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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 due to its convenience and low cost. However, the limited specificity of such markers has been widely recognized. When a tumor marker is detected but the organ specificity of that marker is poor, it is not possible to determine which organ has cancer. For example, CEA is a tumor marker for breast cancer, but it is also expressed at high levels in colorectal cancer, stomach cancer, and pancreatic cancer (Vaidyanathan and Vasudevan 2012). Therefore further tests are required, which results in an increased burden on the patient. Because of these problems, screening for new molecular biomarkers to be used in liquid biopsy is underway: the aim is to develop more accurate methods for early cancer diagnosis.

The discovery of new molecular biomarkers overcomes the drawbacks, thanks to the intimate association of these se biomarkers with cancer-related genetic alterations. The proteins on the surfaces of exosomes are potential cancer biomarkers that can allow early tumor diagnosis (Table 3.2). Glypican-1 (GPC1) (+) exosomes could distinguish healthy subjects from patients with pancreatic cancer (Melo et al. 2015). The percentage of GPC1 (+) exosomes was markedly increased in plasma from patients with pancreatic cancer; however, they were decreased to normal levels after surgery. It was also reported that the exosome protein survivin-2B was a good biomarker for breast cancer diagnosis, and the level of TRIM3 protein in serum exosomes was decreased in gastric cancer patients (Khan et al. 2012; Fu et al. 2018).

Since exosomes are surrounded by a lipid bilayer membrane, they are not easily degraded even in those body fluids that contain many digestive enzymes. Therefore an EV-associated RNA is highly stable and makes an ideal noninvasive biomarker for cancer diagnosis. The most abundant type of RNA in exosomes is miRNA; consequently liquid biopsies using miRNAs in exosomes as cancer biomarkers have been developed in various cancer types (Table 3.3).

      Increasing evidence has accumulated that EV-associated miRNAs may also prove clinically useful other than as cancer biomarkers—namely as biomarkers for non-cancer diseases. These diseases include pathogenic inflammation, infection, and chronic diseases. However, the disease-specific exosome cargo remains to be fully elucidated and validated.

      Recently EV-associated miRNAs were identified as biomarkers for Alzheimer’s disease and Parkinson’s disease.

Table 3.3 Summary of exosomal proteins for early tumor diagnosis.

Type of cancer	Protein	Reference
Colorectal cancer	Copine III (CPNE3)	(Sun et al. 2019)
	CD147	(Tian et al. 2018)
	CD147	(Yoshioka et al. 2014)
Gastric cancer	HER-2/neu, EMMPRIN, MAGE-1, C-MET	(Baran et al. 2010)
	TRIM3	(Fu et al. 2018)
Lung adenocarcinoma	CD91, CD317, ITA2B	(Ueda et al. 2014)
	EGFR, KRAS, claudins and RAB-family proteins	(Clark et al. 2016)
	CD151, CD171 and tetraspanin 8	(Sandfeld-paulsen et al. 2016)
Melanoma	Caveolin	(Logozzi et al. 2009)
	Met	(Peinado et al. 2012)
Pancreatic ductal adenocarcinoma	GPC-1	(Melo et al. 2015)
Prostate cancer	PSA	(Mizutani et al. 2014)
	ephrinA2	(Li et al. 2018)
	Survivin	(Khan et al. 2012)
Renal cell carcinoma	MMP-9, DKP4, EMMPRIN, PODXL	(Raimondo et al. 2013)

Table 3.3 Summary of exosomal miRNAs for early tumor diagnosis.

Type of cancer	Protein	Reference
Breast cancer	miR-101, miR-372, miR-373	(Eichelser et al. 2014)
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