The role of extracellular vesicles in cholangiocarcinoma

According to the current knowledge of their biogenesis, EVs can be broadly divided into two main categories: microvesicles and exosomes [30, 31]. Microvesicles (MVs, 100–1000 nm in diameter) are secreted by the shedding or outward budding of the plasma membrane [32]. The formation of microvesicles is the result of the dynamic interaction of phospholipid redistribution and cytoskeletal protein contraction [33]. Since little information about biogenesis and MV release is understood, we focused on the function of exosomes in CCA.

In 1983, Johnstone et al. first isolated exosomes from the supernatant of sheep reticulocyte culture medium [34]. However, due to the role of exosomes being unclear at that time, it is thought to be a nonfunctional particle in the cell. Whereas exosomes are 40–150 nm, endosome-derived, small EVs which are secreted by most cells [35]. RNA, DNA and proteins are reported to be actively and selectively incorporated into intraluminal vesicles, which reside within multivesicular endosomes and are the precursor of exosomes [36, 37]. The release of exosomes into the extracellular space is facilitated by the fusion of multivesicular bodies (MVBs) limiting the membrane with the plasma membrane [35, 38]. Then, exosomes can be carried away by extracellular fluid, such as saliva, urine, blood, semen, amniotic fluid, ascites, alveolar lavage fluid, milk, synovial fluid and cerebrospinal fluid, and taken up by other cells [39‐41]. Besides, bile included [42]. The function of exosomes depends on the type and contents of their parent cells. Exosomes from normal cells play a role in maintaining stability in vivo, while tumor cell-derived exosomes are associated with tumor progression [40, 43].

In addition, we need to identify exosomes that can distinguish them from other EVs by their size and proteins markers. For instance, MVB-associated proteins (tumor susceptibility gene 101 (TSG101) and ALIX) [44, 45], fusion proteins, membrane transport proteins (flotillin-1) [46], tetraspanins (CD63, CD81and CD9) [36], and heat shock proteins (HSP70.1 and HSP20) [42, 47] are often used as protein markers to recognize exosomes in scientific research. Currently, with the identification of a large number of cargo molecules in exosomes, their functions, including regulating immune function, enhancing metastasis, and modulating intercellular communication, have also been explored in tumor cells [48‐50]. Some research shows that exosomes from highly metastatic breast cancer cells (4T1 and EO771 cells) can modulate the favorable microenvironment for lung and liver metastasis colonization [51]. In addition, various immune cell-derived exosomes, such as natural killer cells, dendritic cells, macrophages, neutrophils, mast cells and myeloid-derived suppressors, can act on tumor cells, modulating the growth, metastasis and response to chemotherapy [48, 52]. Next, we mainly elucidate the role of exosomes in CCA.

Cholangiocytes can be actively involved in the development of bile duct disease by stimulating the recruitment and activation of inflammatory cells in the bile duct microenvironment [58]. Katsumi et al. found that activated bile duct cells are involved in activating the proinflammatory polarization of damage-associated molecular patterns (DAMPs) through the receptor for advanced glycation end products (RAGE) signaling pathway by releasing DAMPs as EV cargo [59]. Prior to this, Masyyuk et al. verified exosomes in rat bile [60]. Meanwhile, in the process of studying the cilium interaction of bile duct cells, exosomes were found to induce intracellular signals and functional responses, verifying that bile exosomes participate in intercellular signal communication [60]. This laid a foundation for the influence of bile duct carcinoma exosomes on tumor progression.

In the study of the tumor microenvironment of CCA, Haga et al. exposed marrow mesenchymal stem cells (MSCs) to CCA cell-derived EVs, which enhanced expression of alpha-smooth muscle actin mRNA and release of cytokines/chemokines such as IL-6, thus regulating the tumor microenvironment and promoting the growth of CCA. In addition, CCA cell-derived EVs can contribute to the formation of the tumor stroma through the fibroblast differentiation of MSCs [61]. This further revealed the effect of tumor cell-derived EVs on the local microenvironment. However, they did not identify the specific contents of the EVs. Moreover, proteins in exosomes are essential agents for tumor growth [62, 63]. Another study demonstrated the protein spectrum of CCA-derived exosomes and their potential roles. These researchers isolated exosomes from CCA cell lines (KKU-M213 and KKU-100) and incubated them with normal bile duct cells (H69). After proteomics analysis, exosomes were found to be internalized into H69 cells, resulting in migration and invasion of H69 cells, but failing to induce proliferation. In addition, the exosomes of KKU-M213 cells induced the expression of β-catenin and decreased the expression of E-cadherin, suggesting that exosomes might induce the migration and invasion of bile duct cells through the direct transfer of oncogene proteins between cells, thus affecting the specific intracellular mechanism related to CCA carcinogenesis [64]. The proteomics analysis of normal bile duct cells and CCA cells confirmed the differences between these two cell types, and these differences need to be further studied. Moreover, exosomes from another CCA cell line (RBE) could inhibit the antitumor activity of cytokine-induced killer (CIK) cells by downregulating the populations of CD3⁺, CD8⁺, NK (CD56⁺) and CD3⁺CD56⁺ cells and secreting TNF-α and perforin [65]. According to some research, mutations in the IDH1 gene is common in a variety of tumors, including iCCA [66, 67]. Recent studies have shown that the R132C mutation is the most common type of IDH1 mutation in iCCA. The IDH1^R132C mutation leads to the downregulation of P2RX7 expression, which further affects the exosome secretion of tumor cells, ultimately affecting the progression of CCA [68]. However, more functional experiments on P2RX7 are needed.

