Exosome Research

　　Current exosome research covers almost all research areas in biomedical science (immunology, neuroscience, oncology, endocrinology, and cardiovascular research). For example, exosomes derived from immune cells have been shown to contain antigen peptide/MHC complexes and various antigens, which suggests a possibility that exosomes might regulate various immune responses such as activation/inactivation of immune cells in addition to the exchange of antigenic information between immune cells.³⁾ In the nervous system, exosomes have been found to be involved not only in regulation of neural circuits⁴⁾ but also in extracellular release of proteins causing various neurodegenerative diseases for subsequent transmission to other cells, a process that might be deeply involved in disease progression.⁵⁾ Exosomes released by cancer cells contain many biomolecules related to angiogenesis and immune evasion, suggesting that they might contribute to construction of microenvironment optimal for cancer cell growth and promotion of cancer progression.6) In addition, the expression profile of adhesion molecules on the surface of exosomes from cancer cells has been shown to determine the destination of cancer metastasis.⁷⁾ Recently, exosomes released from adipocytes have been reported to regulate gene expression in the liver.⁸⁾ Furthermore, while many viruses leave cells by utilizing the pathway for exosome production, bacteria and parasites infecting cells are likely to regulate activities of bacteria/parasites infecting other cells via exosomes.^{9, 10)} Most of the above-mentioned functions are mediated by secretory cell-derived biomolecules located within exosomes. In particular, since mRNAs and miRNAs of secretory cell origin were identified in exosomes, potential involvement of exosomes in horizontal transmission of gene expression information between cells has been attracting great research interest.¹¹⁾ Since these RNAs are encapsulated within the lipid bilayer membrane of exosomes, they are not susceptible to degradation by RNase and remain intact in blood or other body fluid. Exosomes incorporated into target cells fuse with the endosome membrane to release encapsulated RNAs into the cytosol of target cells. Once released into the cytosol, mRNAs are translated into proteins while miRNAs suppress translation of target genes. Thus, exosomes regulate gene expression within target cells. A single exosome is considered to contain more than several ten-thousands of proteins and more than several thousands of mRNAs and miRNAs. The composition of these biomolecules may vary depending on the type and conditions of a secretory cell which originally harbored the exosome. In addition, the composition of proteins, mRNAs, and miRNAs within an exosome is different from that within the original secretory cell, which suggests the existence of a mechanism selectively loading exosome- specific proteins and mRNAs/miRNAs into exosomes. Such specific composition of exosome RNAs makes them attractive candidates for biomarkers and targets for therapeutic development. While mRNAs within exosomes incorporated into target cells are capable of inducing expression of functional proteins, most miRNA within exosomes are present as precursors of functional miRNA and their physiological significance is under extensive investigation. Thus, since exosomes contain a wide variety of proteins, RNAs, and lipids, construction of an exosome database "ExoCarta" is currently ongoing through claassification by cell types. Furthermore, while large scale analysis of exosomes utilizing cutting-edge methodologies in proteomics, transcriptomics, and system biology are separately conducted in laboratories all over the world, EV plugin for FunRich (a stand-alone functional enrichment analysis tool) is distributed aiming at a common and integrated analysis tool. Sharing information among researchers in a wide variety of research fields is essential for promotion of future exosome research.

Development of exosome-based therapeutic/diagnostic methods

　　In parallel with clarification of exosome functions, efforts to develop therapeutic methods applying exosome functions are being continued in recent years. For example, exosomes released from blood fibrocytes (a population of mesenchymal progenitors) accelerate wound healing by stimulating angiogenesis and inducing migration and proliferation of keratinocytes. Proangiogenic, anti-inflammatory miRNAs as well as a miRNA promoting collagen deposition within these exosomes are reportedly involved in this process.¹²⁾ In addition, exosomes released from dendritic cells in patients with cancer contain a variety of cancer cell-derived proteins and induce intense activation of cancer cell-specific cytotoxic T lymphocytes. Development of cancer immunotherapy based on this mechanism is currently in the early phase of clinical research.¹³⁾ On the other hand, suppression of exosomal functions involved in pathogenesis has also been attempted. For example, apoptosis- inducing TNF-α is accumulated at high concentrations in exosomes released from synovial fibroblasts in patients with rheumatoid arthritis and exacerbates the pathology of rheumatoid arthritis.¹⁴⁾ In addition, since cancer cellderived exosomes contain molecules related to cancer progression and neuron-derived exosomes contain molecules related to neurodegenerative diseases as described above, inhibition or removal of these exosomes may potentially suppress onset of these diseases. Advancement of future research is expected to clarify exosome functions and expand indications of clinically applied exosomes, thereby realizing utilization of exosomes for the treatment of various diseases. Furthermore, delivery of drugs such as siRNAs and anticancer agents to target cells using exosomes has been attempted. Since various cell adhesion molecules are expressed on the surface of exosome membrane and the expression profile of these molecules has been found to determine the target cells for exosome delivery, application of this property to development of a new drug delivery system (DDS) is expected.¹⁵⁾ Exosomes are extremely stable in body fluids, and the exosome lipid bilayer membrane encapsulating proteins and RNAs within vesicles protects them from degradation. Furthermore, exosomes remain relatively intact even in body fluid specimens stored for a long time after collection and are therefore considered as new and promising laboratory biomarkers for diseases. While correlations between exosomes and various diseases have been investigated, cancer cell-derived exosomes released into blood are recently attracting research interest due to difference in constituents from normal cell-derived exosomes and a correlation between constituents of cancer cell-derived exosomes and cancer progression has been extensively investigated as a potential tool for early cancer diagnosis.¹⁶⁾ In addition, exosomes in urine are expected as a new diagnostic marker for renal, prostate, and bladder diseases, while exosomes in cerebrospinal fluid as a new marker for brain tumor and neurodegenerative diseases.

