Cancer is a severe worldwide health problem. In order to improve the survival rate of patients, clinicians and medical researchers have worked on early diagnosis and awareness of risk factors for cancer. In the meantime, we cannot deny the urgent need for novel markers with better efficiency and less invasive features, as these markers could lead to early diagnosis, changing the therapeutic direction for individual patients with a more precise estimation of prognosis. Cell-derived materials are considered as specific markers that reflect definite cellular characteristics, and extracellular vesicles (EVs) in particular are attracting a lot of interest when it comes to cancer diagnosis and treatment since different cell types produce distinctive amounts of EVs. EVs can be divided into a few subgroups based on their biogenesis, size, and biomarkers. In particular, three main group have been described: intraluminal vesicle (ILV)-derived EVs; microvesicles, and apoptotic bodies. It should be highlighted that most studies have not clearly described the origin of the EVs under investigation. It should also be underlined that the subcellular origin of EVs is often not described in most of the published literature; EVs are differentiated based on the size and expression of biomarkers that we now know are often not exclusive to a particular EV class. Due to this, in many cases, it is not possible to assume that these studies were indeed looking at ILV-derived EVs rather than other biogenesis pathways, but were small EVs of an undetermined origin. In this review, we will focus our attention on those EVs with a size between 30 and 150 nm, like exosomes; we will call these exosome extracellular vesicles (EEVs) throughout the text.
The first description of EEVs’ secretion was mentioned in reticulocytes, platelets, dendritic, lymphocytes B and T, mast cells, and macrophage hematopoietic cells [
1,
2,
3,
4,
5,
6,
7,
8,
9,
10,
11]. Additionally, other nonhematopoietic cells can produce EEVs, such as astrocytes, melanocytes, neurons, adipocytes, epithelial, fibroblasts, and tumor cells [
12,
13,
14,
15,
16,
17,
18,
19,
20]. EEVs are present in most of the physiological fluids, including blood, serum, urine, saliva, breast milk, lymph, amniotic fluid, ascites, semen, cerebrospinal fluid, and nasal secretions [
21]. EEVs play a key role in short- and long-distance cell communication, promoting the development and function of multicellular organisms under physiological and pathological conditions. EEVs are deeply involved in long-distance communications to transfer proteins [
22,
23,
24,
25], mRNAs, and miRNAs that could be expressed in target cells [
26,
27]. This mechanism ensures highly efficient secretion, signaling, and communication, in a robust and economic manner, for information exchange between cells [
28]. Depending on the cellular origin, EEVs can contain different profiles of RNA, miRNA, and proteins, including tetraspanins, metalloproteinases, major histocompatibility molecules (MHC), and adhesion molecules [
20,
29,
30]. These molecules can be altered by stress or pathological conditions; thus, molecular profiles of circulating EEVs can be used for theragnostic implications. Cancer-derived EEVs are ideal as biomarkers for the early diagnosis of cancer as they carry specific molecules that reflect the genetics and signaling alterations of parental cancer cells [
31,
32,
33].
As well as being a potential diagnostic tool, EEVs are ideal drug delivery vehicles. A size of about 100 nm has been demonstrated to be optimal for a long circulating time in biological fluids, avoiding fast elimination [
34]. The EEV membrane contains a specific set of lipids and proteins similar to that of the cell of origin, with infinite combinations of targeting possibilities. The membrane structure and composition allow for immune surveillance escape, ideal interaction with cell membranes, and internalization, better than formulated pegylated liposomes [
35,
36,
37,
38]. EEVs are mediators of cell communication [
27,
39,
40] and many research groups, including us, have utilized EEVs to deliver nucleic acids, proteins, or small molecules [
41,
42,
43,
44].
In the first part of this review, after a brief introduction to EEVs’ biology, we point out the attention to cancer-derived EEVs as potential biomarkers, which will probably have a high impact in the near future in terms of facilitating the early detection, monitoring, and prognosis of cancer. In the second part, we focus on the use of EEVs as novel drug delivery systems, depending on their specific features to apply them as anticancer therapeutic vehicles in vitro and in vivo, and discuss the limits and the prospective challenges.
