for coordinated delivery of multiple signals at high concentrations that is not achievable by classical secretion alone. Most of the understanding of the biosynthesis, composition, and function of EVs comes from mammalian systems. Investigation of fungal EVs, particularly those released by pathogenic yeast species, has revealed diverse cargo including proteins, lipids, nucleic acids, carbohydrates, and small molecules. Fungal EVs are proposed to function in a variety of biological processes including virulence and cell wall homeostasis with a focus on host–pathogen interactions. EVs also carry signals between fungal cells allowing for a coordinated attack on a host during infection. Research on fungal EVs in still in its infancy. Here a review of the literature thus far with a focus on proteomic analysis is provided with respect to techniques, results, and prospects.     

细胞外囊泡在治疗膝骨关节炎中的作用与前景

膝骨关节炎（knee osteoarthritis,KOA）是以关节软骨退变为主要病变的退行性疾病。目前,KOA尚无有效治疗药物。细胞外囊泡（extracellular vesicles,EVs）是由细胞释放的脂质双分子层包绕形成的球状膜性囊泡,可在细胞间传递核酸、蛋白质等生物活性分子。与动物来源EVs相比,植物来源EVs因其来源广泛且经济,在药物载体递送研究领域引起广泛关注。通过基因工程等方法改造EVs进行药物递送,可极大提高药物递送效率及其疗效。本文综述了动、植物两种来源的EVs在KOA中的治疗进展,特别聚焦于工程化EVs作为药物递送载体在KOA治疗中的研发现状,旨在为利用EVs治疗KOA提供参考。     

Redefining Tissue Crosstalk via Shotgun Proteomic Analyses of Plasma Extracellular Vesicles

Protein signaling between tissues, or tissue cross‐talk is becoming recognized as a fundamental biological process that is incompletely understood. Shotgun proteomic analyses of tissues and plasma to explore this concept are regularly challenged by high dynamic range of protein abundance, which limits the identification of lower abundance proteins. In this viewpoint article, it is highlighted how a focus on proteins contained within extracellular vesicles (EVs) not only partially addresses this issue, but can also reveal an underappreciated complexity of the circulating proteome in various physiological and pathological contexts. Furthermore, how quantitative proteomics can inform EV mediated crosstalk is highlighted and the importance of high coverage, sensitive proteomic analyses of EVs to identify both the optimal methods to isolate EV subtypes of interest and proteins that characterize them is stressed.     

Extracellular vesicle‐associated organotropic metastasis

Metastasis refers to the progressive dissemination of primary tumour cells and their colonization of other tissues and is associated with most cancer‐related mortalities. The disproportional and systematic distribution pattern of distant metastasis in different cancers has been well documented, as is termed metastatic organotropism, a process orchestrated by a combination of anatomical, pathophysiological, genetic and biochemical factors. Extracellular vesicles (EVs), nanosized cell‐derived membrane‐bound particles known to mediate intercellular communication, are now considered crucial in organ‐specific metastasis. Here, we review and summarize recent findings regarding EV‐associated organotropic metastasis as well as some of the general mechanisms by which EVs contribute to this important process in cancer and provide a future perspective on this emerging topic. We highlight studies that demonstrate a role of tumour‐derived EVs in organotropic metastasis via pre‐metastatic niche modulation. The bioactive cargo carried by EVs is of diagnostic and prognostic values, and counteracting the functions of such EVs may be a novel therapeutic strategy targeting metastasis. Further investigations are warranted to better understand the functions and mechanisms of EVs in organotropic metastasis and accelerate the relevant clinical translation.     

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including ‘microvesicles’ and ‘exosomes’, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.     

综述与专论: 基于核酸适配体的细胞外囊泡分离分析方法

细胞外囊泡通过参与细胞间通讯，在诸多生理病理过程中发挥着重要作用。因此，细胞外囊泡的分离分析对理解其生物学功能以及发展基于囊泡的疾病诊疗方法具有重要价值。细胞外囊泡的高效分离以及高灵敏可靠检测很大程度上取决于识别配体。核酸适配体是一类高效、特异结合其靶标分子的单链寡核苷酸。核酸适配体的易修饰和可程序化设计等特征，使其成为细胞外囊泡分离和分析的理想识别配体。为提高细胞外囊泡的分离效率，研究者们提出多种策略用于提升核酸适配体的亲和力，以及界面与细胞外囊泡的接触几率。此外，分离不同亚型的细胞外囊泡有助于理解细胞外囊泡的生物学意义。在细胞外囊泡分析方面，根据核酸适配体与细胞外囊泡识别信号的转导方式不同，分为电化学、可视化、表面增强拉曼光谱、荧光法等方法。本文综述了核酸适配体的筛选以及其在细胞外囊泡分离和分析中的最新进展、挑战及未来方向。     