Issues and future perspectives of exosome research

　　Although many studies on roles of exosomes have already been reported, experiments providing evidence for these reported phenomena use highly concentrated exosomes purified from body fluids and cell culture supernatants. Accordingly, whether these phenomena actually occur in vivo remains unclear. The sole approach for clarification of physiological actions of exosomes is to clarify the mechanism of exosome release and physiological phenomena induced by exosome release stimulation/inhibition through modulating the mechanism, which is expected to result in further advancement in exosome research. Another important issue to be addressed in future research and development is in vivo kinetics of exosomes (i.e., which exosomes are directed to which target cells). Conventional methods for exosome purification mainly involved ultracentrifugation and various commercial purification kits using polyethylene glycol (PEG) precipitation technique. However, exosome preparations obtained by these methods contain large amounts of contaminants and careful analysis is required to determine whether experimental results obtained are actually due to actions of exosome constituents per se. Furthermore, ultracentrifugation requiring cumbersome manipulation has several disadvantages including inconsistent recovery interfering quantitative analysis and requirement for an expensive instrument not compatible with high-throughput analysis. Conducting exosome research under these circumstances is difficult and development of technology for easy purification of exosomes at a high purity is urgently needed. We focused on Tim4, an exosome receptor expressed on macrophages, and prepared "Tim4 magnetic beads" by immobilizing the extracellular region of Tim4 on magnetic beads.¹⁷⁾ Since Tim4 binds to phosphatidylserine (PS), a phospholipid on the surface of exosome membrane, in a calcium ion-dependent manner, bound exosomes are released from these beads with an elution buffer containing ethylenediaminetetraacetic acid (EDTA), a chelating agent, to obtain highly purified intact exosomes. In fact, when exosomes released from human leukemia cells were purified by the Tim4-affinity method and compared for purity with exosomes purified by ultracentrifugation and PEG precipitation, the Tim4-affinity method yielded exosome preparations with exosome-specific proteins each exhibiting a band intensity over 10-100 times higher than that obtained by other methods and almost free from non-exosome contaminants, thereby demonstrating reproducible recovery of high-purity exosomes. As a result, many previously unidentified exosome proteins and RNAs could be identified from exosome preparations obtained by this method. Furthermore, application of the strong binding affinity of Tim4 toward exosomes realized high-sensitivity detection and assay of exosomes by enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS). On the other hand, while only crude preparations of microvesicles were conventionally obtained because differential centrifugation was the sole purification technique available, the Tim4-affinity method realized purification of microvesicles at a high purity as well. Details of these techniques are described in this guidebook. We expect that usefulness of these Tim4-affinity-based techniques will be appreciated in the world and greatly contribute to clarification of the original physiological functions of exosomes and microvesicles.

　　In addition to difficulties in detection and isolation of exosomes, the existence of various classification systems for exosomes and resulting lack of consensus among investigators regarding which method should be used for purification of the extracellular vesicles to be called "exosomes" make interpretation of experimental data and confirmation of reproducibility difficult. To overcome such situation, the International Society for Extracellular Vesicles (ISEV) has recently been established to nurture a global community of EV researchers and "Minimal Information for Studies of EVs" (MISEV) Guidelines has been published as international standard that investigators who intend to start EV research should consult.^{18, 19)} In addition, as a method for avoiding such confusion, EV-TRACK knowledge database has been constructed to record experimental conditions employed in individual EV-related articles.²⁰⁾ On the other hand, as EV research has attracted global research interest, a number of large-scale research projects have been launched in various countries. In the United States, National Health Institute (NIH) has initiated a strategic large-scale project "Extracellular RNA Communication" and special interest groups on EV research have been organized at prestigious international conferences such as Gordon Conference and Keystone Symposia since 2016. In Europe, research covering EV has already been conducted as a part of CANCER-ID project supported by "Innovative Medicines Initiative (IMI)," a public-private partnership (PPP) for research and development of medicines. In Japan, EV research has been selected as one of the Research and Development Strategic Objectives in 2017 established by the Ministry of Education, Culture, Sports, Science and Technology Japan and acceleration of future research in this field is expected. In any case, development of solid research methodologies and techniques that constitute the basis of EV research is essentially required for future advancement in this research field, and we expect that the Tim4-affinity method will grow up to be one such technique.

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