In addition, noncoding RNA also play a crucial role in tumor development [69]. As a functional regulatory molecule, noncoding RNAs, such as microRNAs (miRNAs) and circular RNAs (circRNAs), could mediate cellular processes, including chromatin, transcription, posttranscriptional modification and signal transduction, and, of course, predict prognosis [69‐71].

With the development of technology, many exosome separation and purification techniques, including ultracentrifugation, ultrafiltration, immunoaffinity capture, size-exclusion chromatography, microfluidic techniques and charge neutralization-based polymer precipitation, have been exploded [89‐91]. Among them, the most common application is ultracentrifugation, which is the gold standard for exosome separation, even though it has some limitations, such as having a low efficiency and being time consuming [92‐94]. In the CCA research process, the EVs in the culture media of CCA were separated mainly by ultracentrifugation [61, 64, 65]. Similarly, EVs are extracted from the serum by ultracentrifugation, and polymer-based precipitation kit [95, 96]. However, there is a study that supports some commercial kits, such as ExoQuick and miR-CURY, as being better than ultracentrifugation, even with a limited quantity of EVs [92]. Bile is a lipid-rich fluid, that is secreted primarily by hepatocytes, and contains almost all body components: lipids, proteins, carbohydrates, vitamins, mineral salts and trace elements [96, 97]. Because of the characteristics and complexity of bile extraction, we mainly summarize the isolation of bile here. Bile samples of CCA are usually collected by endoscopic retrograde cholangiopancreatography (ERCP), percutaneous transhepatic biliary drainage (PTBD) and surgery, and then bile is immediately centrifuged at 4 ℃ to remove the cell debris and filtered through a 200 nm filter. Finally, the supernatant is collected and added to an ultracentrifuge tube, diluted with PBS for further purification, mainly including ultracentrifugation, PEG precipitation, membrane filtration and affinity purification [98‐100]. Some research, however, initially collected bile and then rapidly diluted with PBS for further centrifuge [42]. The isolation of EVs were used immediately or stored at − 80 °C until use (Fig. 3). However, research in this area is limited, and there is still much room for improvement in the separation and purification of exosomes.

MiRNA in EVs is protected the membrane from degrading enzymes, and this determines its highly stable characteristics in the extracellular environment, thus, miRNA is used for the diagnosis of a variety of cancers, including CCA [101, 102]. Li et al. identified and characterized EVs in human bile for the first time, and miRNA-laden EVs in human bile could be used for the diagnosis of CCA. They defined a new biliary vesicle miR-based panel (miR-191, miR-486-3p, miR-1274b, miR-16 and miR-484) for the diagnosis of CCA with a sensitivity of 67% and specificity of 96% [98]. This research initiates studying EVs in bile. Han and coworkers found that the expression levels of miR-30d-5p and miR-92a-3p in the bile of CCA patients were specifically upregulated compared with those in the bile of patients with benign biliary disease [103]. Compared with CEA and CA19-9, miR-30d-5p had the best effect in differentiating CCA and common bile duct disease, with a sensitivity of 81.1%, specificity of 60.5% and area under the curve (AUC) value of 0.730. In addition, the identification of CCA using the combination of the two bile miRNAs and serum CA19-9 levels was weaker than that of miR-30d-5p alone [103].

Long noncoding RNAs (lncRNAs) in bile also play the same role in CCA as miRNAs. Ge et al. conducted a series of experiments on bile exosomes, and showed that the expression of the lncRNAs ENST00000588480.1 and ENST00000517758.1 in the CCA group was significantly increased compared with that of the control group. The AUC of the combined detection of the two lncRNAs was 0.709, and the sensitivity and specificity were 82.9% and 58.9%, respectively [99]. Moreover, with the increase in the tumor TNM stage, the expression levels of ENST00000588480.1 and ENST00000517758.1 were significantly increased, and they could be potential diagnostic markers [99]. The content of EVs in bile can be used as a diagnostic marker, and the concentration of EVs also has potential diagnostic value. Severino et al. found that the accuracy of the bile EV count in the diagnosis of CCA was 100% with a threshold value of 9.46 × 10¹⁴ nanoparticles, with an AUC of 1, and this could correctly distinguish malignant and nonmalignant common bile duct stenoses in this research [42].

