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, reverse-transcribed, and integrated into the chromosomal genome during a retrotransposition event (Ostertag et al. 2000). Successful L1 integration by retrotransposition can be detected by screening for EGFP expression or by performing a polymerase chain reaction (PCR) (Figure 3.6a).
To investigate the transfer of L1-EGFP RNA to recipient cells, MM231 L1-EGFP cells were co-cultured with MM231 wild-type (WT) cells as recipient cells (Figure 3.6b). Transwell inserts allow EVs, but not whole cells, to pass through.
After twenty-one days of co-culture, EGFP-expressing cells were detected in recipient cells that were exposed to L1-EGFP EVs (Figure 3.6), demonstrating that the L1-EGFP reporter was horizontally transferred from MM231 L1-EGFP cells to recipient cells and that retrotransposition events occurred in the recipient cells.
These reports support the occurrence of EV-mediated trans-species gene transfer. Because EVs are reportedly present in all fluids from living animals, the trans-species gene transfer events discovered in many species may be mediated by EVs.
Cancer Biomarkers
Cancer is one of the leading causes of death worldwide. Approximately 10 million people died prematurely as a result of cancer in 2018; that is, every sixth death in the world is due to cancer (Bray et al. 2018). To make progress in the prevention and treatment of cancer, many anticancer drugs are under active development worldwide. Because molecular targeting drugs and immune checkpoint inhibitors have been developed, improvements have been seen in cancer treatment outcomes (Welslau et al. 2014; Zhang 2016; Negrier et al. 2011; Chen and Han 2015; Topalian et al. 2015; Topalian 2017; Sharma and Allison 2015). However, the prognosis of all patients with advanced cancer has not yet been significantly improved. It has been shown that early detection of cancers has a greater therapeutic effect than late detection. Therefore, to extend the healthy lifespans of cancer patients, it is important to detect cancers at early stages.
Liquid biopsies have been regarded as a powerful tool for screening and identifying tumors before symptoms appear. Historically, the detection of a single circulating protein (e.g., CA19–9, carcinoembryonic antigen (CEA), and prostate-specific antigen (PSA)) has achieved prominence
