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.     

Understanding extracellular vesicle diversity – current status

ABSTRACT

Introduction: Extracellular vesicles (EVs) represent an important mode of intercellular communication. There is now a growing awareness that predominant EV subtypes; exosomes from endosomal origin, and shed microvesicles from plasma membrane budding, can be further stratified into distinct subtypes, however specific approaches in their isolation and markers that allow them to be discriminated are lacking.

Areas covered: Knowledge about these distinct EV subpopulations is important including the regulation of composition, release, targeting/localization, uptake, and function. This review discusses the mechanisms of distinct EV biogenesis and release, defining select EV classes (and subpopulations), which will be crucial for development of EV-based functions and clinical applications. We review the dynamics of cargo sorting leading to the mechanisms of EV heterogeneity, their mechanisms of formation, intracellular trafficking pathways, and provide an uptake about biochemical/functional differences. With advances in purification strategies and proteomic-based quantitation, allows significant benefit in accurately describing differences in EV protein cargo composition and modification.

Expert commentary: The advent of quantitative mass spectrometry-based proteomics, in conjunction with advances in molecular cell biology, and EV purification strategies, has contributed significantly to our improved characterization and understanding of the molecular composition and functionality of these distinct EV subpopulations.     

Blood/plasma secretome and microvesicles

A major but hitherto overseen component of the blood/plasma secretome is that of extracellular vesicles (EVs) which are shed from all blood cell types. These EVs are made up of microvesicles (MVs) and exosomes. MVs, 100 nm–1 μm in diameter, are released from the cell surface, and are a rich source of non-conventionally secreted proteins lacking a conventional signal peptide, and thus not secreted by the classical secretory pathways. Exosomes are smaller vesicles (≤ 100 nm) having an endocytic origin and released upon multivesicular body fusion with the plasma membrane. Both vesicle types play major roles in intercellular cross talk and constitute an important component of the secretome especially in the area of biomarkers for cancer. The release of EVs, which are found in all the bodily fluids, is enhanced in cancer and a major focus of cancer proteomics is therefore targeted at EVs. The blood/plasma secretome is also a source of EVs, potentially diagnostic of infectious disease, whether from EVs released from infected cells or from the pathogens themselves. Despite the great excitement in this field, as is stated here and in other parts of this Special issue entitled: An Updated Secretome, much of the EV research, whether proteomic or functional in nature, urgently needs standardisation both in terms of nomenclature and isolation protocols. This article is part of a Special Issue entitled: An Updated Secretome.     

Urinary extracellular microvesicles: Isolation methods and prospects for urinary proteome

Extracellular microvesicles (EVs) are membranous vesicles, which are released from diverse cells. These EVs have also been found in a wide range of body fluids. The cargo of EVs, including proteins, lipids, carbohydrates, and nucleic acids, can be stably preserved in EVs. Researchers have found that EVs can mediate intercellular communication by shuttling the cargo components. Therefore, EVs can be used for the identification of disease‐specific biomarkers. As one class of EVs, urinary exosomes can reflect the status of the renal system. Moreover, urinary exosome analysis can minimize the interference of high abundant proteins in the whole urine sample. Therefore, urinary exosomes have gained much attention in recent years. In this review, we present a comprehensive summary of urinary exosome studies in recent years, including collection, storage, and isolation methods. The normal and disease proteomic analyses of urinary exosomes are also presented. Thus, this review may provide a valuable reference for future research.     

Extracellular vesicles: Mediators of embryo-maternal crosstalk during pregnancy and a new weapon to fight against infertility

In modern-day life, infertility is one of the major issues that can affect an individual, both physically and psychologically. Several anatomical, physiological, and genetic factors might contribute to the infertility of an individual. Intercellular communication between trophectoderm and endometrial epithelium triggers successful embryo implantation and thereby establishes pregnancy. Recent studies demonstrate that Extracellular vesicles (EVs) are emerging as one of the crucial components that are involved in embryo-maternal communication and promote pregnancy. Membrane-bound EVs release several secreted factors within the uterine fluid, which mediates an intermolecular transfer of EVs’ cargos between blastocysts and endometrium. Emerging evidences indicate that several events like imbalance in the release of endometrial or placenta-derived EVs (exosomes/MVs), uptake of their content, failure of embryo selection might lead to implantation failure. Here in this review, we have discussed the current knowledge of the involvement of EVs in maternal-fetal communications during implantation and also highlighted the EVs’ rejuvenating ability to overcome infertility-related issues. We also discussed the alteration of the EVs’ cargo in different pathological conditions that lead to infertility. Therefore, this review would give a better understanding of EVs’ contribution in successful embryo implantation, which could help in the development of new diagnostic tools and cell-free biologics to improve the in vivo reproductive process and to treat infertility by restoring normal reproductive functions.     

