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

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 contained in tumor-derived EVs [76].

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 epithelial-to-mesenchymal transition [111].

## **5. Extracellular vesicles and metastatic niches**

Tumor microenvironment was described to undergo series of molecular and cellular changes to form the metastatic-designated sites, called pre-metastatic niche [115, 116]. The formation of pre-metastatic niche requires the cross talk between primary tumor-derived components, and the microenvironment of the host stromal components and of the tumor-mobilized bone-marrow-derived cells [117].

Interestingly, the role of EVs in metastatic niches can be exploited in novel therapeutic approaches. Indeed, technologies based on exosomes, separated from the ascitic fluid of ovarian cancer patients, embedded in a 3D scaffold metastatic trap, were successfully tested in murine models of ovarian metastasis in order to improve survival [118]. Numerous studies indicated that tumor-derived exosomes might play a role in promoting angiogenesis and modulation of the immune system [119, 120]. Moreover, exosomes derived from cancerous tumor are capable of remodeling the surrounding parenchyma, thus supporting tumor progression and the generation of the pre-metastatic niche [121, 122].

## **6. Role of extracellular vesicles in metastasis**

EVs have been described to play an essential role in the local and distant communication between cancer cells and their environment and in contributing to the progression of metastasis [123]. Although the function of EVs in metastasis is not completely understood, studies show that miRNAs isolated from EVs are actively involved in complex metastatic processes, including local invasion, angiogenesis, immune modulation, metastatic niche preparation, colonization, and dormancy [123].

EVs play an essential role in the tumor metastasis by ensuring the cross talk between tumor and the adipose tissue, and obesity was described to influence the metastatic behavior of tumors, especially in melanoma, breast, and ovarian cancers [124].

In breast cancer, metastatic exosomes creating a facilitating local environment for metastasis was demonstrated, and annexin II contained in these exosomes contributes to this process by promoting angiogenesis [125].
