1. Introduction
Intercellular communication is mostly thought to be mediated by direct cellular interaction or through the secretion of soluble factors [
1]. Recently, extracellular vesicles (EVs) are proposed as a novel mode of intercellular communication for both short and longer-range signaling events [
2,
3,
4]. EVs (
Figure 1) carry a rich cargo of DNA, RNA, proteins, lipids and metabolites reflective of their cellular origin and are released into the extracellular space by multiple cell types during both physiological and pathological conditions [
4,
5]. Whilst the role of EVs in normal physiology is poorly understood, their role in pathological conditions is relatively well characterized [
6]. EVs have been isolated from many biological fluids, including blood, milk, saliva, malignant ascites, amniotic fluid and urine [
7,
8,
9]. Though the presence of proteins in EVs was reported alongside the discovery of EVs [
10], the existence of RNA in EVs was only demonstrated during the past decade.
Figure 1.
Schematic representation of subtypes of extracellular vesicles (EVs) released by a cell. Three subtypes of EVs, namely exosomes, shedding microvesicles or ectosomes and apoptotic bodies, are known to be secreted by a cell into the extracellular space. Exosomes are released by exocytosis, whereas shedding microvesicles or ectosomes are secreted by outward budding of the plasma membrane. Apoptotic bodies are released by dying cells during the later stages of apoptosis so that cell debris can easily be eliminated by neighboring and immune system cells. MVB: multivesicular body.
Figure 1.
Schematic representation of subtypes of extracellular vesicles (EVs) released by a cell. Three subtypes of EVs, namely exosomes, shedding microvesicles or ectosomes and apoptotic bodies, are known to be secreted by a cell into the extracellular space. Exosomes are released by exocytosis, whereas shedding microvesicles or ectosomes are secreted by outward budding of the plasma membrane. Apoptotic bodies are released by dying cells during the later stages of apoptosis so that cell debris can easily be eliminated by neighboring and immune system cells. MVB: multivesicular body.
In 2007, Valadi
et al. were the first to confirm the presence of RNA inside EVs and also showed that mRNA inside EVs could be translated into proteins
in vitro [
11]
. Interestingly, the secretion of EVs is conserved in multiple species and thus EVs from one species have the potential to regulate cellular processes in another species, either by inducing benefit (e.g., cow milk exosomes in humans—at least infants) or mediating disease/infection (e.g., fungal exosomes in plants/humans) [
12]. In addition, EVs were shown to carry single-stranded DNA (ssDNA), amplified oncogene sequences, transposable elements and mitochondrial DNA [
3,
13]. Though the presence of mitochondrial DNA has not been validated by other groups, double stranded DNA (dsDNA) in tumor-derived EVs was also discovered and reported recently by several research groups [
14,
15,
16]. This unparalleled horizontal transfer of multiple gene and protein products among cells was until recently considered impossible because some researchers argued that such transfers violate the cell’s autonomy [
17,
18,
19].
EVs can broadly be divided into three categories based on the current state of knowledge of their biogenesis. Discrete biogenesis pathways result in subsets of EVs namely: (i) exosomes; (ii) ectosomes or shedding microvesicles (SMVs); and (iii) apoptotic bodies (ABs), as schematically depicted in
Figure 1. A common feature in all the three EV subtypes is a lipid bilayer membrane that surrounds a specific cargo of biomolecules, e.g., proteins, RNA, or cellular debris. However, their size and buoyant densities vary significantly [
20]; albeit that both size and buoyant density ranges for the various EV subtypes have been heterogeneously reported in the literature. Nonetheless, exosomes are thought to be around 30–150 nm in diameter and have a buoyant density of 1.10–1.14 g/mL. Furthermore, exosomes display cup-like morphology when observed under the transmission electron microscopy [
20,
21,
22]. When discovered more than three decades ago, exosomes were initially thought to be a mechanism of discarding plasma membrane (PM) proteins in maturing reticulocytes [
10,
23]. These small membranous vesicles are formed by inward budding of endosomal membranes, resulting in the progressive accumulation of intraluminal vesicles (ILVs) within large multivesicular bodies (MVBs) as shown in
Figure 2. MVBs can either traffic to lysosomes for degradation (degradative MVBs) or, alternatively, to the PM where, upon fusion with the PM, they release their contents (the ILVs) into the extracellular space (exocytic MVBs). ILVs released into the extracellular space are referred to as “exosomes” (
Figure 2). Among the EV subtypes, exosomes have been and are extensively studied [
5,
20]. While multiple studies have implicated Alix, TSG101, CD63 and CD9 as exosomal markers [
20], it is becoming clear that these molecules are enriched in exosomes, but are not markers
per se as considered previously [
24]. In agreement with this, Keerthikumar
et al. identified enrichment of Alix, TSG101, CD9 and CD63 in exosomes compared to ectosomes [
22]. Their study further confirmed that CD81 might distinctly be utilized as an exosomal marker which was further supported by Minciacchi
et al. [
25].
