II. Inhibiting EV release.

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].

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

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

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].

turn facilitates adherence of cancer cells to the vessel wall [27].

IV. Metastasis.

94 Tumor Metastasis

**4.1. EVs in cancer therapeutics**

their uptake by recipient cells [1].

*4.1.1. Inhibition of EVs*

I. Inhibiting EV formation.

**Figure 4.** Flowchart showing the variety of roles of EVs in cancer therapeutics.

Many proteins have been shown to be associated with the secretion of EVs, but again the exact mechanism of regulated EV release remains unclear and probably varies between different cells. However, it has been shown that some small GTPases in some tumour cells, such as RAB27A73, can be a promising therapeutic target (by RNAi) for reducing tumour exosomemediated signalling to inhibit neutrophils that support tumour growth [73, 74]. This approach was used in two independent studies and it showed a significant reduction in the growth rate of primary metastatic carcinoma and in metastasis progression [73, 75]. Other GTPases such as RAB11 and RAB35 might serve as alternative targets for inhibiting the release of exosomes by weakening and loosening the docking and/or fusion of multi-vesicular bodies with the plasma membrane [74].
