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      Figure 3.4 Schematic representation of the major components of exosomes. Common exosome markers include tetraspanins (CD9, CD63, and CD81), integrins, TSG101, and Alix. Exosomes also contain other proteins, different species of RNA, and DNA.

It is biologically important to determine the organ where metastasis might be facilitated by such a mechanism. Accumulating evidence suggests that exosome-mediated activities play an important role in various diseases, especially cancer.

      Physiological and Cellular Functions of EVs

      Angiogenesis is the physiological process through which new blood vessels are formed from pre-existing vessels (Birbrair et al. 2014; Birbrair et al. 2015). Angiogenesis is regulated by angiogenic factors, extracellular matrix components, and endothelial cells (ECs) (Carmeliet and Jain 2000).

      Angiogenesis is also promoted by the uptake of cancer cell-derived EVs that are rich in matrix metalloproteinases, especially MMP-2, MMP-9 and MMP-13. These proteins have been found in glioblastoma-, melanoma-, myeloma- and nasopharyngeal carcinoma-derived exosomes (Skog et al. 2008; Ekstrom et al. 2014; You et al. 2015; Wang et al. 2016; Giusti et al. 2016; Chan et al. 2015). It has been recently reported that metastatic breast cancer-derived EVs, which express high levels of the proangiogenic protein annexin II (Anx II), promote angiogenesis and that head and neck squamous cell carcinoma-derived EVs regulate angiogenesis through ephrin-B reverse signaling (Sato et al. 2019).

      EV-associated miRNAs are also known to promote angiogenesis. EV-associated miR-23a, derived from hypoxic lung cancer cells, promotes angiogenesis by targeting prolyl hydroxylase and the tight junction protein ZO-1 (Hsu et al. 2017). It also promotes angiogenesis in hypoxic HCC (Sruthi et al. 2018). Gastric cancer (GC) cell-derived EVs containing miR-155 promote angiogenesis by enhancing the expression of vascular endothelial growth factor (VEGF) (Deng et al. 2020). In this way EVs play an essential role in the promotion of angiogenesis, which suggests that they could promote carcinogenesis by promoting cancer metastasis. EVs are also important for the crosstalk between cancer cells and fibroblasts through EVs.

      The interactions between cancer cells and cancer-associated fibroblasts (CAFs) vary and are considered to be important for carcinogenesis (Xouri and Christian 2010; Rasanen and Vaheri 2010; Kalluri 2016; Shiga et al. 2015; Kalluri and Zeisberg 2006; Tommelein et al. 2015). Moreover, the epithelial–mesenchymal transition (EMT), which enables tumor cells to acquire resistance to anticancer drugs, was reported to be facilitated by the presence of CAFs (Shan et al. 2017; Yu et al. 2014; Zhuang et al. 2015). There have been several reports that the interactions between cancer cells and CAFs are mediated by EVs. Cancer cells educate fibroblasts through EVs, which leads to the progression of metastasis (De Wever et al. 2014). Cancer-derived EVs are involved in the reprogramming of normal stromal fibroblasts to activated CAFs in chronic lymphocytic leukemia, hepatocellular carcinoma (HCC), and melanoma (Paggetti et al. 2015; Fang et al. 2018; Gener Lahav et al. 2019). These studies revealed that the CAF transition leads to metastatic niche formation and promotes cancer metastasis.

      EVs facilitate not only cancer-related functions but also many other physiological activities. For example, it has been recently reported that EVs mediate horizontal gene transfer.

      To date, there have been two reports of exosome-mediated trans-species horizontal gene transfer events in mammals: Ono et al. (2019) and Kawamura et al. (2019).

Approximately 40% of the mammalian genome is derived from retrotransposons, which have long been considered “junk.” Recently accumulated evidence has demonstrated that mammalian ancestors acquire retrotransposons as endogenous genes. A chromodomain (chromatin organization modifier) is a protein structural domain. Chromodomains are highly conserved in chromoviruses, and SCAN domains might originate from GYPSYDR-1 retrotransposons. Sirh-family genes, which are conserved in mammals, contain a gag-like domain from the Ty3/Gypsy-type retrotransposon of fugu fish (Ono et al. 2001; Ono et al. 2003; Ono et al. 2006; Sekita et al. 2008; Naruse et al. 2014; Irie et al. 2015). In particular, in the case of Sirh-family genes, the mechanism of the horizontal gene transfer from fugu to the mammalian ancestor was a mystery (Ono et al. 2011). Recent in silico analyses demonstrated horizontal transfer of BovB (non-LTR retrotransposon from Bos taurus) and L1 retrotransposons (Bos taurus) in eukaryotes (Ivancevic et al. 2018).

In mouse NIH-3T3 cells, most DNA double-strand breaks (DSBs) introduced by CRISPR-Cas9 are repaired through non-homologous end joining (NHEJ) without homologous DNA sequences for homologous recombination (HR) (Wang et al. 2013). NHEJ-mediated repair of DSBs is prone to error, causing small indels (Wang et al. 2013). It was reported that DSBs introduced by CRISPR-Cas9 can be repaired through the capture of non-target (i.e., unintended) sequences (Ono et al. 2015). These non-target sequences may include retrotransposon sequences, reverse-transcribed spliced mRNA sequences, and CRISPR-Cas9 vector sequences (Figure 3.5a, b, c). Although most of the captured retrotransposons were derived from murine endogenous retrotransposons, horizontal gene transfer from bovines to mice was certainly confirmed (Figure 3.5d; see Ono et al. 2019).

      Figure 3.5 Horizontal gene transfer mediated by EVs. Three types of capture of non-target sequences associated with genome editing (a, b, c). Distribution of the captured retrotransposons at CRISPR-Cas9-induced DSB sites in NIH-3T3 cells cultured using DMEM containing 10% FBS; 12% of the reads that captured retrotransposon sequences, including genomic, Short interspersed nuclear element (SINE), and satellite DNA sequences (d), were from Bos taurus (bovine). Distribution of the captured retrotransposons at CRISPR-Cas9-induced DSB sites in NIH-3T3 cells cultured using DMEM containing 10% exosome-free FBS; none of the reads that captured retrotransposon sequences, including genomic, SINE, and satellite DNA sequences (e), were from Bos taurus (bovine).

      As it was possible that these horizontal genes were transferred from the cell culture medium, we repeated these experiments using exosome-free 10% FBS (DMEM). Then most of the bovine DNA insertions were abolished by culture with exosome-free 10% FBS/DMEM (Figure 3.5e). Furthermore, it was reported that bovine retrotransposons were enriched in FBS-derived exosomes (Ono et al. 2019). These data demonstrate that trans-species gene transfer is mediated by exosomes (Ono et al. 2019).

      It was also reported that LINE-1 retrotransposons underwent horizontal transfer mediated by exosomes by using the engineered L1-EGFP reporter assay (Figure 3.6a).

      Figure 3.6 (a) Schematic representation of the engineered L1-EGFP reporter assay. (b) L1-EGFP RNA is transferred to recipient cells by using a co-culture system.

      The human cancer cell line MDA-MB-231-D3H2LN (MM231) was transfected with an L1-EGFP reporter cassette as described previously (Farkash et al. 2006; Garcia-Perez et al. 2010; Ostertag et al. 2000; Coufal et al. 2009). In this construct, the EGFP reporter gene is interrupted by an intron and inserted in the opposite transcriptional orientation into the 3ʹUTR of a retrotransposition-competent human L1. Thus EGFP is expressed only when the L1 transcript is spliced,

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