Figure 2.
Pathways involving various types of vesicles. In the classical secretory pathway, vesicles with protein cargo, sorted and packed in the Golgi apparatus, transport their cargo to the plasma membrane (PM). By fusing with the PM, both membrane proteins and secretory proteins are effectively transported to their intended destinations. Various types of cargo, e.g., proteins, RNA, can also be transported into the extracellular space via outward PM budding and formation of shedded vesicles (ectosomes). Cargo is taken up by the cell via endocytosis (receptor-mediated and free uptake) and formation of early endosomes. In early endosomes, proteins are either recycled to the PM or sequestered into the intraluminal vesicles (ILV) of MVBs. Formation of exosomes starts with inward budding of the early endosome’s membrane and subsequent formation of MVBs. In the exocytic pathway ①, MVBs fuse with the PM to release their contents (exosomes) into the extracellular space; In the degradative pathway ②, the MVBs are trafficked to lysosomes for enzyme-assisted degradation. This pathway is particularly important for restricting signaling by activated growth factor receptors. Exosomal cargo delivery to the recipient cell can occur through various mechanisms, i.e., direct fusion with the recipient cell’s membrane, pinocytosis/phagocytosis, or ligand–receptor binding.
Figure 2.
Pathways involving various types of vesicles. In the classical secretory pathway, vesicles with protein cargo, sorted and packed in the Golgi apparatus, transport their cargo to the plasma membrane (PM). By fusing with the PM, both membrane proteins and secretory proteins are effectively transported to their intended destinations. Various types of cargo, e.g., proteins, RNA, can also be transported into the extracellular space via outward PM budding and formation of shedded vesicles (ectosomes). Cargo is taken up by the cell via endocytosis (receptor-mediated and free uptake) and formation of early endosomes. In early endosomes, proteins are either recycled to the PM or sequestered into the intraluminal vesicles (ILV) of MVBs. Formation of exosomes starts with inward budding of the early endosome’s membrane and subsequent formation of MVBs. In the exocytic pathway ①, MVBs fuse with the PM to release their contents (exosomes) into the extracellular space; In the degradative pathway ②, the MVBs are trafficked to lysosomes for enzyme-assisted degradation. This pathway is particularly important for restricting signaling by activated growth factor receptors. Exosomal cargo delivery to the recipient cell can occur through various mechanisms, i.e., direct fusion with the recipient cell’s membrane, pinocytosis/phagocytosis, or ligand–receptor binding.

Contrary to exosomes, ectosomes (SMVs) are large vesicles ranging from 100–1000 nm in diameter [
26], ubiquitously assembled at and released from the PM through outward protrusion or budding (
Figure 2). Ectosomes were first defined by Stein and Luzio when they observed ectocytosis and shedding of PM vesicles in stimulated neutrophils [
27]. The rate of ectosome shedding has been observed to be variable between various cell types, but even resting cells shed ectosomes at a low rate. Unlike exosomes, the molecular composition of ectosomes is still largely unknown, but matrix metalloproteinases (MMPs) [
28,
29,
30,
31], glycoproteins, e.g., GPIb, GPIIb–IIIa and P-selectin [
32,
33,
34,
35], and integrins, e.g., Mac-1, [
35,
36] seem to be enriched in ectosomes, depending on the cell type. Recent studies also suggest that MMP2 might be utilized as a marker of ectosomes [
22,
37]. However, ectosomal enriched proteins are largely cell type dependent. For instance, the epithelial cell marker CK18 was enriched in ectosomes [
22] and oncosomes [
25] secreted by epithelial cells and hence cannot be utilized as markers of ectosomes secreted by fibroblasts. Oncosomes are larger vesicles ranging from 1 to 10 μm in diameter that are thought to follow the biogenesis pathway of ectosomes and are extensively studied by Di Vizio and colleagues [
25,
38]. Though abundance of large oncosomes in patient plasma and tissue biopsies are shown to be correlated with tumor progression, until now, these large oncosomes are exclusively shown to be released by prostate cancer cells and are poorly characterized in comparison to exosomes [
25,
39].
Apoptotic bodies (
Figure 1) are heterogeneous vesicles that are known to be released from cells undergoing apoptotic cell clearance [
40,
41] and are thought to be around 50–5000 nm in diameter [
20]. Apoptosis or programmed cell death [
41], first introduced by Kerr and co-workers in 1972 [
42], and the subsequent phagocytic corpse removal are essential during embryonic development, growth, and maintenance of multicellular organisms. Furthermore, apoptosis ensures the selective removal of aged, damaged, infected or aberrant cells from healthy tissues. Essentially, apoptosis is the coordinated dismantling of the cell and cellular debris is packed into ABs. These vesicular structures have external features that trigger phagocytosis; the final step in cell dismantling and recycling of biomolecule building blocks.
