were originally discovered in 1983, as membrane vesicles of approximately 100 nm in diameter with a lipid bilayer structure. These vesicles are secreted by reticulocytes (Harding and Stahl 1983; Pan and Johnstone 1983). They were initially thought to be a “trash bag” in which cells could eliminate excess proteins; however, from 2007 on great advances in exosome research were made, as it was shown that microRNAs (miRNAs) in exosomes could be transferred between cells and played important roles in many physiological and pathological processes (Valadi et al. 2007; Pegtel et al. 2010; Mittelbrunn et al. 2011; Hergenreider et al. 2012; Montecalvo et al. 2012).
Exosomes are secreted into body fluids from various types of cells and organs. Recent studies have demonstrated that exosomes secreted by tumor cells are transported to surrounding cells and participate in metastasis and infiltration. Since these tumor-derived exosomes containing cancer cell-specific miRNAs and mRNAs are secreted into body fluids such as blood, urine, ascites, amniotic fluid, bronchoalveolar lavage, and tears, the miRNAs and mRNAs in cancer cell-derived exosomes have come to be used as diagnostic biomarkers for early stage cancers (Thery et al. 2002; Logozzi et al. 2009; Lu et al. 2009; Rabinowits et al. 2009; Montecalvo et al. 2012; Peinado et al. 2012). In parallel, miRNAs contained in exosomes released into the blood from tissues and organs in response to adverse events, such as exposure to chemical substances and drugs, are expected to be useful as novel biomarkers for toxicity assessment.
In this chapter we will introduce the latest findings on exosomes, including the newly appreciated biological significance of exosomes, and will explain the potential use of exosomes as biomarkers of toxicity.
The Classification of Extracellular Vesicles (EVs)
Various particles are secreted by cells and released into body fluids such as blood, urine, ascites, amniotic fluid, bronchoalveolar lavage, and tears; the International Society for Extracellular Vesicles (ISEV) has named these particles “extracellular vesicles” (EVs) (Thery et al. 2002; Witwer et al. 2017). EVs are divided into three subtypes: exosomes, microvesicles (MVs), and apoptotic bodies. This classification is based on their size and on the biosynthetic pathways by which they are generated (see Figure 3.1 and Table 3.1; also Colombo et al. 2014).
Figure 3.1 Schematic illustration of extracellular vesicles (EVs). EVs include exosomes, microvesicles and apoptotic bodies. MVB: multivesicular body.
Exosomes are thought to derive from intraluminal vesicles through the fusion of an intermediate endocytic compartment, the multivesicular body (MVB), with the plasma membrane (Witwer et al. 2013; Cufaro et al. 2019). The MVB contains vesicles that bind either to lysosomes, to degrade their contents, or to the plasma membrane. The latter produces the release of intraluminal vesicles defined as exosomes into extracellular space (Harding and Stahl 1983; Pan et al. 1985).
As already mentioned, exosomes are approximately 100 nm in diameter; hence they are smaller than MVs (Witwer et al. 2013). Exosome membrane proteins are enriched in heat shock proteins (HSP70, HSP90), integrins (LFA-1), proteins involved in MVB formation (ALIX, TSG101), tetraspanins (CD9, CD63, CD81, CD82, and CD151), immunostimulatory molecules (MHC class I/II proteins), lipid-related proteins, and phospholipases (Conde-vancells et al. 2008; Subra et al. 2010).
Table 3.3 Main characteristics of exosomes, microvesicles (MV), and apoptotic bodies.
| Exosomes | Microvesicles | Apoptotic Bodies | |
|---|---|---|---|
| Origin | Endocytic pathway | Plasma membrane | Plasma membrane |
| Size | 40–120 nm | 50–1000 nm | 500–2000 nm |
| Function | Intercellular communication | Intercellular communication | Facilitate phagocytosis |
| Markers | Alix, Tsg101, tetraspanins | Selectins, integrins, CD proteins, DNA, RNA40 ligand | Histones, annexin V |
| Contents | Protein, DNA, RNA | Protein, DNA, RNA | Nuclear fractions, cell organelles |
By contrast, MVs range from a few nanometers to a few microns in diameter and derive from outward budding of the plasma membrane (Witwer et al. 2013; Kalluri 2016). In some lipids and phosphatidylserine they are enriched.
Apoptotic bodies are released by apoptotic cells. They are the largest EVs, with a diameter of 1 to 5 μm, and contain several intracellular fragments, cellular organelles, membranes, and cytosolic contents (van der Pol et al. 2012; Cufaro et al. 2019). However, in practice it remains difficult to distinguish between the different subtypes of EVs. Therefore the ISEV recommends, by consensus, using the general term “EV” in the nomenclature (Gould and Raposo 2013; Thery et al. 2018). In this chapter we use this term to refer to all the subtypes of vesicles present in the extracellular space, alongside small EVs (sEVs) for exosomes.
Additional components are found in EVs, including a wide variety of genetic molecules such as DNA and coding or non-coding RNAs (Nawaz et al. 2018).
Components of Exosomes (EVs)
RNAs
Most of the RNA present in normal cells is ribosomal RNA, which is reported to account for 80–85% of total cellular RNA. Among its subunits, the 28S and 18S have the highest abundance; therefore, when electrophoresis of total RNA extracted from mammalian cells is performed, the two main bands represent the 28S and 18S subunits (Figure 3.2; see Imbeaud et al. 2005).
Figure 3.2 Gel electrophoresis of mouse liver total RNA (left panel) and mouse serum EV-associated RNAs (right panel). Black triangles represent size markers.
Both mRNAs and small RNAs in normal cells have low abundance by comparison with ribosomal RNAs. Small RNAs include small nuclear RNAs (snRNAs), miRNAs, small nucleolar RNAs (snoRNAs), and piwi-interacting RNAs (piRNAs) (Watson et al. 2019).
In contrast, small RNAs, especially miRNAs, are enriched in sEVs (Figure 3.2; see Valadi et al. 2007). MicroRNAs (miRNAs) were originally discovered in Caenorhabditis elegans and are found in most eukaryotes, including humans and mice. These molecules are 21–25 base (nt)-long single-stranded RNAs that are involved in the post-transcriptional regulation of gene expression in eukaryotes (Lee et al. 1993; Ambros 2004). To date, 1974 hairpin precursors of miRNAs and 2654 mature miRNA sequences have been annotated in the human genome (Kozomara et al. 2019). The latest information about miRNAs is available in the miRBase database (
