**3. Extracellular vesicles in the pathology of cancers**

The importance and the role played by the tumor microenvironment on tumor development and progression has been established in recent years [51]. EVs are known to influence the tumor microenvironment either through a direct impact on the tumor or from a distant site which promote future metastasis of circulating cancer cells [51]. Due to these characteristics, key processes involved in cancer developments such as angiogenesis, thrombosis, oncogenic transfer, immune modulation and pre-metastatic niche formation have seen an up-regulation of EVs [52–57]. Compared to non-malignant cells, tumor cells are known to release higher amounts of EVs. In this regard increased levels ESCRT components as well as heparanase and syntenin have been expressed in various cancers [58–60]. Specifically, in colorectal cancer and pancreatic carcinoma, hyperactivity of RalB has been observed and in non-smallcell lung cancer YKT6 overexpression coupled with elevated Rho-ROCK signaling expressed in various type of cancers may contribute to EVs generation in tumor cells [61–64]. On the basis that tumorigenesis occurs due to accumulation of genetic alterations, the metastatic traits of EVs are expressed through the transfer of their oncogenic cargo. Tumor derived EVs through the co-transfer of protein crosslinking enzymes (tissue transglutaminase) and fibronectin, are able to import transformed characteristics of cancer cells on to recipients endothelial cells and fibroblast [57]. Both the cell-intrinsic and environmental signals may influence EV release in tumor cells. EVs production in tumor cells may be induced by the activation of H-RASv12 and EGFRvIII oncogenic signal pathways [65–67]. Again, the level (de)regulation of the machinery, which aid in plasma membrane fusion could also influence the release of EVs in tumor cells. For example, it has been demonstrated that EV secretion could be enhanced when PKM2 (a glycolytic enzyme associated with the Warburg effect) is over expressed leading to phosphorylating tSNARE SNAP23 [68]. Also SRC, a proto-oncogene, through the phosphorylation of the cytosolic domains of syntenin and syndecan is able to stimulate the syntenin exosome biogenesis pathway [69]. On the other hand, in some cancers such as colon cancer cells, mutant proto-oncogene, KRAS could be transferred via EVs to increase the population of recipients colon cancer cells expressing the wild-type KRAS [70]. Further an increase in levels of tissue factor (TF) bearing EVs are known to mediate thrombosis occurrence in cancer patients. Available evidence indicates a possible role of tumor-derived EVs in thrombosis occurrence among cancer subjects [54]. Specifically, P-selectin glycoprotein ligand-1 (PSGL-1) and TF have been implicated in cancer associated thrombosis [71]. In mice with induced pancreatic tumor, formation of thrombosis was high compared to cancer free mice [72]. A major hallmark of tumor growth and development is increased angiogenesis. That is to say for the development of the tumor beyond its minute size an adequate supply of oxygen and nutrients is essential for its survival. Thus, numerous studies have established that besides the cell's intrinsic mechanisms, the release and regulation of exosomes and microvesicles could be due to enhanced prevailing hypoxic microenvironmental conditions [73–75]. In hypoxic glioma cells, an induction of a pro-angiogenic process mediated by derived EVs was able to influence the vasculature surrounding cell [55]. Another specific example where EVs promotes angiogenesis is reported in squamous carcinoma. It was reported that in A431 squamous carcinoma cells, angiogenesis was induced as a results of a direct transfer oncogenic epidermal growth factor receptor (EGFR) from the derived EVs to endothelial cells [76].

RNAs are a major important cargo incorporated into EVs. Cancer cells promote an increase in the release of EVs containing varying amount and types of proteins and RNAs compared to normal cells [77, 78]. There exist an EV-RNA mediated crosstalk within tumors and also between tumors and stroma which could modify the malignant behavior of cancer cells [79]. EV-RNAs derived from tumor may be implicated in the devolvement of oncogenic, pro-angiogenic, and pro-metastatic processes as well as stromal cell differentiation in the tumor microenvironment. Also it is known that normal and tumor cells subpopulations are likely to be driven towards malignant phenotypes aided by tumor derived EVs [79]. Some EV-RNAs are known to actively mediate proliferation, migration, invasion, apoptosis, dormancy and therapy resistance of cancer cells. There seems to be a dual function of EV-RNAs in cancer pathology. Whiles some are known to promote the malignant characteristics of cancer cells, it also possible for some EV-RNAs to inhibit the malignant characteristics of cancer cells. In this regard various studies have reported the ability of EV-RNAs to inhibit mechanisms that favor tumor growth. In order to establish homeostasis, various non tumor cells can produce miRNAs which could suppress the malignant phenotypes of adjacent cancerous cells [80]. This is due to the fact that a natural competition exists between cancerous and adjacent non-cancerous cells during the development of cancer [81, 82]. In hepatocellular carcinoma, EV-miRNAs released from liver stem cells were able to promote apoptosis whiles inhibiting cell proliferation *in vitro and in vivo* [83]. Again, in pancreatic ductal adenocarcinoma cells, tumor-associated stroma cells derived EV-miR-145 inhibited cancer cell viability whiles promoting apoptosis [84]. Similarly, EV-miE-145 derived from adipose tissue-derived mesenchymal stem cells promoted apoptosis and inhibited proliferation in prostate cancer cells [85]. Another important tumor modulatory role influenced by EVs is the immune system modulation [86–88]. Examples of cancer derived EVs in immune-modulation have been reported in the peripheral circulation of oral squamous carcinoma patients in which Fas ligand positive EVs were able to induce apoptosis of effector cytotoxic T cells [89]. Other studies have demonstrated that various Treg regulatory mechanisms such as Treg expansion promotion, Treg induction, Treg suppressor functional upregulation and others have been promoted by cancer-derived EVs (**Figure 1**) [90].

#### **Figure 1.**

*Tumor cells release EV-RNAs. These EV-RNAs mediate many functions including sustaining proliferation, migration, invasion and metastasis, evading growth suppression, dormancy and therapy resistance of tumor cells, which promote growth.*
