Mass spectrometry-based proteome profiling of extracellular vesicles and their roles in cancer biology

1Extracellular vesicles (EVs) are membrane-surrounded vesicles released by numerous cell types into the extracellular microenvironment–. EVs are involved in cell–cell communication, coagulation, inflammation, immune response modulation, and disease progression,–. Although EVs vary in size, biological function, and components, their significance in cancer progression and the potential use of EV molecules as novel cancer biomarkers has gradually increased. Cancer cells actively release EVs into neighboring tissues, and these EVs play dynamic roles in cancer progression and metastasis, invasion, angiogenesis, tumorigenesis, and immune modulation–. EVs released by cancer cells are usually chosen as a gateway in the search for biomarkers for a specific cancer type. Recent results pertaining to EV-cargo molecules, including proteins and miRNAs, are summarized in EVpedia (http://evpedia.info), an integrated and comprehensive database of EVs.

2The main focus of this review is proteome profiling of EVs using mass spectrometry (MS)-based proteomic approaches. We discuss the mass spectral characterization of isolated EV proteins from different cancers and the use of these proteins as predictive cancer biomarkers. Additionally, we summarize the key characteristics of enriched proteins in cancer-associated EVs as potential therapeutic targets and provide novel information on their roles in cancer development and progression. Information provided in this review may help in understanding recent progress regarding EV biology and the prospective roles of EVs as new noninvasive biomarkers and therapeutic targets, as well as emerging therapeutic opportunities and associated challenges.

3EVs are small spherical vesicles that are secreted into the extracellular milieu by many cell types. The term “EV” was invented by the International Society of Extracellular Vesicles (ISEV) and is used to define all phospholipid bilayer-bound vesicles that are secreted by cells into the extracellular microenvironment, regardless of the differences in biogenesis, size, and composition,. The roles of EVs in different physiological and pathological processes have made them a novel field of research. EVs are categorized into several subtypes based on their size, density, shape, subcellular origin, function, and molecular cargo. The four major subtypes of EVs are exosomes, microvesicles, apoptotic bodies, and oncosomes (Table 1 and Fig. 1). Exosomes are 30–200-nm-sized homogeneous membrane vesicles, and they form through the endosomal trafficking pathway,,. Exosomes contain late endosomal markers, even though biochemically indistinguishable vesicles can bud directly from the plasma membrane,. They play critical roles in cell–cell communications, such as that occurring during the regulation of cell and tissue homeostasis, as well as in pathological conditions. Microvesicles are 100–1000-nm-sized heterogeneous membrane vesicles that originate via outward budding and the fission of the plasma membrane due to dynamic interactions during phospholipid redistribution. Phospholipid distribution is controlled by aminophospholipid translocases,– and cytoskeletal protein contraction. Microvesicles are released mostly under cellular stress or in pathological processes. Like exosomes, microvesicles transfer bioactive molecules into target cells. Apoptotic bodies (> 1 µm) are released by cells that undergo the apoptosis process or programmed cell death,, and they can be characterized by cellular organelles and DNA. Finally, the vesicles named “oncosomes” are much larger than most other EV types characterized to date (1–10 μm). Owing to their unusual size, large oncosomes might have unique properties in vivo and would provide novel opportunities for tumor profiling.

4EVs contain proteins, lipids, metabolites, and RNAs. However, the mechanisms by which these components enter EVs remain obscure. EVs are shed from almost all cell types and are present in biological fluids and conditioned cell culture media. EVs are involved in cell–cell communication, coagulation, inflammation, immune response modulation, and disease progression–. The functional roles of EVs in intercellular communication have made them of major interest in many scientific fields. The biomolecular composition of EVs could play a significant role in disease progression in several neurodegenerative diseases as well as in cancer.

5EV proteome analysis is a novel approach and is part of the growing interest in proteomics cancer research. Over the past three decades, many proteomics studies performed on EVs have elucidated their diverse roles. Large-scale proteomics datasets and protein-interaction networks have established significant relationships between EV proteins, which improves the understanding of vesicle biogenesis and pathophysiological roles,,. Proteomic studies on EVs from different origins have also suggested a controlled protein-sorting mechanism and the random packaging of EV proteins from various cell types that contain common vesicular proteins. Furthermore, proteomic studies of EVs have produced a high-throughput vesicular proteome dataset from various cell types and body fluids. Since EVs are normally isolated in small amounts, better sensitivity is required for their analysis. Liquid chromatography (nanoscale or ultra-high performance)–electrospray ionization tandem mass spectrometry (LC/ESI–MS/MS) is the most popular and versatile analytical technique to study the molecular contents of EVs. In particular, nano-ESI–MS/MS provides high sensitivity and resolution, allowing the detection, identification, characterization, and quantification of thousands of proteins from even a single EV sample. Similar to other biological fields, LC–MS/MS-based technological platforms have become the most popular fundamental tools for elucidating the structural and functional architecture of EVs. The fragment ions from ESI (positive- and negative-ion) tandem MS experiments provide the composition, unambiguous structural characterization, and proper identification of proteins present in various biological samples. Due to the high sensitivity and small initial sample volumes required for MS, MS-based proteomic analysis has increased the understanding of EV protein content. Several investigators,– have used ESI tandem MS experiments in combination with chromatographic methods (HPLC, UHPLC, UPLC, and nano LC) to profile and structurally characterize proteins in various cancer cells, tissues, biofluids, and biological samples, which have been summarized in Table 2.