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.     

Matrix vesicles from chondrocytes and osteoblasts: Their biogenesis,properties, functions and biomimetic models

BackgroundMatrix vesicles (MVs) are released from hypertrophic chondrocytes and from mature osteoblasts, the cells responsible for endochondral and membranous ossification. Under pathological conditions, they can also be released from cells of non-skeletal tissues such as vascular smooth muscle cells. MVs are extracellular vesicles of approximately 100–300 nm diameter harboring the biochemical machinery needed to induce mineralization.Scope of the reviewThe review comprehensively delineates our current knowledge of MV biology and highlights open questions aiming to stimulate further research. The review is constructed as a series of questions addressing issues of MVs ranging from their biogenesis and functions, to biomimetic models. It critically evaluates experimental data including their isolation and characterization methods, like lipidomics, proteomics, transmission electron microscopy, atomic force microscopy and proteoliposome models mimicking MVs.Major conclusionsMVs have a relatively well-defined function as initiators of mineralization. They bind to collagen and their composition reflects the composition of lipid rafts. We call attention to the as yet unclear mechanisms leading to the biogenesis of MVs, and how minerals form and when they are formed. We discuss the prospects of employing upcoming experimental models to deepen our understanding of MV-mediated mineralization and mineralization disorders such as the use of reconstituted lipid vesicles, proteoliposomes and, native sample preparations and high-resolution technologies.General significanceMVs have been extensively investigated owing to their roles in skeletal and ectopic mineralization. MVs serve as a model system for lipid raft structures, and for the mechanisms of genesis and release of extracellular vesicles.     

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.     

Fourier-Transform InfraRed (FT-IR) spectroscopy to show alterations in molecular composition of EV subpopulations from melanoma cell lines in different malignancy

BackgroundMelanoma cells release extracellular vesicles (EVs) subpopulations which differ in size, phenotype and molecular content. Melanoma derived EVs play a role in the development and progression of cancer by delivering surface receptors and bioactive (proteins, lipids, nucleic acids) or signaling molecules to target cells.MethodsWe applied Fourier Transform Infrared Spectroscopy (FTIR) to compare infrared spectra of absorption for different subpopulations of EVs originating from normal human melanocytes, primary cutaneous melanoma (WM115) and metastatic cutaneous melanoma (WM266-4).ResultsFTIR results showed that exosome and ectosome populations differ in content of protein and lipid components. We obtained higher lipid to protein ratio for ectosomes in comparison with exosomes what confirms that exosomes are very densely packed with protein cargo. We identified the lowest value of saturated fatty acids/unsaturated fatty acids parameter in the metastatic WM266-4 cell line and ectosomes derived from WM266-4 cell line in comparison with normal melanocytes and the primary WM115 cell line. We identified the alterations in the content of secondary structures of proteins present in EV subpopulations originating from melanocytes and melanoma cells in different malignancy.ConclusionsObtained results revealed differences in the molecular composition of melanoma derived EVs subtypes, including protein secondary structure, and showed progressive structural changes during cancer development.     

Proteomics and personalized medicine: a focus on kidney disease

ABSTRACT

Introduction: Inter-individual variability in response to drug treatment has induced an increased demand for decisions via personalize medicine. Also, the contribution of proteomics to the era of personalized medicine would seem to be vital in improving therapeutic outcomes.

Areas covered: We review validated biomarkers discovered by proteomics techniques and their use in personalized medicine with the focus on kidney diseases. We discuss this topic with a special emphasis on recent publications and relevant initiatives and depict some limitations that remain for personalized medicine.

