Proteomic Analysis of Extracellular Vesicles for Cancer Diagnostics

Extracellular vesicles (EVs) including exosomes and microvesicles are lipid bilayer‐encapsulated nanoparticles released by cells, ranging from 40 nm to several microns in diameter. Biological cargoes including proteins, RNAs, and DNAs can be ferried by EVs to neighboring and distant cells via biofluids, serving as a means of cell‐to‐cell communication under normal and pathological conditions, especially cancers. On the other hand, EVs have been investigated as a novel “information capsule” for early disease detection and monitoring via liquid biopsy. This review summarizes current advancements in EV subtype characterization, cancer EV capture, proteomic analysis technologies, as well as possible EV‐based multiomics for cancer diagnostics.     

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。     

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.     

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

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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.     

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.     

Studying the Fate of Tumor Extracellular Vesicles at High Spatiotemporal Resolution Using the Zebrafish Embryo

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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.     

胞外和胞内菌胞外囊泡的功能及应用

细胞外囊泡(Extracellular Vesicles,EVs)是从细胞膜上脱落或者分泌的双层膜结构的囊泡状小体.真核生物、细菌、古细菌和支原体等具有细胞结构的生物均能够释放EVs.细菌分泌的EVs含有DNA、RNA及蛋白质等多种成分,其在细菌毒力保持、免疫逃逸、细菌间物质运输、宿主细胞免疫调节、宿主转录基因调节、耐...     

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.     

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.     

Extracellular vesicles as modulators of cell-to-cell communication in the healthy and diseased brain

Homeostasis relies heavily on effective cell-to-cell communication. In the central nervous system (CNS), probably more so than in other organs, such communication is crucial to support and protect neurons especially during ageing, as well as to control inflammation, remove debris and infectious agents. Emerging evidence indicates that extracellular vesicles (EVs) including endosome-derived exosomes and fragments of the cellular plasma membrane play a key role in intercellular communication by transporting messenger RNA, microRNA (miRNA) and proteins. In neurodegenerative diseases, secreted vesicles not only remove misfolded proteins, but also transfer aggregated proteins and prions and are thus thought to perpetuate diseases by ‘infecting’ neighbouring cells with these pathogenic proteins. Conversely, in other CNS disorders signals from stressed cells may help control inflammation and inhibit degeneration. EVs may also reflect the status of the CNS and are present in the cerebrospinal fluid indicating that exosomes may act as biomarkers of disease. That extracellular RNA and in particular miRNA, can be transferred by EV also indicates that these vesicles could be used as carriers to specifically target the CNS to deliver immune modulatory drugs, neuroprotective agents and anti-cancer drugs. Here, we discuss the recent evidence indicating the potential role of exosomes in neurological disorders and how knowledge of their biology may enable a Trojan-horse approach to deliver drugs into the CNS and treat neurodegenerative and other disorders of the CNS.     

Extracellular-vesicle type of volume transmission and tunnelling-nanotube type of wiring transmission add a new dimension to brain neuro-glial networks

Two major types of intercellular communication are found in the central nervous system (CNS), namely wiring transmission (WT; point-to-point communication via private channels, e.g. synaptic transmission) and volume transmission (VT; communication in the extracellular fluid and in the cerebrospinal fluid). Volume and synaptic transmission become integrated because their chemical signals activate different types of interacting receptors in heteroreceptor complexes located synaptically and extrasynaptically in the plasma membrane. In VT, we focus on the role of the extracellular-vesicle type of VT, and in WT, on the potential role of the tunnelling-nanotube (TNT) type of WT. The so-called exosomes appear to be the major vesicular carrier for intercellular communication but the larger microvesicles also participate. Extracellular vesicles are released from cultured cortical neurons and different types of glial cells and modulate the signalling of the neuronal–glial networks of the CNS. This type of VT has pathological relevance, and epigenetic mechanisms may participate in the modulation of extracellular-vesicle-mediated VT. Gerdes and co-workers proposed the existence of a novel type of WT based on TNTs, which are straight transcellular channels leading to the formation in vitro of syncytial cellular networks found also in neuronal and glial cultures.     

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.     

Scalable Production and Isolation of Extracellular Vesicles: Available Sources and Lessons from Current Industrial Bioprocesses

Potential applications of extracellular vesicles (EVs) are attracting increasing interest in the fields of medicine, cosmetics, and nutrition. However, the manufacturing of EVs is currently characterized by low yields. This limitation severely hampers progress in research at the laboratory and clinical scales, as well as the realization of successful and cost‐effective EV‐based products. Moreover, the high level of heterogeneity of EVs further complicates reproducible manufacturing on a large scale. In this review, possible directions toward the scalable production of EVs are discussed. In particular, two strategies are considered: i) the optimization of upstream unit operations and ii) the exploitation of well‐established and mature technologies already in use in other industrial bioprocesses.