综述: 胞外囊泡表面糖缀合物研究进展

胞外囊泡(extracellular vesicles,EVs)是一类由细胞分泌到胞外的能够被受体细胞摄取的膜性囊泡小体,直径在20～ 1 000 nm．近年来,越来越多的研究者发现胞外囊泡在疾病诊断、预后评估以及药物递送等方面具有重要的生物学作用．胞外囊泡可以直接参与细胞间信息的传递以及物质的运输,其携带的核酸(mRNA,microRNA和lncRNA)和蛋白质可以影响受体细胞的生理状态．大量研究表明,胞外囊泡是被糖基化修饰的,胞外囊泡表面覆盖了大量的聚糖以及糖结合蛋白,而已知聚糖类物质在调控细胞黏附、细胞-细胞之间的信息传递、细胞和细胞外基质相互作用、免疫调节和肿瘤转移等方面发挥重要的作用．本文综述了近年来细胞外囊泡表面糖缀合物修饰的前沿研究,以期更好地理解聚糖在胞外囊泡的合成、释放以及运输过程及其生物学功能中的作用．     

Embryonic Stem Cell-Derived Extracellular Vesicles Maintain ESC Stemness by Activating FAK

1. Download : Download high-res image (109KB)
2. Download : Download full-size image

     

Extracellular vesicles in urologic malignancies—Implementations for future cancer care

Extracellular vesicles (EVs), a heterogeneous group of vesicles differing in size and shape, cargo content and function, are membrane‐bound and nano‐sized vesicles that could be released by nearly all variations of cells. EVs have gained considerable attention in the past decades for their functions in modulating intercellular signalling and roles as potential pools for the novel diagnostic and prognostic biomarkers, as well as therapeutic targets in several cancers including urological neoplasms. In general, human and animal cells both can release distinct types of EVs, including exosomes, microvesicles, oncosomes and large oncosomes, and apoptotic bodies, while the content of EVs can be divided into proteins, lipids and nucleic acids. However, the lack of standard methods for isolation and detection platforms rein the widespread usage in clinical applications warranted furthermore investigations in the development of reliable, specific and sensitive isolation techniques. Whether and how the EVs work has become pertinent issues. With the aid of high‐throughput proteomics or genomics methods, a fully understanding of contents contained in EVs from urogenital tumours, beyond all doubt, will improve our ability to identify the complex genomic alterations in the process of cancer and, in turn, contribute to detect potential therapeutic target and then provide personalization strategy for patient.     

Application of Extracellular Vesicles Proteomics to Cardiovascular Disease: Guidelines,Data Analysis,and Future Perspectives

Extracellular vesicles (EVs) are a heterogeneous population of vesicles composed of a lipid bilayer that carry a large repertoire of molecules including proteins, lipids, and nucleic acids. In this review, some guidelines for plasma‐derived EVs isolation, characterization, and proteomic analysis, and the application of the above to cardiovascular disease (CVD) studies are provided. For EVs analysis, blood samples should be collected using a 21‐gauge needle, preferably in citrate tubes, and plasma stored for up to 1 year at ?80°, using a single freeze–thaw cycle. For proteomic applications, differential centrifugation (including ultracentrifugation steps) is a good option for EVs isolation. EVs characterization is done by transmission electron microscopy, particle enumeration techniques (nanoparticle‐tracking analysis, dynamic light scattering), and flow cytometry. Regarding the proteomics strategy, a label‐free and gel‐free quantitative method is a good choice due to its accuracy and because it minimizes the amount of sample required for clinical applications. Besides the above, main EVs proteomic findings in cardiovascular‐related diseases are presented and analyzed in this review, paying especial attention to overlapping results between studies. The latter might offer new insights into the clinical relevance and potential of novel EVs biomarkers identified to date in the context of CVD.     

Extracellular vesicles: pathogenetic,diagnostic and therapeutic value in traumatic brain injury

Introduction: Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Accurate classification according to injury-specific and patient-specific characteristics is critical to help informed clinical decision-making and to the pursuit of precision medicine in TBI. Reliable biomarker signatures for improved TBI diagnostics are required but still an unmet need.

