Development of extracellular vesicle (EV)-based diagnostics and therapeutics
Article ID: 2020-016
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Article ID: 2020-016
The term “Mi-byo” is increasingly used nowadays like a buzzword since people are afraid of diseases that may eventually fall on themselves with aging. In addition, it is also known that one out of two Japanese has cancer. Thus, it is necessary to identify the marker used to judge the health condition of humans, which is represented by the word “pre-disease”. Pre-disease is a condition in which no special abnormality is found even after undergoing an examination, and a specific illness is not diagnosed, but it cannot be said that the person is healthy. Many studies state that it is likely that it will cause illness if left unchecked. Thus, persons suffering from conditions such as hyperlipidemia, diabetes, and hypertension, can be considered as “undiseased”. However, it is important to develop new biomarkers or tools to quickly find the signs of these diseases and restore them to their original state. Therefore, in this review, I will overview the recent progress of a novel approach to diagnostic and therapeutic needs using small particles named extracellular vesicles (EVs).
Japan is moving toward a super-aging society at an unmatched high speed in the world. In addition, one in two people is now suffering from “cancer” in Japan. Under such circumstances, it is expected that the enormous and ever-increasing medical expenses will exceed 56 trillion JPY in 2025, and we have requested that various measures be taken at the national level. However, now that human life expectancy is 100 years old and further increase in life-span is not far away. Tthe demand for smart life care to extend healthy life expectancy will increase. Although the word “Mi-byo:unillness or pre-disease state” is prevalent, there is no indicator to accurately judge whether the person is healthy or one step before having the disease. In the current situation, even if the realization of a disease-free society is aimed, it would not be easy to achieve. In this article, we have considered the possibility of using extracellular vesicles (EVs), which is expected to be an indispensable approach in the science of pre-disease, and outlined the new drug discovery constructed by EV technology.
Exosomes, a subtype of EVs, are small particles with a diameter of approximately 50–150 nm that are secreted by all cell types. Many signaling substances, such as mRNA, microRNA, and proteins, are present inside exosomes, surrounded by a lipid bilayer [1]. These vesicles are released extracellularly by a mechanism that uses endosomes as origin, or if there are slightly larger vesicles released in a form that involves the shedding of the cell membrane, then they are known as microvesicles. Exosomes are not formed directly from the cell membrane but are formed intracellularly and then secreted extracellularly. Exosomes bud from the cytoplasm to the interior of early endosomes, and it is thought that the endosomal sorting complex required for transport (ESCRT) and tetraspanin are involved in the formation. The endosome containing many exosomes is called a multivesicular body (MVB). Since the lipids that comprise the exosome membrane are mostly ceramide, sphingomyelin, cholesterol, etc. and are similar to lipid rafts, they are considered to be formed from regions such as lipid rafts of MVB. Kosaka et al. have already demonstrated that overexpression of nSMase2, a ceramide synthase, increases exosome secretion [2]. MVB has the property of being fused to lysosomes and cell membranes, and this process is regulated by multiple Rab family small G proteins. Exosomes are secreted extracellularly only when fused to the cell membrane by the action of the SNARE (soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors) protein.
Tetraspanin is suggested to be involved in adhesion to cells that receive exosomes. The uptake of exosomes into cells has been shown to go through various endocytic pathways. They are clathrin-dependent, independent pathways, caveolin-mediated uptake, macropinocytosis, phagocytosis, and lipid raft-mediated uptake [3]. Exosomes are also transported to the periphery of the nucleus in microtubules when it is taken up directly to the cell membrane [4]. The inclusion of exosomes is released by fusion with endosomes [3], but it has also been observed that proteins are taken up by lysosomes, and their membrane parts are returned to the cell surface [4].
In addition to exosomes, apoptotic bodies and microvesicles are also EVs. Apoptotic bodies are 800–5,000 nm in diameter and are formed directly from the cell membrane when cells undergo apoptosis. Microvesicles are 50–1,000 nm in diameter and are formed directly from cell membranes. In addition to the difference in the formation process, the composition of RNA contained in these three types of EVs is different. Apoptotic bodies mainly contain ribosomal RNA (rRNA), whereas microvesicles contain very little RNA. A feature of RNA in exosomes is that they contain a large amount of small RNA and contain almost no rRNA [5].
Currently, ultracentrifugation is the conventional method used for the collection of three types of EVs, but it is impossible to separate each EV completely. In addition, the nature of exosomes varies depending on the secreting cells, so it is still difficult to clearly define exosomes [6]. At present, the definition of “exosome” differs among different studies; hence, it is necessary to pay attention to the definition and separation methods in the experiments conducted by these studies.
It is an interesting question whether intracellular substances are selectively encapsulated in exosomes. The proportion of substances in exosomes seems to reflect the amount present in their host cells to some extentMany studies have already shown that miRNA, protein, and mRNA overexpressed in cells are encapsulated highly within exosomes. On the other hand, there are also reports that they are selectively incorporated. For example, although ribosomal RNA (rRNA) occupies the majority of RNA in cells, almost no rRNA is detected in exosomes. In breast cancer cells MCF-7, the ratio of miR-720, which is highly abundant in cells, in exosomes is only 2%, and miR-451 and miR-107, which are less abundant in cells, have higher concentrations in exosomes [7]. However, the mechanism of exosome-specific inclusion of miRNAs has not yet been clarified.
The transport mechanism of exosomes to specific cells is one of the unsolved questions. For example, some miRNAs are unidirectionally transported from T cells to antigen-presenting cells via exosomes, and at least specific transport seems to be present [8]. However, it is not clear how generalized this process is, and at present, the factors that determine specificity have not been identified. Currently, studies are being conducted to present ligands on the surface of exosomes by gene recombination and specifically transport exosomes using receptor-ligand interactions [9]. If the specific transport mechanism of exosomes is elucidated, a drug delivery system (DDS) applying exosomes may be possible.
