**4. Involvement of EVs in cancer**

III. Embryonic development.

[15].

90 Tumor Metastasis

IV. Tissue repair.

V. Liver homeostasis.

**3.2. Pathological actions of EVs**

T-cell activity [21–24].

EVs are likely to be involved in the regulation of embryonic development, including mainte‐ nance of morphogen gradients, collective cell migration and tissue polarity. However, this still remains an emerging field with many unanswered questions, which need further investigation

EVs derived from human adult mesenchymal stem cells (MSCs) have been found to prevent ischaemia-reperfusion kidney injury and improve survival in a model of lethal acute kidney injury [16]. MSC-derived EVs are reported to modify the expression of miR29c and miR150 and upregulate the expression of SDF-1, CXCR4, CXCR7, CCL2 and ANGPTL4, which are

A comprehensive study of hepatocyte-derived EVs showed the presence of several members of cytochrome P450, uridinediphosphate-glucuronosyl-transferase (UGTs) and glutathione Stransferase (GST) protein families, supporting a role of these vesicles in the metabolism of endogenous and xenobiotic compounds [18]. Recently, it has been shown that EVs from hepatocytes were able to activate stellate cells to mediate a response to liver damage [19] and many studies support an important role of these vesicles in maintaining liver homeostasis.

Given their essential role in regulating biological processes, it is not surprising that EVs have a significant influence in disease pathogenesis. This has been most extensively studied in tumour biology. Several reports have indicated that EVs may be an important means of driving the formation of a pre-metastatic tumour [12, 20]. EVs can promote proliferation of their target cells, stimulate angiogenesis, induce metastasis and promote immune escape by modulating

Prior to the discovery of EVs, it was known that the vesicles secreted by tumour cells retained procoagulant activity, linking cancer progression with EV-induced thrombosis [25–27]. In addition, a direct link between EVs and tumour invasion of healthy tissues was reported in 2008 [28]. It was shown that the mRNA expression of an activated mutated epidermal growth factor receptor (EGFRvII) in glioma cells can enhance vesiculation significantly and intercel‐ lular transfer of this oncoprotein to adjacent tumour cells, leading to the production of

Similar results were reported in another study by Skog et al. [22] showing that various miRNAs that stimulate tumour growth and angiogenesis in addition to EGFR can be transferred by human primary glioblastoma cell-derived EVs. Moreover, EVs derived from tumour cells were shown to transfer activated EGFR to endothelial cells, inducing VEGF expression and resulting in VEGF receptor activation to stimulate angiogenesis [29]. Many of the previously mentioned

angiogenic mediators such as vascular endothelial growth factor (VEGF) [28].

known to play essential roles in acute and chronic wounding [17].

Tumour EVs (oncosomes) are associated with many types of cancers [37–39], with elevated concentration in the plasma of cancer patients compared to healthy controls [40]; this can be up to 10-fold more than the approximately 1011 MVs per ml of serum measured in healthy individuals [41, 42]. Tumour EVs contain lipids and proteins as well as RNAs, genomic DNA and cellular metabolites, which can be transferred between cells [43], thus regulating the bioactivities of recipient cells. Production of EVs seems to be highly regulated. Several studies have characterised tumour EV components to identify useful cancer biomarkers [44]. For example, in two breast cancer cell lines, MCF-7 and MDA-MB 231, the cell-derived EVs show different profiles; 59 proteins were identified in MCF-7-derived EVs and 88 in EVs from MDA-MB 231, with 27 proteins common between the two exosome-like vesicle types [45]. Among all of these molecules that can be transferred from one cell to another through EVs, miRNAs have attracted most attention because of their newly recognised regulatory role in modulating gene expression. As some profiling studies have shown, miRNAs are not randomly incorpo‐ rated into exosomes. According to previous studies, there exists a class of miRNAs that are preferentially sorted into exosomes, such as miR-320 and miR-150. Members of the miR-320 family are widely distributed in exosomes derived from both normal tissue and tumours [22, 46, 47] Moreover, some reports have shown that exosomal miRNA expression levels are altered under different physiological conditions. Exosomal miR-105 released from the breast cancer cell line MDA-MB-231 reduced *ZO*-*1* gene expression in endothelial cells and enhanced metastases to the lung and brain [48]. Exosomal miR-214, derived from the human microvas‐ cular endothelial cell line HMEC-1, stimulated migration and angiogenesis in neighbouring HMEC-1 cells [49]. Thus, it is attractive to speculate that EVs may 'export' the ability of the producer cells to metastasise, to other cells.

