**3.1 Nucleic acids**

Circulating DNA can be found in free form or contained in EVs and is thought to be the future in cancer diagnosis and treatment monitoring. This will be possible because the DNA fragments contained in EVs are relatively intact (average 15 kbp) by comparison with the circulating cell-free one (average 130 bp) due to the protection offered by the lipid bilayer [79]. Vagner et al. showed in a very recent study that the majority of the extracellular DNA is contained in large oncosomes, rather than in exosomes, both in vitro and in the patients' plasma and has all cancer-specific genomic alterations [80]. The majority of the DNA contained

in tumor-derived exosomes is double stranded and represent the whole genomic DNA, suggesting its usefulness in identifying mutations present in parental tumor cells, as it was indicated by Thakur et al. [81]. Wyatt et al. showed that cancerderived DNA is sufficient to identify the DNA alterations from metastatic tissue and is very important because it integrates somatic information from more than one metastatic lesion [82].

The presence of retrotransposons, cDNAs, and ncRNAs has also been reported in EVs and appears to be a unique feature of tumor cells [83]. Several studies reported a correlation between increased retrotransposon activity and tumorigenesis [84]. For example, LINE-1 hypomethylation in various human cancers was intensively studied since it is considered to be an early event in tumorigenesis and to be linked with the induction of proto-oncogenes [85]. Loss of LINE-1 methylation was found to associate with more aggressive progression of colorectal cancer [86]. Moreover, LINE-1 hypomethylation level can be considered as an important epigenetic process, which became a potential prognostic factor for ovarian multistep carcinogenesis [87].

miRNAs represent potential candidates responsible for influencing the tumor microenvironment; however, little is known about the mechanism by which they produce changes in the transcriptome of target cells [88].

#### **3.2 Proteins**

Proteins exported in EVs are signaling molecules that interfere in a whole series of processes such as cell metabolism, cell invasion and growth, angiogenesis, and mRNA processing [89]. Among these, it is worth mentioning that the epidermal growth factor receptor vIII (EGFRvIII), mutant Ras family members, or c-Met have been proposed as cancer biomarkers [90, 91]. Other proangiogenic regulators, such as VEGF and bFGF, are harbored in EVs shed from cancerous cells promoting new blood vessel formation [92].

Moreover, EVs also transfer proteases such as MMP-2, MMP-9, and MT1-MMP and become responsible for the partial degradation of the extracellular matrix [93]. The content is released in an acidic environment after the vesicle is stabilized in the extracellular matrix with the aid of β1 integrin adhesion molecules [94].

#### **3.3 Lipids**

Naturally, EVs also contain a lipid component, which consists of the main membrane lipids: sphingomyelin, phosphatidylserine, and glycosphingolipids and cholesterol, but they also carry polyunsaturated fatty acids PUFAs, mainly arachidonic acid and linoleic acid [95, 96]. Sphingomyelin, as a component of the EVS, was firstly reported by Kim et al. who described its angiogenic properties [97] . In addition, it has been shown that the lipid content of exosomes suppresses critical cancer survival pathways such as notch leading to cancer cell death of human pancreatic tumoral SOJ-6 cells [98]. Moreover, other important signaling mediators, such as prostaglandins, arachidonic acid, phospholipase A2, and phospholipase C and D, are also found in EVs [99]. The prostaglandins found in breast-cancer-derived exosomes, such as PGE2, are responsible for promoting tumor growth by inducing the release of pro-inflammatory cytokines such as IL-6 and VEGF, which induce the accumulation of myeloid-derived suppressor cells capable to differentiate into macrophages in the tumor microenvironment [100, 101]. PGE2 indeed makes the connection between cancer and macrophages and can promote tumorigenesis by enhancing the expression of programmed cell death protein ligand 1 (PD-L1) responsible for tumor escape from immune system during cancer progression [102].