Exosomes were discovered a surprisingly long time before, dating back more than 30 years ago. It was first discovered in 1981 by two groups almost simultaneously in a reticulocyte study [10, 11]; however, the EVs contained in the prostatic fluid were named prostasomes. It was given the name “exosome” a little later in 1987 [12]. Exosomes have long been considered as “trash cans” that allow the disposal of unwanted substances from cells. However, in 1996, an antigen presented on the surface of B cell-derived exosomes was involved in T cell activation [13]. In 2007, a group led by Professor Jan Lötvall of Gothenberg, Sweden, found that miRNAs were present in exosomes [14]. Thus, the possibility of using exosomes for exchanging information between cells resulted in them to attract more attention.
The second international conference on EVs was held in Paris in 2011. At this conference, in contrast to the first time organized by Professor Rose Johnston of McGill University, Canada, many researchers from all over the world gathered, and the researchers who participated in the conference shared the real moment when a new history began. In 2012, Professor Lötvall and others led the establishment of the International Society for Extracellular Vesicles (ISEV), an international society of EVs. In Japan, the Japanese Society for Extracellular Vesicles (JSEV) was established in 2014, and it has become a lively group for many young researchers. Furthermore, in 2019, the ISEV meeting was held for the first time in Asia in Kyoto, Japan.
There are various types of signaling substances present in exosomes. In addition to mRNA and miRNA, various non-coding RNAs such as snoRNAs, Y RNAs, piRNAs, and long non-coding RNAs have been detected as extracellular RNA (exRNA) species. Since RNase is contained in body fluids such as blood, it is degraded by RNA alone, but exRNAs contained in exosomes are protected from degradation and stable in body fluids [15]. Proteins common to many cell-derived exosomes include actin, tubulin, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), endosome sorting proteins (ESCRT 0-III, Alix, Syntenin, Tsg101), heat shock proteins (HSP70, HSP90), membrane transport, and fusion. It also includes RABs and annexins [16, 17]. In addition, tetraspanins, such as CD9, CD63, and CD81, are localized on the surface. These proteins are used as exosome marker proteins. Most exosomal DNA (exDNA) secreted by cancer cells seems to be double-stranded DNA derived from the genome. The detected DNA sequences are scattered throughout the genome, and no DNA sequences that are particularly susceptible to inclusion have been found [18].
In any case, the endoplasmic reticulum functions as a communication tool between cells, and in particular, in the cancer microenvironment, as a means of survival of cancer cells, they are cancer-specific to their own alternation, exosomes. Thus, the information is delivered and transmitted to various surrounding cells. By controlling the surrounding cells in this way, cancer cells can reign at the top of the microenvironment. This is because cancer cells use exosomes to escape immune cells that attack themselves. Furthermore, attention has been focused on the function of exosomes in neurological diseases. It has been determined that they can even cause Alzheimer’s dementia and Parkinson’s disease and are involved in the maintenance of pathological conditions, and, thus, have various functions.
As described above, research on exosomes, which is a typical example of EVs, is fiercely competed in the world, mainly in the field of cancer, from the elucidation of disease mechanisms to diagnosis and treatment. On the other hand, in recent years, many studies have revealed that exosomes secreted from mesenchymal stem cells (MSCs) have therapeutic effects against various diseases, and their development as new therapeutic agents for diseases as a cell-free therapy has garnered attention [19]. As the concept of therapeutic exosomes spreads, the global market is expected to develop exosomes, and the exosome-related industry is contemplated to reach 240 million USD by 2025, or nearly 26 billion JPY. In particular, in the field of regenerative medicine, more than 100 clinical trials of exosomes derived from MSCs, tissue stem cells, and immunocompetent cells have been performed, and new treatments from these drug pipelines based on cell-free therapy are expected to come closer to clinical practice. The function of exosomes released due to disease is Malum (evil). Cancer cells have mechanisms such as metastasis and drug resistance, as described above and survive in the patient’s body by functioning brilliantly through these exosomes. The new molecular mechanisms of cancer metastasis that cause death in patients have begun to be understood through exosomes, and research to prevent the secretion and functioning of exosomes in cancer cells, which are Malum, are increasing. Thus, steadily linking this trend to drug discovery, can bring us one step closer to realizing a society that coexists with cancer. The possible applications of exosomes in several research and industrial aspects are shown in Fig. 1.
Possible applications of exosomes in several research and industrial aspects. Exosomes have potential not only for basic research but also for industrial applications in various fields such as biomarker analysis, drug development for therapeutics, vaccine production for infectious diseases, nutraceuticals, and food and cosmetics.
Humans have dug into the cause of the disease down to the genome and epigenome level, and have come to elucidate the mechanism of its onset deeply. It is also very recently understood that non-coding RNAs, such as microRNAs, trigger many diseases. In addition, the ring connecting the exosome and the pre-disease was faintly visible. Research on understanding the basic “food” of humans at the microRNA level is also underway worldwide [20]. Until now, medical care that cures illness has been the mainstream, but henceforth, it is time to focus more on medical care and research to prevent illness. The study of EVs or exosomes holds the key to what humanity needs to consolidate its wisdom to confront disease-free life care and depict the true picture of healthy longevity.
The authors have nothing to disclose.
This study was supported by the “Development of Diagnostic Technology for Detection of miRNA in Body Fluids” grant from the Japan Agency for Medical Research and Development (AMED) and the Foundation for Promotion of Cancer Research. Department of Extracellular Vesicle Science, Industrial-Academic Collaboration, Tokyo Medical University, supported by Theoria Science, Inc., Japan.