Areas covered: Extracellular vesicles (EVs) represent a new class of biomarker candidates in TBI. These nano-sized vesicles have key roles in cell signaling profoundly impacting pathogenic pathways, progression and long-term sequelae of TBI. As such EVs might provide novel neurobiological insights, enhance our understanding of the molecular mechanisms underlying TBI pathophysiology and recovery, and serve as biomarker signatures and therapeutic targets and delivery systems.

Expert commentary: EVs are fast gaining momentum in TBI research, paving the way for new transformative diagnostic and treatment approaches. Their potential to sort out TBI variability and active involvement in the mechanisms underpinning different clinical phenotypes point out unique opportunities for improved classification, risk-stratification ad intervention, harboring promise of predictive, personalized, and even preemptive therapeutic strategies. Although a great deal of progress has been made, substantial efforts are still required to ensure the needed rigorous validation and reproducibility for clinical implementation of EVs.     

Unique Protein Profiles of Extracellular Vesicles as Diagnostic Biomarkers for Early and Advanced Non‐Small Cell Lung Cancer

Extracellular vesicles (EVs) mediate intercellular communication via transferring proteins and other biological molecules and have been recently investigated as biomarkers of disease. Sensitive and specific biomarkers are required for lung cancer diagnosis and prognosis. The present study screens for abnormal EV proteins in non‐small cell lung cancer (NSCLC) using a quantitative proteomics strategy involving LC‐MS/MS to identify ideal biomarkers for NSCLC diagnosis. EVs are enriched from the sera of early and advanced NSCLC patients and healthy controls and from cell culture supernatants of lung adenocarcinoma and bronchial epithelial cell lines. In the sera and supernatants, 279 and 632 differentially expressed proteins, respectively, are associated with signaling pathways including extracellular membrane–receptor interaction, focal adhesion, and regulation of the actin cytoskeleton. Thirty‐two EV proteins are identified at the intersection of differentially expressed proteins between the NSCLC groups and cell lines. Based on bioinformatics analysis, in silico immunohistochemical, and PRM verification, fibronectin is selected for following in vitro studies and validation with an independent cohort. Fibronectin on EVs is estimated to perform well in the diagnosis of NSCLC patients based on AUC, showing great potential for clinical use and demonstrating the efficacy of this method for EV‐associated biomarker screening.     

Application and Molecular Mechanisms of Extracellular Vesicles Derived from Mesenchymal Stem Cells in Osteoporosis

Osteoporosis (OP) is a chronic bone disease characterized by decreased bone mass, destroyed bone microstructure, and increased bone fragility. Accumulative evidence shows that extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) (MSC-EVs), especially exosomes (Exos), exhibit great potential in the treatment of OP. However, the research on MSC-EVs in the treatment of OP is still in the initial stage. The potential mechanism has not been fully clarified. Therefore, by reviewing the relevant literature of MSC-EVs and OP in recent years, we summarized the latest application of bone targeted MSC-EVs in the treatment of OP and further elaborated the potential mechanism of MSC-EVs in regulating bone formation, bone resorption, bone angiogenesis, and immune regulation through internal bioactive molecules to alleviate OP, providing a theoretical basis for the related research of MSC-EVs in the treatment of OP.     

Proteomic and Post‐Translational Modification Profiling of Exosome‐Mimetic Nanovesicles Compared to Exosomes

Issues associated with upscaling exosome production for therapeutic use may be overcome through utilizing artificial exosomes. Cell‐derived mimetic nanovesicles (M‐NVs) are a potentially promising alternative to exosomes for clinical applicability, demonstrating higher yield without incumbent production and isolation issues. Although several studies have shown that M‐NVs have similar morphology, size and therapeutic potential compared to exosomes, comprehensive characterization and to what extent M‐NVs components mimic exosomes remain elusive. M‐NVs were generated through the extrusion of cells and proteomic profiling demonstrated an enrichment of proteins associated with membrane and cytosolic components. The proteomic data herein reveal a subset of proteins that are highly abundant in M‐NVs in comparison to exosomes. M‐NVs contain proteins that largely represent the parental cell proteome, whereas the profile of exosomal proteins highlight their endosomally derived origin. This advantage of M‐NVs alleviates the necessity of endosomal sorting of endogenous therapeutic proteins or RNA into exosomes. This study also highlights differences in protein post‐translational modifications among M‐NVs, as distinct from exosomes. Overall this study provides key insights into defining the proteome composition of M‐NVs as a distinct from exosomes, and the potential advantage of M‐NVs as an alternative nanocarrier when spontaneous endosomal sorting of therapeutics are limited.