**95**

*Extracellular Vesicles in Cancer*

*DOI: http://dx.doi.org/10.5772/intechopen.85117*

Cancer stem cells (CSCs) are held directly responsible to promote cancer initiation and progression. Also, there are several studies showing their importance in therapy resistance, recurrence, and metastasis [103]. CSCs themselves do not exist as a static population, their stemness being supported by the mesenchymal stem cells, endothelial cells, fibroblasts, or immune cells by paracrine signaling [104]. For example, in breast cancer, the overexpression of the chemokines CXCL14 and CXCL12 in myoepithelial cells and myofibroblasts favors the metastasis [105]. Cancer-associated fibroblasts (CAF) release exosomes, which induce the stemness of breast cancer cell lines, developing an aggressive cancer cell phenotype [106]. Also, the ECM molecules are relevant for breast cancer colonization, which contribute to the control of CSC. In this sense, tenascin C, a protein in the ECM, contributes in the formation of the stem niche by protecting CSC from immune surveillance [107]. In breast cancer, high levels of tenascin C are associated with poor

clinical outcome in breast cancer due to lung cancer metastasis [108].

The role of EVs in tumorigenesis was described in various types of cancer, including ovarian [109–111] and breast cancers [112, 113]. EVs undergo several alterations in tumorigenesis, including changes in their biogenesis, release rate and/ or protein content, incorporation of oncogenic and mutant macromolecules, mediated release of genomic DNA, and uptake of tumoral cells [114]. The transfer of DNA between apoptotic tumoral cells and other cells is important in tumorigenesis. In vitro, it was shown that apoptotic bodies derived from cancer cells are responsible for triggering the expression of oncogenes in fibroblasts due to the information

In ovarian or breast cancer, investigating the content of EVs might give important

informations on tumorigenesis. To detail, exosomes released by IGROV1 ovarian cancer cells (with high content of RNA-binding proteins, such as LIN28A or LIN28B), but not by OV420 ovarian cancer cells, were taken up by HEK293 cells, contributing to the tumor development [109]. Moreover, in hypoxia conditions, SKOV3 human epithelial ovarian cancer cells release exosomes with high content of miR-940 that stimulate the M2 macrophage phenotype, and in turn, M2 subtype macrophages stimulate the tumor cell migration and proliferation [111]. The majority of circulating miRNA, packed in EVs, can be used as biomarkers in ovarian cancer, but their use is not only limited for diagnosing the existence of the cancer, but also being reliable markers for monitoring the tumor histology, stage, or the patient outcome [110]. The content of EVs released from two human breast cancer cell lines, MCF-7 (less invasive) and MDA-MB-231 (more invasive), was analyzed, and approximately, 270 proteins were identified [113]. In circulating EVs, epidermal growth factor-like repeats and discoidin I-like domains 3 (EDIL3) are the extracellular matrix (ECM) protein that was described to play a critical role in tumorigenesis by the activation of integrin-focal adhesion kinase (FAK) signaling cascade [113]. Breast-cancer-derived EVs (e.g., exosomes) present a cell-independent microRNA biogenesis from pre-miRNAs (like Dicer, AGO2, or TRBP) to mature miRNAs [112]. In particular, exosomes detected in the cells and sera of patients affected by breast cancer were shown to stimulate tumorigenesis in nontumoral epithelial cells by a Dicer-dependent mechanism [112]. It was also demonstrated that in the breast tumor microenvironment, there is a downregulation of the tumor suppressor p85α, which is clinically relevant in tumorigenesis, and the mechanism involves the loss of p85α expression in stromal fibroblasts promoting breast cancer progression by the

**4. Role of extracellular vesicles in tumorigenesis**

contained in tumor-derived EVs [76].

epithelial-to-mesenchymal transition [111].

#### *Extracellular Vesicles in Cancer DOI: http://dx.doi.org/10.5772/intechopen.85117*

*Extracellular Vesicles and Their Importance in Human Health*

produce changes in the transcriptome of target cells [88].

metastatic lesion [82].

step carcinogenesis [87].

blood vessel formation [92].