There are also some diagnostic markers of CCA in serum EVs. The new proteomic features found by Ander Arbelaiz in serum EVs of primary sclerosing cholangitis (PSC), CCA and HCC patients have potential diagnostic value [104]. For example, fibrinogen gamma chain (FIBG), alpha-1-acid glycoprotein 1 (A1AG1) and S100A8 (S10A8) proteins have the strongest differential diagnoses of CCA and PSC, with AUC values of 0.796, 0.794 and 0.759, respectively [104]. Similarly, using proteomic methods, Weeraphan et al. studied the exosomal phosphoproteome of M213 and M213D5 in CCA cells, and showed that Ser255 of HSP90B was highly phosphorylated in tissues of CCA patients with a low TNM stage (I and II) compared to those with a TNM stage of III or IV. ROC analysis showed that HSP90B-S255 was a new potential biomarker for metastatic CCA with an AUC value of 0.936 (sensitivity 87.27%, specificity 97.62%) [105]. Moreover, Shen et al. studied exosomal miRNA in peripheral blood samples from CCA patients and healthy controls. The results showed that the serum extracellular miR-200 family, especially miR-200c-3p, had the strongest diagnostic ability for CCA than that in serum CA19-9, with an AUC of 0.93, which is worth further study [82]. Meanwhile, positive of Annexin V, epithelial cell adhesion molecule (EpCAM), asialoglycoprotein receptor 1 (ASGPR1) and tumor-associated microparticles found in serum EVs were shown to have the potential to differentiate HCC and CCA from tumor-free individuals; after tumor resection, the number of these microparticles decreased, which proved a correlation with the presence of the tumor [95].

The studies above indicate that miRNAs, lncRNAs and proteins in blood and bile could be used as diagnostic indicators of CCA (summarized in Table 3). It can be concluded that although there are many substances in EVs, exosomes are the main diagnostic agents. Exosomes coated with lipid bilayers are more stable and more suitable as diagnostic markers [8]. The concentration of EVs in bile may increase as a result of bile flow disorder caused by bile duct stenosis or obstruction in CCA patients [106]. In addition, potential tumor-derived biomarkers may be secreted directly into bile by adjacent CCA cells, and local sampling may be more likely to detect candidate biomarkers directly related to the tumor [106]. Therefore, compared with circulating serum sampling, bile sampling can improve diagnostic performance [103]. However, the study of the EVs in CCA is still not complete. For example, the mechanism of EVs in bile duct cancer is not clear and how to efficiently extract and detect EVs in body fluids remains unclear.

Since the tumor microenvironment promotes the progression and invasion of CCA, targeting the microenvironment and related cells is a strategy for the treatment of CCA [110, 111]. Chen et al. found that RBE-derived exosomes could inhibit the antitumor activity of CIK cells [65]. This suggests that the effect of CIK cell-based immunotherapy is related to EVs, which may be a potential therapeutic target. In addition, circ-0000284 may be a therapeutic target for CCA. Wang et al. proved that the knockdown of exosomal circ-0000284 inhibited CCA growth and metastasis in vivo through animal experiments [86]. Besides, according to Zhang’s research, mutations in the IDH1 gene alter the function of IDH1 and affect the development of CCA by promoting exosome release [68]. Interestingly, IDH1 inhibitors have been reported. For example, the safety and clinical efficacy of mutant IDH1 inhibitor ivosidenib in the recurrence or refractory IDH1-mutated acute myeloid leukemia were demonstrated [112]. At present, inhibitors of IDH1 (AG120 and IDH305) are being tested in iCCA patients [113]. This provides a potential treatment of CCA. Furthermore, one study reported that inhibition of miR-205-5p in the exosomes of CCA could reduce the invasion and migration of CCA, and the miR-200 family was associated with drug resistance [73, 114].

EVs are natural membrane vesicles involved in intercellular communication; accumulating evidence has revealed that EVs have the characteristics of stability and low immune reactivity, and exosomes in EVs can effectively transport a variety of different types of cargo to target cells [109, 115]. As a result, EVs can also be selected as a therapeutic tool to modulate the function of CCA cells by delivering cargo media [35, 116]. Stromal derived EVs are suitable for the delivery of materials to CCA cells, and this property can be exploited for delivering antitumor therapy to CCA cells [117]. Li et al. proved that EVs could transport miR-195 from fibroblasts to cancer cells. In addition, fibroblasts-derived EVs loaded with miR-195, play a key role in the CCA rat model, by reducing the size of tumors and improving survival in the treated rats [77]. Moreover, after incubating the miR-30e-enriched EVs with CCA cells, Zhang found that the expression of miR-30e in receptor CCA cells increased, which ultimately regulated the invasion and migration of cells. This demonstrates that miR-30e-enriched EVs can be ingested by recipient cells as a means of transferring miR-30e [81]. A recent study showed that methotrexate-loaded tumor-cell-derived microvesicles were injected into the bile duct lumen of patients with extrahepatic CCA, which mobilized and activated neutrophils and alleviated biliary obstruction [118]. These studies suggest that EVs carrying designed cargo can be used as media carriers to manage the progression of CCA.