1.1. EEVs’ Biogenesis
During their life cycle, eukaryotic cells periodically engulf small amounts of intracellular fluids and form small intracellular bodies called endosomes [
29,
45]. As the early endosome is maturing into a late endosome, it forms ILVs in the lumen of endosomes. ILVs have a range of 30–100 nm of diameter and are formed by inward budding of the endosome’s membrane. Portions of the cytosol and incorporated transmembrane/peripheral proteins are engulfed into the invaginating membrane. This phenomenon can be detected in late endosomes by following the changes in their location and shapes: early endosomes are located in the outer part of the cytoplasm with a tube-like shape, whereas late endosomes are present near to the nucleus with a spherical shape [
29]. Late endosomes containing ILVs are also called multivesicular bodies (MVB) [
46,
47]; usually, they fuse with lysosomes, followed by degradation of their contents mainly by hydrolysis. It has been hypothesized that MVBs’ content can be divided based on their function: the proteins found in the ILVs are destined for lysosomal degradation, while functional proteins with a biological role are imported into the ILVs of MVB [
48]. Until now, the mechanism that describes this process has not been completely understood, and there are different theories about protein sorting [
49]. MVBs may fuse with the plasma membrane instead of fusion with lysosomes and therefore release ILVs to the extracellular space (
Figure 1); these released vesicles are then called EEVs [
46,
50].
The uniformity of EEVs originating from similar cell types reveals the presence of a sorting mechanism behind EEVs’ development. The protein sorting of ILVs in MVBs is thought to involve different participants such as an endosomal sorting complex required for transport (ESCRT) components (e.g., Alix, Tsg101, and clathrin), lipids, and/or tetraspanin-enriched microdomains present in EEVs. The sorting mechanism started with the recognition of the ubiquitinated cargo proteins by an ESCRT complex, which then recruits the ESCRT-I subunit Tsg-101, activating AIP/Alix [
51]. All these sequential interactions drive the cargo into the budding vesicles. It appears that there is also an ESCRT-independent mechanism involved in protein sorting to MVBs [
52]. The sorting of cytosolic proteins does not require the ESCRT machinery and can be explained by a “random” engulfment of small portions of cytosol during the inward budding process and/or by their transient association with transmembrane proteins [
53]. Moreover, the sequestration of glycophosphatidylinositol-anchored proteins and other “raft”-associated proteins in EEVs reflects the presence of lipid-like domains in EEVs’ membranes. The lipid microdomains could themselves be involved in the generation of ILVs, or in concert with other proteins, with affinity for “raft-like domains” such as tetraspanins [
46,
54]. The biogenesis of EEVs indicates that their macromolecule composition, including proteins, lipids, mRNAs, and microRNAs, can be varied depending on the cell of origin. Protein analysis of EEVs from different cell types including dendritic cells (DC) [
47], B-lymphocytes [
7], and epithelial intestinal cells [
55] revealed that there are common, as well as cell-type-specific, proteins within EEVs. Common proteins shared by various EEVs are Annexins I, II, V, and VI, which could be involved in cytoskeleton dynamics and membrane fusion [
10,
56]. The Ras superfamily of monomeric G proteins (Rab) also contains common proteins that could act in EEVs’ docking and on the ability to fuse with membranes of other cells [
10,
57]. Adhesion molecules [
10], apoptosis proteins, heat shock proteins (Hsc73 and Hsc90), tetraspanins (CD9, CD63, CD81, and CD82 [
47,
55,
58,
59]), GTPases, and cytoskeletal proteins (actin, synenin, moesin, and albumin) [
60] are classically found within EEVs of different origins. Other main EEV proteins are reported in
Table 1.