**3.2 Proteins**

**3.3 Lipids**

in tumor-derived exosomes is double stranded and represent the whole genomic DNA, suggesting its usefulness in identifying mutations present in parental tumor cells, as it was indicated by Thakur et al. [81]. Wyatt et al. showed that cancerderived DNA is sufficient to identify the DNA alterations from metastatic tissue and is very important because it integrates somatic information from more than one

The presence of retrotransposons, cDNAs, and ncRNAs has also been reported

miRNAs represent potential candidates responsible for influencing the tumor microenvironment; however, little is known about the mechanism by which they

Proteins exported in EVs are signaling molecules that interfere in a whole series of processes such as cell metabolism, cell invasion and growth, angiogenesis, and mRNA processing [89]. Among these, it is worth mentioning that the epidermal growth factor receptor vIII (EGFRvIII), mutant Ras family members, or c-Met have been proposed as cancer biomarkers [90, 91]. Other proangiogenic regulators, such as VEGF and bFGF, are harbored in EVs shed from cancerous cells promoting new

Moreover, EVs also transfer proteases such as MMP-2, MMP-9, and MT1-MMP and become responsible for the partial degradation of the extracellular matrix [93]. The content is released in an acidic environment after the vesicle is stabilized in the

Naturally, EVs also contain a lipid component, which consists of the main membrane lipids: sphingomyelin, phosphatidylserine, and glycosphingolipids and cholesterol, but they also carry polyunsaturated fatty acids PUFAs, mainly arachidonic acid and linoleic acid [95, 96]. Sphingomyelin, as a component of the EVS, was firstly reported by Kim et al. who described its angiogenic properties [97] . In addition, it has been shown that the lipid content of exosomes suppresses critical cancer survival pathways such as notch leading to cancer cell death of human pancreatic tumoral SOJ-6 cells [98]. Moreover, other important signaling mediators, such as prostaglandins, arachidonic acid, phospholipase A2, and phospholipase C and D, are also found in EVs [99]. The prostaglandins found in breast-cancer-derived exosomes, such as PGE2, are responsible for promoting tumor growth by inducing the release of pro-inflammatory cytokines such as IL-6 and VEGF, which induce the accumulation of myeloid-derived suppressor cells capable to differentiate into macrophages in the tumor microenvironment [100, 101]. PGE2 indeed makes the connection between cancer and macrophages and can promote tumorigenesis by enhancing the expression of programmed cell death protein ligand 1 (PD-L1) responsible for tumor escape from immune system during cancer progression [102].

extracellular matrix with the aid of β1 integrin adhesion molecules [94].

in EVs and appears to be a unique feature of tumor cells [83]. Several studies reported a correlation between increased retrotransposon activity and tumorigenesis [84]. For example, LINE-1 hypomethylation in various human cancers was intensively studied since it is considered to be an early event in tumorigenesis and to be linked with the induction of proto-oncogenes [85]. Loss of LINE-1 methylation was found to associate with more aggressive progression of colorectal cancer [86]. Moreover, LINE-1 hypomethylation level can be considered as an important epigenetic process, which became a potential prognostic factor for ovarian multi-

**94**

Cancer stem cells (CSCs) are held directly responsible to promote cancer initiation and progression. Also, there are several studies showing their importance in therapy resistance, recurrence, and metastasis [103]. CSCs themselves do not exist as a static population, their stemness being supported by the mesenchymal stem cells, endothelial cells, fibroblasts, or immune cells by paracrine signaling [104]. For example, in breast cancer, the overexpression of the chemokines CXCL14 and CXCL12 in myoepithelial cells and myofibroblasts favors the metastasis [105]. Cancer-associated fibroblasts (CAF) release exosomes, which induce the stemness of breast cancer cell lines, developing an aggressive cancer cell phenotype [106]. Also, the ECM molecules are relevant for breast cancer colonization, which contribute to the control of CSC. In this sense, tenascin C, a protein in the ECM, contributes in the formation of the stem niche by protecting CSC from immune surveillance [107]. In breast cancer, high levels of tenascin C are associated with poor clinical outcome in breast cancer due to lung cancer metastasis [108].