Stressful stimuli, such as hypoxia, acidosis, oxidative stress, radiation and cytotoxic drugs, activate signalling pathways that can trigger exosome production and secretion [50]. The p-53 regulated gene product, TSAP6 [51], as well as ceramide [52] have been documented as

**Figure 3.** Extracellular vesicles (EVs) are potential carriers of stress-mediated tumour progression (Adapted with per‐ mission from Ref. [50]).

triggers. Stressful conditions can change both the molecular content and function of EVs, allowing for cancer progression through any of the processes displayed in **Figure 3**. For example, thermal and oxidative stress on leukemia/lymphoma T and B cells has been shown to induce the release of exosomes rich in Natural Killer Group 2 and member D (NKGD2) ligands that confer immunosuppressive properties to the exosomes. In addition, aggressive Bcell lymphoma cells that have been exposed to rituximab, which is an anti-CD20 chimeric antibody, started secreting CD20-poitive exosomes that protected the lymphoma cells from antibody and complement-dependent cytolysis. It is known that the phenotype of metastatic cells is a result of an accumulation of stress conditions on tumour cells. It has also been reported that while EVs derived from primary tumour cells can contain cell-adhesive proteins, those from metastatic cells are loaded with proteins that are responsible for cancer progression, invasion, metastasis and multidrug resistance. Thus, EVs can act as conveyors of stressmediated tumour progression. Like cancer cells, stromal cells could release EVs with modu‐ lated function upon exposure to stress. As an example, mesenchymal stem cells exposed to hypoxic conditions released microvesicles with angiogenic effects [50]. The horizontal transfer of bioactive molecules by EVs can influence the different aspects of tumour progression, which include angiogenesis, decrease of immune surveillance, ECM degradation, metastasis and chemoresistance. The following sections discuss the influence of EVS on the processes that are vital for tumour progression, through horizontal transfer of bioactive molecules.

#### I. Neoangiogenesis.

Fibrin, the end product of the coagulation process, plays an important role in tumour growth as tumour cells can be coated with fibrin to escape immune surveillance; at the same time, the fibrin matrix enhances the outgrowth of new blood vessels. In several studies, it has been shown that EVs support coagulation through various mechanisms. They expose negatively charged phospholipids, which enable binding of coagulation factors and hence formation of prothrombinase complexes [53, 54]. In cancer, tissue factor vesicles are present in the peripheral blood [27, 55]. A part of these MVs originates from cancer cells and usually participate in thrombus formation equally to leukocyte-derived vesicles. Those MV-exposed tissue factors can promote coagulation by adhering at the site of vascular damage [56, 57].

In addition, tissue factor also plays a more direct role in angiogenesis, which is induced through cytoplasmic domain phosphorylation of the tissue factor and subsequent downstream signalling events. Consequently, thrombin will be generated through the activation of coagulation by tissue factor, which cleaves several protease-activated receptors (PARs), in order to initiate angiogenesis [58].

Besides, platelet-derived vesicles stimulate mRNA expression of angiogenic factors in cancer cells and then cancer cell-derived vesicles will contain mRNA for growth factors, such as VEGF and hepatocyte growth factor [59]. It has been showed that such vesicles fuse with monocytes, conveying their nucleic acids content and altering their biologic activity [60]. It is believed that cancer cell-derived MVs transfer mRNA to other cancer cells, enhancing their malignant potential, and it has been reported, as mentioned previously, that intercellular transfer of oncogenic growth factor receptor by cancer cell-derived EVs modify the phenotype of these cells [28].

II. Escape from immune surveillance.

triggers. Stressful conditions can change both the molecular content and function of EVs, allowing for cancer progression through any of the processes displayed in **Figure 3**. For example, thermal and oxidative stress on leukemia/lymphoma T and B cells has been shown to induce the release of exosomes rich in Natural Killer Group 2 and member D (NKGD2) ligands that confer immunosuppressive properties to the exosomes. In addition, aggressive Bcell lymphoma cells that have been exposed to rituximab, which is an anti-CD20 chimeric antibody, started secreting CD20-poitive exosomes that protected the lymphoma cells from antibody and complement-dependent cytolysis. It is known that the phenotype of metastatic cells is a result of an accumulation of stress conditions on tumour cells. It has also been reported that while EVs derived from primary tumour cells can contain cell-adhesive proteins, those from metastatic cells are loaded with proteins that are responsible for cancer progression, invasion, metastasis and multidrug resistance. Thus, EVs can act as conveyors of stressmediated tumour progression. Like cancer cells, stromal cells could release EVs with modu‐ lated function upon exposure to stress. As an example, mesenchymal stem cells exposed to hypoxic conditions released microvesicles with angiogenic effects [50]. The horizontal transfer of bioactive molecules by EVs can influence the different aspects of tumour progression, which include angiogenesis, decrease of immune surveillance, ECM degradation, metastasis and chemoresistance. The following sections discuss the influence of EVS on the processes that are

**Figure 3.** Extracellular vesicles (EVs) are potential carriers of stress-mediated tumour progression (Adapted with per‐

vital for tumour progression, through horizontal transfer of bioactive molecules.