The “Focus on extracellular vesicles” series of reviews highlights recent developments in EV research and their role in normal physiology, degenerative and cancerous diseases, and as emerging novel therapeutics [
43,
44,
45,
46,
47]. The following sections of this introductory review offer a compact overview of various aspects of extracellular vesicles—
THE NEXT SMALL BIG THING.
5. Function of EVs and Development of EV-Based Technologies
Although the exact physiological functions of EVs are poorly understood, when generalizing for all classes of EVs, these all function as transport vehicles of some sort. Exosomes have been shown to contain molecules, predominantly from an endosomal and cytosolic origin, for intercellular communication over a short range. Ectosomes contain ubiquitous cargo and are believed to also be involved in cell-cell communication, whereas ABs function to transport and present cellular debris from intentional cell suicide to phagocytic cells for further dismantling and recycling of building-blocks. Furthermore, increasingly evidence accumulates that cells modify the content of EVs in response to extrinsic stressors such as heat shock, hypothermia, hypoxia, oxidative stress, and infectious agents. These results suggest that the EVs are connected to intracellular signaling and are part of the global intricate mechanism to maintain physiological homeostasis; the levels of which we are just beginning to understand. It also suggests that perturbation of the roles that EVs play in homeostasis potentially results in disease and a link can indeed be established between EVs and various diseases. Consequently, EVs have also become of interest with regard to their pathophysiology, the development of novel therapeutic modalities, and because particularly exosomes are ubiquitously present in bodily fluids, exosomes are deemed ideal as diagnostic biomarkers. In this focus edition, Iraci and co-workers provide an extensive overview of the physiological roles of EVs and their signaling properties [
44].
Tumor cells have been reported to secrete increased amounts of exosomes [
165]. Since these tumor-derived exosomes carry the tumor-specific genomic and proteomic signatures, tumor-derived exosomes are ideal and unique targets for cancer detection. However, the fact that tumor-derived exosomes carry the hallmark properties for tumorigenicity also means that these exosomes might aggravate the tumorigenic potential already present in cells [
133]. Indeed a number of studies seem to confirm that exosomes secreted by tumor cells play a role in the growth and dissemination of tumor cells [
166,
167,
168,
169,
170]. For instance, Lázaro-Ibáñez
et al. recently showed that the various prostate cancer cell-derived EVs subgroups carried different fractions of genomic DNA (gDNA) fragments of
MLH1,
PTEN, and
TP53 genes, including mutations [
158]. Their results suggest that nucleic acids are selectively and cell-dependently packed into the various EV subtypes and that circulating EVs potentially contribute to both pre-metastatic niche formation and tumor metastasis. On the other hand, some investigations report quite the opposite,
i.e., anti-tumorigenic properties, such as tumor cell apoptosis induction in pancreatic carcinoma or enhancement of anti-tumor immunity [
171,
172]. Furthermore, even if tumor-derived EVs are found in the circulation of cancer patients, this must not necessarily mean that EVs are actively involved in tumor progression, but could simply be the result of tumor expansion and thus enhanced EV secretion. Nonetheless, tumor-derived EVs show both the potential as cancer biomarkers as well as the possibility to develop novel anti-cancer therapeutics. In this focus edition, Ciardiello
et al. discuss the current state of the art regarding the EV-cancer connection [
43], whereas Ohno and Kuroda focus on the development of EV-based therapeutics [
45].
Similarly, EVs have drawn the attention of researchers investigating degenerative brain disorders, such as Alzheimer’s dementia and Parkinson’s disease, ischemic stroke, neuro-inflammation, and epilepsy. However, the study of EVs of neuronal origin in neurological disorders is still challenging due to technical and ethical limitations;
in vivo sampling of brain material cannot readily be performed, apart from biopsies for diagnostic purposes, and repetitive sampling of cerebrospinal fluid is overall considered unethical, but still recent research results from brain tumors seem to be promising. For instance, Skog
et al. showed that nested-PCR-based detection of the tumor-specific epidermal growth factor receptor EGFRvIII transcript in serum-purified exosomes allows diagnosis of a glioblastoma sub-set [
170]. Overall, particularly exosomes are implicated to facilitate the spread and accumulation of key disease-causing neuronal proteins, such as β-amyloid [
173,
174,
175] and α-synuclein [
176,
177,
178]. Here, Vella and co-workers review the role of exosomes in protein trafficking with respect to Alzheimer’s and Parkinson’s disease and not only highlight recent advances but also the remaining challenges [
46].