It was demonstrated in vitro and in vivo that EEVs are produced in both normal and pathological conditions [
64]. EEVs have been involved in numerous physiological processes, including the removal of unnecessary proteins from cells, but their principal role is in cell‒cell communication, both locally and systemically, by transferring their contents, including protein, lipids, and RNAs, between cells [
65]. The role of EEVs in immune system stimulation was extensively investigated [
66]; it was also proposed a new approach to vaccine development [
67]. EEVs were described as playing a pivotal role during normal development and the physiology of the nervous system, acting as cell–cell communicators and playing functional roles not only during development but also during the regeneration of normal neurons [
68]. Besides their physiological role, EEVs were depicted as a Trojan horse in neurodegenerative processes due to their capability to transfer “toxic” cargos from unhealthy to normal neurons [
69,
70]. Recently, it was demonstrated that EEVs are involved in a wide range of cardiovascular physiological and pathological processes, with beneficial or pathological activity [
71,
72]. In this review, we discuss the EEVs’ role in cancer. Cancer cells have shown a higher secretion rate of EEVs compared to normal cells [
73,
74]. The secretion of EEVs in cells could follow two different mechanisms: the constitutive secretion involving the trans-Golgi network (TGN) and/or inducible release, depending on the cell type and on the activation state of the cell [
75]. The constitutive pathway does not require a specific stimulus, although it is controlled by cell activity (intracellular signaling, cell growth, differentiation, DNA damage, etc.). Proteins destinated to be secreted into the extracellular medium or to the cell surface can be routed from the TGN, where EEVs are transported within vesicles containing only one or two EEVs. However, the inducible release of EEVs requires specific stimuli such as hypoxia or toxic stress, causing DNA damage and leading to vesicular trafficking [
29]. Thery et al. have demonstrated that Rab proteins such as Rab27 isoforms play a regulatory role in EEVs’ secretion [
76]; in addition, Rab35 family members have been shown to be an essential part in the regulation of EEVs’ secretion due to the interaction with the TBC1 domain of GTPase-activating protein and 10A-C (TBC1D10A-C) family members [
77,
78]. It is known that Rab proteins are usually mutated (constitutively active) or overexpressed in tumor cells [
79]. Furthermore, other studies have shown that, due to the activation of the tumor suppressor protein p53, EEVs’ secretion rate is stimulated by regulating the transcription of different genes like TSAP6 and CHMP4C [
75,
80,
81]. Once the cells suffer from stresses like toxicity or hypoxia, damage may occur at the DNA level; then, a response of the p53 protein is generated by regulating the transcription of different genes [
82]. This process is also called the “bystander effect”, in which cells communicate with the microenvironment by the secretion of specific proteins in order to compensate for a response to stress [
83]. Evidence of this process was seen during the irradiation of human prostate cancer cells, which led to DNA damage that could induce an increase in EEV production due to cell activation [
84]. Many other mechanisms are correlated to EEVs’ secretion in several cell types, including K
+ depolarization of neuronal cells, intracellular Ca
2+ level, pH variation, and CD3 crosslinking with T cells [
29,
46,
83,
85].
Various functions are carried out by EEVs once they are secreted from the cells of origin and might be transferred to other cells [
86]. It has been shown that some EEVs, such as those secreted by tumor cells, were found to carry phosphatidylserine (PS) on their surface as signal transduction, allowing their uptake by appropriate cells via specific mechanisms [
87,
88]. Once EEVs are taken up by other cells, they can be either endocytosed via clathrin-coated pits or could release their contents in the cell and remain joined with the plasma membrane [
89]. EEVs contain various molecules derived from the cell surface, which allows them to recognize different cell receptors at the same time [
90]; furthermore, the EEV‒cell interaction favors the intercellular exchange of several materials like lipids, proteins, carbohydrates, and pathogens. Recent data from 286 studies on the ExoCarta database show that 9769 proteins, 3408 mRNA, 2838 miRNA, and 1116 lipids (07/10/2015 Exocarta update published in
J Molecular Biology [
91];
http://www.exocarta.org/) are associated with EEVs, thus demonstrating the complexity of EEVs.