Fibrin, the end product of the coagulation process, plays an important role in tumour growth as tumour cells can be coated with fibrin to escape immune surveillance; at the same time, the fibrin matrix enhances the outgrowth of new blood vessels. In several studies, it has been shown that EVs support coagulation through various mechanisms. They expose negatively charged phospholipids, which enable binding of coagulation factors and hence formation of prothrombinase complexes [53, 54]. In cancer, tissue factor vesicles are present in the peripheral blood [27, 55]. A part of these MVs originates from cancer cells and usually participate in

I. Neoangiogenesis.

mission from Ref. [50]).

92 Tumor Metastasis

Collective data suggest a relationship between stressful conditions due to the tumour envi‐ ronment and immunological tolerance of tumours [50]. There are many mechanisms, either direct or indirect, that have been suggested which can facilitate escape from immune surveil‐ lance. For example, cancer cells may employ vesiculation as a means to efficiently deceive the immune system and survive [13]. Another study also showed that under the pressure of oxidative stress, tumour cells release NKG2DL-expressing tumour exosomes, which facilitate tumour escape from cytotoxic immune attack [61]. Further, exosomes from various cancer cells were shown to expose Fas ligand (FasL, CD95L) of the death receptor Fas (CD95), to trigger T-cell death and to diminish the function of adaptive immune cells [62].

Tumour-associated EVs may also enhance the function of regulatory T (TReg) cells, weaken natural cytotoxic responses mediated by natural killer cells, downregulate dendritic cell differentiation from monocytes and turn these cells into immunosuppressive cells [24, 63, 64]. In addition, cancer cells can integrate with EVs derived from non-cancer cells, for example, platelets, by this means receiving lipids and transmembrane proteins, which would protect them from immune surveillance [59]. Additionally, cancer cells may hide from the immune system by mimicking the host environment.

III. Environmental degradation.

It has been shown that degradation of the ECM is needed for tumour growth [65]. EVs expose and contain several proteases, including matrix metalloproteinase (MMP)-2 and MMP-9 and urokinase-type plasminogen activator (uPA). uPA catalyzes the conversion of plasminogen into plasmin, whereas MMPs degrade basement membrane collagens. Plasmin, which is a serine protease, degrades numerous components of the ECM, including fibrin, and activates various MMP zymogens [66].

When Ginestra et al. [67] analyzed vesicle content in ascites fluids from 33 women with different gynaecologic pathologies, they found that malignant tumour fluids contained higher amounts of vesicles compared to benign proliferative cells. Moreover, they showed that the EVs from benign serous cysts had only minimal lytic activity, whereas those from cancer ascites contained active metalloproteases [67]. Furthermore, a link was found between the malignant potential of tumours and the MV-associated MMP-2 activity [68]. Another study reported an increase in numbers of vesicles in late stage ovarian cancer ascites and showed that MMP-2, MMP-9 and uPA activities were mainly concentrated within the MVs. Further, the MMP-2, MMP-9 or uPA inhibition using antibodies almost eliminated the ability of these MVs to enhance tumour invasion capacity, which highlights the significance of this pathway [69].

#### IV. Metastasis.

Metastasis necessitates an increase in cellular survival and invasiveness, which are both enhanced by MVs. Some evidence suggests that MVs may favour lymphogenous and haema‐ tological spread as the expression of Fas ligand by cancer cell-derived MVs plays a role in lymph node infiltration [70]. Furthermore, as mentioned above, activation of platelets by tissue factor-derived vesicles supports the haematological spread of cancer cells. Since the cancer cells will be surrounded by platelets, this would afford them some protection from immune surveillance and enhance their attachment to the vessel wall [59]. In addition, the procoagulant properties of cancer cell-derived MVs further support intravascular fibrin formation, which in turn facilitates adherence of cancer cells to the vessel wall [27].

#### **4.1. EVs in cancer therapeutics**

Since MVs appear to contribute significantly to cancer development, it is not surprising that much effort is being focused on trying to find ways of utilizing them in therapy as well. There are at least four strategies that could potentially be used to oppose EV-driven disease by inhibiting various aspects of EV function; these are summarised in **Figure 4**. The most obvious approach is to get rid of them and this can be achieved by blocking their biogenesis, by interfering with their release from the cell, removing them from the circulation or inhibiting their uptake by recipient cells [1].

#### *4.1.1. Inhibition of EVs*

#### I. Inhibiting EV formation.

Various cellular components are known to be vital for EV formation but until now no clear inhibition strategy has been forthcoming although many are under investigation. However, some studies showed that inhibition of ceramide formation (which is essential for endosomal sorting and exosome biogenesis) using small molecule inhibitors of neutral sphingomyelinase or through treatment with the blood pressure-lowering drug amiloride (which decreases endocytic vesicle recycling) can reduce EV formation [52, 71]. Another interesting study emphasised the importance of syndecan proteoglycans and their cytoplasmic adaptor syntenin, in regulating exosome formation and release, directly interfering with this interac‐ tion either by RNA interference (RNAi) or using small molecule inhibitors [72].