6Proteomic analysis of EVs has revealed significant changes in protein expression under various physiological and pathological conditions,,. Characterization of these proteomic profiles may be useful in understanding disease pathogenesis and assisting in the discovery of new biomarkers for different diseases. The secretion of EVs from several types of tumor cells is a significant means of conditioning and altering the tumor microenvironment by malignant cells,. Multiple studies have reported that the secretion of EVs from cancer cells contributes to angiogenesis, metastasis, tumor formation, and disease progression,,,. EVs are more attractive sources of biomarkers because of their biological consequences and relatively noninvasive accessibility in a wide range of biological fluids. EVs have been studied in relation to numerous cancers, such as colorectal,, bladder, prostate, pancreatic, breast, gastric, lung, blood, ovarian, cholangiocarcinoma, hepatocellular carcinoma, and oral squamous cell carcinoma (Table 2), as well as cardiovascular diseases and malignancies of the central nervous system. The proteomic analysis of EVs, specifically the analysis of their protein composition, may be helpful for further understanding the mechanisms of their biogenesis and their functional roles. Molecular communication between cancer cells and their stromal microenvironment is a key factor for cancer progression,. In conjunction with typical secretory pathways, it was proposed that these small membranous vesicles are alternate mediators of intercellular communication. EVs carry an effector-rich proteome with the ability to control different functional properties of the recipient cell. The protein composition of EVs from different sources was studied previously by using MS,–, providing a robust basis for the identification of biomarker proteins in EVs for the purpose of quality control research. A thorough understanding of the protein composition of EV subtypes and the extent to which EV composition reflects the source cell composition is essential for further development of diagnostics and therapeutics. Although EVs are secreted by almost all cell types, some available data suggest the enhanced release of EVs under pathological conditions, such as cancer. It is reasonable to expect that these vesicles may also play key roles in tumorigenesis since they can facilitate distant intercellular communication. Tumor-derived EVs typically carry tumor antigens, and functional proteins can be transferred to recipient cells through EVs,,. A better understanding of the molecular bases underlying cancer invasion and metastasis is necessary to develop effective targets for therapy.

7EV proteins from many cancers have similar biological processes and functions. To understand the functions of differentially expressed proteins (DEPs) in cancer, we performed gene ontology analysis on a variety of DEPs. As expected, the EV–DEPs from different cancer types were implicated in similar biological processes, such as cell adhesion, migration, and transport. Considering that EVs are potential metastasis factors, those proteins appear to be relevant for cancer metastasis or cancer cell proliferation. Of the 12 different cancers evaluated, we observed that DEPs that overlapped more than five times were primarily related to cancer metastasis or cancer cell proliferation, and many of the DEPs had strong interactions with each other (Fig. 2). Even though the selection of these DEPs from different cancers was biased, the roles of EVs in different cancers focused mainly on cell adhesion and cell migration.

8In this review, we summarized different EV studies to discuss the potential of EVs in cancer treatment. All studies discussed in this review indicated that the specific protein composition of various EVs has high potential for identifying different cancers. The majority of these studies revealed the relationship of cancer with changes in the protein contents of various body fluids. Moreover, we have highlighted the emerging roles of EVs in cancer, specifically their role in metastasis, which opens the possibility of the rapid translation of EV research for clinical applications in diagnosis, prognosis, and treatment. Ultimately, the majority of the investigations discussed in this review need further verification in large-cohort, multicenter clinical studies. In the future, highly reliable EV proteome data could be combined with well-developed current popular genomic and other “omics-” studies to provide extended knowledge of EVs from the perspective of systems biology approaches.

9There are many perspectives on the potential contribution of EV research for the development of cancer therapeutics and diagnosis. EVs could play key roles in intercellular communication during cancer development, which may offer new therapeutic strategies for various cancers. EV protein composition in different body fluids reveals the overall condition of the patient and is also useful for screening the efficacy and toxicity of anticancer treatments. Additionally, EVs could be used as cancer vaccines and drug delivery components. Moreover, the inhibition of intercellular communication through EVs might provide opportunities to suppress tumor progression. In the near future, clinical applications of EVs could contribute to cancer management and treatment. However, before EV-targeted therapy can be applied in cancer, the identification of cancer-specific genes or molecules that are crucial for EV communication is necessary.