Analysis of Extracellular Vesicles: New Avenues for Signaling Biology and Biomarker Discovery

Abstract

The broad term “extracellular vesicles” summarizes a group of nanometer sized, membranous vesicles secreted from a variety of cell types. The most commonly studied vesicles include exosomes, microvesicles, prostasomes and apoptotic bodies. These vesicles differ in size, biogenesis, marker composition and molecular cargo such as DNA, RNA, proteins and lipids. Although the first manuscripts on exosomes date back nearly 30 years, the field of extracellular vesicle research has witnessed a significant surge in interest in recent years. For example in 2012, >300 manuscripts were published using a PubMed query containing the keywords “exosomes AND 2012”. This rapid expansion in extracellular vesicle research is likely fueled by several complementary research areas that have resulted in a better understanding of: i) molecular mechanisms of intercellular communication/signaling; ii) detailed analyses of extracellular vesicle cargo in the context of biomarker discovery; and iii) mechanisms of vesicle biogenesis. The current Focus Issue in PROTEOMICS centers on exosomes and related extracellular vesicles, providing interested readers with a comprehensive overview of current exosome research. The issue is completed by three contributions on new research developments, which highlight the multiple research areas, described above. Below, I provide a brief summary of all articles published in this Focus Issue, while attempting to categorize them into broader categories that might be useful for the readership. In the last section of this editorial I attempt to speculate on future developments/directions of this exciting research area. Choi et al. provide a broad overview of current omics technologies used for the molecular characterization of various cargo molecules (RNA, protein, lipids) of exosomes and ectosomes (also known as microvesicles). This review covers technical aspects of isolation procedures, biological implications and potential diagnostic applications. Dear and colleagues deliver a manuscript on urinary exosome research in the context of kidney disease and biological function. Exosome isolation for down-stream biomarker discovery, in the context of intercellular signaling and cell-cell communication along the nephron, are covered in this review. In recent years, extracellular vesicles have generated considerable excitement, especially in the area of cancer research. Currently, the majority of manuscripts are focused on diagnostic and/or biomarker applications. The idea here is that extracellular vesicles carry cell-type specific cargo that can be readily isolated from biofluids. This could potentially overcome sensitivity, dynamic range and stability problems faced by current biofluid analysis pipelines. Alternatively, recent studies in breast cancer 1 and melanoma 2 have impressively linked tumor-derived extracellular vesicles to detailed molecular mechanisms in cancer biology. In this Focus Issue Fontana et al. provide a comprehensive overview of extracellular vesicles in cancer biology and biomarker discovery, nicely setting the stage for tumor-derived extracellular vesicles. Rak and colleagues review the literature of extracellular microvesicles with a specific focus on brain tumors, while Principe et al. covers tumor-derived vesicles in the context of head and neck cancer. The review component of this Focus Issue is completed by a series of articles that cover more “boutique” areas of extracellular vesicles research, providing a primer for more extensive investigations in the future. The group of Rama Khokha provides a detailed manuscript on metalloproteinases (MMPs and ADAMs) commonly detected in extracellular vesicles. These vesicular proteases are thought to be involved in a complicated cross-talk of tumor cells and the microenvironment, contributing to invasion, progression and metastasis. Future studies are required to carefully dissect their molecular mechanisms in the context of tumor biology. Katsuda et al. review the therapeutic role of mesenchymal stem cell-derived extracellular vesicles. The authors summarize the literature and provide a detailed discussion of current efforts in advancing the therapeutic efficiency of mesenchymal stem cell-derived extracellular vesicles. Gramolini and colleagues review the literature of the currently poorly studied cardiovascular exosomes. The authors also comment on future research questions to investigate intercellular communication via exosomes in the heart. In recent years the group of Willem Stoorvogel has made significant contributions to the molecular characterization of prostasomes; a sub-type of vesicles specifically released by prostate epithelial cells. Prostasomes are directly linked to sperm function and immune processes during fertilization. In the current article, this group extends on their previous discovery of two distinct prostasome sub-populations. Briefly, lipid extracts were prepared from prostasomes and analyzed by liquid chromatography mass spectrometry (LC-MS). Interestingly, unusual lipid compositions were reported for both populations, including quantitative differences in sphingomyelin and hexosylceramides. Based on these data, potential implications in the context of prostasome formation and function are discussed. In a related study, Drake and colleagues report an in-depth proteome resource of exosomes isolated from expressed prostatic secretions in urine (EPS-urine), a biofluid enriched in prostasomes with clinical relevance for prostate cancer biomarker discovery. Next-generation LC-MS approaches were used to report a large repertoire of protein cargo from a relatively small volume of EPS-urine. Finally, the group of Richard Simpson, one of the pioneers of exosome proteomics/biology, reports on a quantitative proteomic comparison of isogenic colorectal cancer cell lines established from the primary tumor and the metastasis, using highly purified exosomes. Quantitative differences that provide novel insights into metastatic factors and signaling pathways related to tumor progression are reported. The current Focus Issue, which covers the rapidly growing field of extracellular vesicle research, provides a broad range of manuscripts covering all aspects of extracellular vesicle research. It is reasonable to speculate considerable growth of this research area in the coming years with a particular focus on biomarker discovery and detailed mechanistic studies on the roles of extracellular vesicles in horizontal signaling, cell-cell communication and disease progression. To accomplish these goals and achieve impact, both in terms of translational science and basic research, several steps must be taken. In the context of clinical diagnostics, detailed protocols and quality control criteria must be established for the rapid and reproducible collection of extracellular vesicles from diverse human biofluids. Ideally, these protocols must be applicable to small volumes (<5ml) to be compatible with most current bio-banking efforts. In addition, multi-institutional studies, addressing specific clinical needs, must be initiated using richly annotated clinical cohorts. These studies will provide unambiguous proof for the utility of extracellular vesicles in clinical diagnostics to mirror tissue alterations in easily accessible biofluids (i.e. a concept often referred to as “liquid biopsy”). In an ideal scenario, such studies should simultaneously quantify various vesicular cargo molecules (i.e. DNA, RNA, proteins and lipids). In the context of cancer diagnostics, it would be reasonable to suggest integration with current large-scale cancer genomics profiling efforts, to specifically monitor for known genomic alterations. Although in recent years the functional relevance of cancer-associated extracellular vesicles has been impressively demonstrated in breast cancer and melanoma, additional mechanistic studies are required to dissect the exact mechanism of vesicle biogenesis, cell-type specific cargo incorporation and intercellular signaling mechanism using well-established model systems. In summary, I hope you enjoy this Focus Issue and that it might serve as a primer for future research programs with a specific focus on extracellular vesicles. I would like to thank all authors for their contributions, the reviewers for their invaluable insights and suggestions throughout the submission process and Drs. Dunn and Kraus for their support and patience in preparing this Focus Issue. Thomas Kislinger