Expert opinion: The development of highly accurate biomarkers is essential for optimizing the management of kidney diseases. Various biomarkers of kidney diseases have been identified using proteomic techniques. However, only a few of these biomarkers showed the potential to be used in clinical practice concerning personalized medicine. Therefore, it becomes evident that the combination of multiple biomarkers confers higher accuracy and the ability to depict complex pathophysiological conditions, a prerequisite for personalized treatment. CKD273, a multimarker panel for early CKD detection may serve as a first example for personalized medicine in nephrology. Based on this successful example, proteomics is expected to develop into the key technology to guide personalized intervention.     

Qualitative differences in T‐cell activation by dendritic cell‐derived extracellular vesicle subtypes

Exosomes, nano‐sized secreted extracellular vesicles (EVs), are actively studied for their diagnostic and therapeutic potential. In particular, exosomes secreted by dendritic cells (DCs) have been shown to carry MHC‐peptide complexes allowing efficient activation of T lymphocytes, thus displaying potential as promoters of adaptive immune responses. DCs also secrete other types of EVs of different size, subcellular origin and protein composition, whose immune capacities have not been yet compared to those of exosomes. Here, we show that large EVs (lEVs) released by human DCs are as efficient as small EVs (sEVs), including exosomes, to induce CD4⁺ T‐cell activation in vitro. When released by immature DCs, however, lEVs and sEVs differ in their capacity to orient T helper (Th) cell responses, the former favouring secretion of Th2 cytokines, whereas the latter promote Th1 cytokine secretion (IFN‐γ). Upon DC maturation, however, these functional differences are abolished, and all EVs become able to induce IFN‐γ. Our results highlight the need to comprehensively compare the functionalities of EV subtypes in all patho/physiological systems where exosomes are claimed to perform critical roles.     

Exosomes at the crossroad between therapeutic targets and therapy resistance in head and neck squamous cell carcinoma

Head and neck squamous cell carcinomas (HNSCCs) are aggressive and clinically challenging tumours that require a multidisciplinary management approach. Despite significant therapy improvements, HNSCC patients have a poor prognosis with a 5-year survival rate of about 65%. As recently recognised key players in cancer, exosomes are extracellular vesicles (EVs) with a diameter of nearly 50–120 nm which transport information from one cell to another. Exosomes are actively involved in various aspects of tumour initiation, development, metastasis, immune regulation, therapy resistance, and therapeutic applications. However, current knowledge of the role of exosomes in the pathophysiological processes of HNSCC is still in its infancy, and additional studies are needed. In this review, we summarise and discuss the relevance of exosomes in mediating local immunosuppression and therapy resistance of HNSCC. We also review the most recent studies that have explored the therapeutic potential of exosomes as cancer vaccines, drug carriers or tools to reverse the drug resistance of HNSCC.     

Emerging MS-based platforms for the characterization of tumor-derived exosomes isolated from human biofluids: challenges and promises of MudPIT

Introduction: Exosomes are small extracellular vesicles of endosomal origin that are produced and released by several type of cells. These vesicles contain different macromolecules: proteins, mRNA, miRNA, mitochondrial DNA, and lipids. Exosomes play an important role in cell-to-cell communication, also promoting cancer progression.

Areas covered: Various proteomic approaches have been applied to study exosomes isolated from different human biofluids in search of possible cancer biomarkers. The results of these studies are reported, and pros and cons of each employed technique are described. Gel-free and gel-based mass spectrometry systems are discussed, giving particular emphasis on the innovative multidimensional protein identification technology (MudPIT).

Expert commentary: Proteomic studies on exosomes as candidate cancer biomarkers from urine and other body fluids in cancer have shown the potential of MS-based techniques. In particular, MudPIT is a promising tool to be applied in clinical proteomics of cancer.     

Utilizing proteomics to understand and define hypertension: where are we and where do we go?

Introduction: Hypertension is a complex and multifactorial cardiovascular disorder. With different mechanisms contributing to a different extent to an individual’s blood pressure, the discovery of novel pathogenetic principles of hypertension is challenging. However, there is an urgent and unmet clinical need to improve prevention, detection, and therapy of hypertension in order to reduce the global burden associated with hypertension-related cardiovascular diseases.