EVs have been shown to be secreted by stem cells, which in itself is not surprising given their undifferentiated nature and the potential that stem cells carry. The fact that stems cells are the “mother of all cells” and potentially can produce any cell type, stem cell therapy has been heralded as the ultimate regenerative therapy. However, the results from various experimental and clinical studies have not produced the expected results for multiple reasons. It is known that stem cells secrete a myriad of biomolecules in order to communicate with the cells in the surrounding tissue. Consequently, researchers tried to determine the factors involved, but no single biomolecule or combination could induce the desired therapeutic effects of stem cell transplantation. Since EVs are involved in intercellular communication and may contain all the signals required for successful communication, even at multiple levels and via multiple pathways, EVs have attracted the attention of researchers in the stem cell therapeutics field. Stem-cell derived EVs might themselves constitute potent therapeutics against various degenerative diseases. Recent research already validates this assumption, since various groups have found encouraging results from various stem cell types, e.g., mouse embryonic stem cells EVs enhanced survival and expansion of hematopoietic progenitor cells [
179], endothelial progenitor cells-derived EVs protect against angiotensin II-induced cardiac hypertrophy [
180], and mesenchymal stem cell EVs reduce infarct size in a mouse model of myocardial ischemia/reperfusion injury [
181]. Focusing on the effects of EVs, Zhang and colleagues briefly review the current advances in the stem cell therapeutics field [
47].
Some indications that ABs are not just garbage bags advertising “eat me” signals to phagocytic cells, but rather might have more intricate roles, both positive and negative ones, come from recent research. Kogianni and co-workers showed that osteocyte ABs were able to initiate
de novo osteoclastic bone resorption on quiescent bone surfaces
in vivo, which suggests a physiological signaling role of ABs in directed osteocyte apoptosis in damaged bone [
182]. Phagocytosis of HepG2-derived ABs by hepatic stellate cells (HSC) activates JAK1/STAT3 and, to a lesser extent, PI3K/Akt/NF-κB survival pathways, upregulating Mcl-1 and A1 anti-apoptotic proteins, which leads to HSC survival and propagation of liver fibrosis [
183]. That ABs can be used in a therapeutic setting was recently demonstrated by Marin-Gallen
et al. [
184]. These authors showed that tolerogenic dendritic cells (DCs) could be generated that reestablished peripheral tolerance in type 1 diabetes by pulsing DCs
in vitro with ABs from β cells. Consequently, treated DCs diminished the expression of the co-stimulatory molecules CD40 and CD86 and reduced secretion of proinflammatory cytokines, thereby reducing autoimmunity towards β cells and thus insulitis. Furthermore, Schiller and co-workers observed that active packing of immunogenic molecules into ABs occurred early during apoptosis, well before DNA degradation [
185]. These results indeed suggest that formation of ABs might follow a distinct “plan” and thus a significant level of control by the cell might be present.
Finally, the positive and negative modulation of the immune response by both immune and non-immune cell-derived EVs is one of the best established (patho)physiological functions of EVs. Exosomes have been shown capable of direct antigen presentation since they preserve the topology of the antigen-presenting cell (APC) from which they originate and directly stimulate CD8
+ and CD4
+ T cells through surface MHC-I and II molecules [
186,
187]. Exosomes have also been shown to be involved in indirect antigen presentation either through transfer of antigenic peptides to APCs [
188,
189] or by cross-dressing APCs [
188,
190,
191]. Not surprisingly, EVs have been shown to carry a variety of antigens from various origins, including the aforementioned tumor-derived antigens, pathogens-derived antigens, e.g., antigens from
Cytomegalovirus [
192] or
Mycobacterium bovis bacillus Calmette-Guérin [
193], and B cell-derived antigens [
194,
195]. Besides the tolerogenic effect elicited by ABs through DC modulation, epithelial cells of the small intestine have been shown to release MHC class II
+ exosome-like structures, called “tolerosomes”, which induce specific tolerance to orally administered antigen ovalbumin [
196]. Lastly, recent research suggests that EVs not only transfer antigens to APCs, but also signals that induce transformation of recipient cells into immunogenically competent APCs [
197]. Notwithstanding these important results, it is imperative to emphasize that the majority of results to date have been derived from
in vitro experiments on immune cells or lab animals treated with
in vitro purified EVs. A significant gap exists between the knowledge gained from these experiments and the potential
in vivo immunomodulatory roles of EVs, especially in humans. Nonetheless, as our understanding of the roles that EVs play in immune regulation develops, new therapeutic options will certainly become available that might allow inhibition of tumor-derived EVs and modulation of the tumor microenvironment, modification of the release of endogenous immunosuppressive EVs, or even specifically engineered EVs as novel therapeutics. In this focus edition, Ohno and Kuroda discuss the potential of EV-based therapeutics [
45], whereas comprehensive reviews covering the role of EVs in immune system-related processes were recently provided by Robbins and Morelli [
198] and Théry
et al. [
124].