1.2. EEVs’ Isolation Methods
Size, protein and lipid contents are usually used to characterize EEVs and related microvesicles [
28]. The complete separation and purification of each type of vesicles would help to exploit their benefits for clinical use; however, this is extremely hard if not impossible [
37,
92]. Researchers are trying to overcome these limits, although the absolute separation and definition of various extracellular vesicles and EEVs based on their size or biogenesis is still to be determined because no markers could distinguish the origin of the EEVs [
93], and there are difficulties in sorting them due to their heterogeneous biochemical composition [
94]. EEVs could be isolated from body fluids or from processed cell culture media through different techniques (
Table 2), based on EEV surface markers such as tetraspanins, integrins, and cell adhesion molecules [
95], or lipid composition, as they are rich in cholesterol, phosphatidylcholine, and phosphatidylethanolamine [
96]. These techniques include immunoaffinity or size-exclusion chromatography (SEC), differential centrifugation, filtration coupled with centrifugation, microfluidic technologies, and polymer-based precipitation [
28]. With biological fluids or cell culture supernatants that may be the source of EEV, the initial volumes can be scaled up or down according to the number of vesicles required for further analysis. Although biological fluids contain a large number of exosomes, they also have large numbers of soluble proteins and aggregates content, which could lead to contamination issues during EEV isolation methods.
Differential ultracentrifugation and density-gradient centrifugation are the most important methods for isolating EEVs [
97]. In the differential ultracentrifugation method, different centrifugal forces are applied sequentially to a solution containing EEVs, eliminating cells, dead cells, cellular debris, intact organelles, and, finally, EEVs. One limit of this technique is that other vesicles and proteins can be deposited. Moreover, even in this sequentially extended ultracentrifugation, contaminants from bovine small RNAs could be miscategorized as human RNA. Density-gradient centrifugation can help to overcome this limitation: using a sucrose density gradient, contaminants may be separated from EEVs, resulting in a more uncontaminated fraction [
98]. Another technique is immunoaffinity chromatography, which depends on antibody recognition of EEV proteins: antibodies are covalently attached to beads, binding specifically to surface proteins or antigens on the EEV surface, which helps to minimize antibody contamination and buffers interference [
93].
Polyethylene glycol (PEG) solutions have been used to precipitate and isolate viruses and other macromolecules for more than five decades. EEVs are typically isolated using a precipitation solution consisting of PEG with a molecular weight of about 8 kDa. This solution is mixed with the source containing EEVs and then centrifuged at a low speed to form a pellet containing EEVs, which still carry a risk of contamination by proteins, before collecting the pellet from the upper aqueous phase [
99].
Size-exclusion chromatography is a different method used to separate heterogeneous populations of different vesicle size in a solution. Biological materials with a small radius can penetrate through pores, whereas larger components, such as EEVs, are unable to pass through the pores [
100]. Evidence showed that for the lipidomics profiling of plasma- or serum-derived EEVs, size-exclusion chromatography could be the most suitable method due to their porous structure reducing the risk of contamination with undesired circulating lipids and biological fluids [
101].
Immunoaffinity approaches using antibodies specific for EEVs surface proteins could be integrated with microfluidic technologies. These antibodies are covalently bound to the chip for the separation of EEVs from other contaminants [
102]. This allows for the rapid production and isolation of EEVs, but not in sufficient quantities to enable their use in the clinic. Chip-based immunoaffinity was developed to isolate EEVs and microvesicles, allowing for quantitative and high-throughput analysis of EEVs’ contents [
103]. A microfluidic device formed by a porous silicon nanowire on the micropillar structure can trap EV-like lipid vesicles while filtering out proteins and debris. Extracellular vesicles are sieved through a porous membrane with a specific size, and collected by filtering the biofluid through a membrane; the filtration is then driven by either pressure or electrophoresis to assist EEVs’ separation from contaminants. Proteins are less affected by the electric field due to their lower negative charge compared to phospholipidic vesicles [
104]. A porous ciliated silicon microstructure could selectively trap particles of 40–100 nm in size. This technique offers benefits in terms of both diagnostic and therapeutic applications [
105]; however, stronger collaborations between microfluidic engineers and clinicians would be able to take advantage of microfluidic technology to isolate EEVs from body fluids [
102]. It is well known that EEVs have significant advantages for disease diagnostics and monitoring because of their abundance, stability, and unique molecular cargos [
106]. Since the challenges of EEVs’ isolation could be addressed by the integration of a microfluidics platform, clinicians must contribute to point-of-care testing and treatment options by focusing on these potential players.