Areas covered: Proteomic techniques have been applied in reductionist experimental models including angiotensin II infusion models in rodents and the spontaneously hypertensive rat in order to unravel mechanisms involved in blood pressure control and end organ damage. In humans proteomic studies mainly focus on prediction and detection of organ damage, particularly of heart failure and renal disease. While there are only few proteomic studies specifically addressing human primary hypertension, there are more data available in hypertensive disorders in pregnancy, such as preeclampsia. We will review these studies and discuss implications of proteomics on precision medicine approaches.

Expert commentary: Despite the potential of proteomic studies in hypertension there has been moderate progress in this area of research. Standardized large-scale studies are required in order to make best use of the potential that proteomics offers in hypertension and other cardiovascular diseases.     

Cancer biomarker discovery and translation: proteomics and beyond

Introduction: Cancer is often diagnosed at late stages when the chance of cure is relatively low and although research initiatives in oncology discover many potential cancer biomarkers, few transition to clinical applications. This review addresses the current landscape of cancer biomarker discovery and translation with a focus on proteomics and beyond.

Areas covered: The review examines proteomic and genomic techniques for cancer biomarker detection and outlines advantages and challenges of integrating multiple omics approaches to achieve optimal sensitivity and address tumor heterogeneity. This discussion is based on a systematic literature review and direct participation in translational studies.

Expert commentary: Identifying aggressive cancers early on requires improved sensitivity and implementation of biomarkers representative of tumor heterogeneity. During the last decade of genomic and proteomic research, significant advancements have been made in next generation sequencing and mass spectrometry techniques. This in turn has led to a dramatic increase in identification of potential genomic and proteomic cancer biomarkers. However, limited successes have been shown with translation of these discoveries into clinical practice. We believe that the integration of these omics approaches is the most promising molecular tool for comprehensive cancer evaluation, early detection and transition to Precision Medicine in oncology.     

Proteomic approaches to identify blood-based biomarkers for depression and bipolar disorders

Introduction: Major depressive disorder (MDD) and bipolar disorder (BD) are leading causes of disability worldwide, yet many people remain undiagnosed, are misdiagnosed, and/or ineffectively treated. Diagnosis relies on the clinical assessment of symptoms, and there is currently no molecular or brain-imaging diagnostic test available. Identifying and validating protein biomarkers could provide a more accurate and objective means of diagnosis.

Areas covered: Proteomics is a powerful tool that enables the identification and quantification of novel candidate biomarkers of disease. In this review, we discuss the role of proteomic technologies in biomarker discovery and validation, peripheral blood as a source of protein biomarkers, statistical methods for analyzing proteomic data, and some existing challenges in the field. We also review a selection of previously published studies focused on identifying blood-based diagnostic protein biomarkers of MDD and BD within a ten-year period.

Expert commentary: Proteomic studies have led to the identification of numerous potential biomarkers of MDD and BD. However, clinical validation and translation into clinical practice have not yet been achieved. Conducting large-scale validation studies and addressing various factors that limit the reproducibility of the proteomic findings are key to ensure that robust and reliable biomarker tests are developed and clinically validated.     

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.     

Concepts and strategies of soybean seed proteomics using the shotgun proteomics approach

ABSTRACT

Introduction: The last decade has yielded significant developments in the field of proteomics, especially in mass spectrometry (MS) and data analysis tools. In particular, a shift from gel-based to MS-based proteomics has been observed, thereby providing a platform with which to construct proteome atlases for all life forms. Nevertheless, the analysis of plant proteomes, especially those of samples that contain high-abundance proteins (HAPs), such as soybean seeds, remains challenging.

Areas covered: Here, we review recent progress in soybean seed proteomics and highlight advances in HAPs depletion methods and peptide pre-fractionation, identification, and quantification methods. We also suggest a pipeline for future proteomic analysis, in order to increase the dynamic coverage of the soybean seed proteome.

Expert opinion: Because HAPs limit the dynamic resolution of the soybean seed proteome, the depletion of HAPs is a prerequisite of high-throughput proteome analysis, and owing to the use of two-dimensional gel electrophoresis-based proteomic approaches, few soybean seed proteins have been identified or characterized. Recent advances in proteomic technologies, which have significantly increased the proteome coverage of other plants, could be used to overcome the current complexity and limitation of soybean seed proteomics.