The significance of exosomes in the development and treatment of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third most common malignancy in the world, accounting for 85–90% of primary liver cancer [1‐3]. The annual incidence of new cases with HCC is estimated to be about 841,000 [4]. Hepatitis B virus (HBV) infection is considered as the main risk factor for HCC development in China [5]. Despite new breakthroughs in imaging technology, chemotherapy, interventional radiology, surgical techniques, and liver transplantation in the recent years, the prognostic rate of patients with advanced liver cancer still remained poor, and there is no effective treatment method till date for it [6‐8]. Currently, the 5-year survival rate of HCC is not more than 20% [9, 10] and early diagnosis can significantly reduce the mortality in patients with HCC [11]. Therefore, the major methods to improve the total survival rate of HCC include improvementof the early diagnostic rate and explore the detailed formation mechanisms of HCC.

Exosomes are small nano-sized vesicles that transport biologically active molecules between cells, regulate microenvironment and immune system between cells by a variety of biomolecules (such as proteins, RNA and DNA) [4, 12, 13], and the genetic and epigenetic mechanisms of cells [14]. Studies have shown that exosomes not only initiates the downstream signals to target cells, but also transfers the genetic material to downstream cells, providing intercellular communication with a new mechanism [4]. It has been reported that different types of exosomal compositions provided by databases such as Vesiclepedia, EVpedia, and Exocarta were found in different cells with similar physiological or pathological conditions [15‐17]. In addition, exosomes are also involved in angiogenesis, metastasis of tumor cells and transformation of normal cells into tumor cells [18]. Many types of cells such as mesenchymal cells, immune cells, and tumor cells may induce the release of exosomes, and increase in these cells indicate that exosomes are involved in tumorigenesis, development, metastasis, immune escape, and drug resistance [19]. Moreover, tumor-derived exosomes contain a large number of cancer-related serological markers, such as miRNAs, which could be used for detecting early HCC [20]. Despite several breakthroughs in the field of exosomes, the concrete biological role of exosomes has not been fully figured out. In this review, the source, structure, isolation of exosomes, and their impact and clinical application in HCC were described.

Exosomes were first discovered by Johnstone RM et al. in 1983 in mature sheep reticulocytes and named them as exosomes. They were initially perceived as cellular “debris” [21]. In 1996, Raposo et al. have confirmed the significant role of exosomes in antigen presentation from B cells, causing T cells responses [22]. So, the exosomes have gradually drawn the attention of several researchers, and is now considered to play an important role in the diagnosis and therapy of tumors. Exosomes are small phospholipid bilayer membrane nanovesicles that are formed by segregation of intracellular poly vesicles with cell membranes, followed by releasing into the extracellular space [23]. The detailed formation of exosomes is presented as follows. To begin with, the inward budding of cell membrane confirms the early endosomal stage [24], followed by the generation of multivesicular bodies (MVBs) by further inward budding of early endosomes and several miRNAs, proteins and other selected substances [25]. Finally, the MVBs either fuse with cell membrane, leading to the inclusion of extracellular DNA [26, 27], or fuse with lysosome, inducing the degradation of biological information containers in MVBs [28]. The endosomal sorting complex required for transport (ESCRT) mainly guides special molecules into the exosomes of MVBs, and is regarded as an important mechanism of synthesis [29, 30]. The ESCRT mainly contains4 core ingredients (ESCRT 0, I, II, and III), wherein the primary function is to provide ubiquitinated proteins to induce lysosomal degradation and protein reusing [31]. In the above process, ALG2-interacting protein X (ALIX), an accessory protein, plays an important role in interacting with ESCRT-III subunit SNF7, and then combines with MVB [32]. There are other mechanisms discovered by the researchers and are considered as ESCRT-independent, as they play a big role in this procedure [24]. Due to less understanding onESCRT-independent mechanisms, a great effort should be made to outline the detailed role of ESCRT-independent mechanism. The concrete mechanism of the formation of exosomes is clearly presented in Fig. 1.

Exosomes can be synthesized by any cells, such as B lymphocytes, T cells, mast cells, dendritic cells (DC), etc. and might be secreted to enter into other cells to carry out their function [34, 35]. Exosomes contain biologically active substances including proteins, RNA, DNA, cholesterol, ngosine, and so on, which range in size from 50 to 140 nm [36‐38]. The density of exosomes is about 1.13~1.19 g/ml [39]. Exosomes are abundant in human body, and can be found in biological fluids including urine, tears, plasma, breast milk, and cell culture supernatants [40‐42]. Emerging evidence indicates that tumors during growth processing can secret exosomes. For example, Yang L illustrated a new function of long noncoding ribonucleic acid (lncRNA HOTAIR) that induces MVBs for transporting them to plasma membrane, further propelling the release of exosomes from HCC [43].

The isolation of exosomes mainly involves5 methods, which include differential ultracentrifugation [44, 45], polyethylene glycol (PEG) precipitation [46, 47], sucrose and iodixanol density ultracentrifugation [38], immunoaffinity (IAC) capture [48], and size exclusion chromatography [49, 50]. In this review, we mainly introduced differential ultracentrifugation, as it is considered as gold standard for isolating exosomes [51]. Firstly, centrifugation at 300 g for 10 min is used to remove cellular debris. Secondly, the microvesicles were removed by centrifugation at 10,000 g for 30 min. Thirdly, ultracentrifugation is performed at 1,00,000 g, for 90 min at 4 °C. Fourthly, the supernatant is discarded, followed by the addition of exosomal pellet to 1× PBS, and then washing it by ultracentrifugation at 1, 00,000 g for 90 min at 4 °C. Finally, the exosomes are resuspended in 500 μl 1 × PBS and stored at - 80 °C for further use [52]. But all these methods have their own limitations, and so further research of a more efficient method for isolating exosomes is needed.

Several normal liver cells (such as hepatocytes,stellate cells and immune cells) can secrete exosomes, and meanwhile, these extracellular vesicles mediate normal communication between liver cells, maintaining liver homeostasis [53]. According to Liu WH et al., the amount of serous exosomes in the cirrhotic stage, early HCC stage and late HCC stage is significantly higher than normal liver,liver degeneration and liver fibrosis, indicating that exosomes have high capability in the early detection of HCC [54]. Emerging evidence indicates that exosomes participate in the spread, immune regulation and antiviral response when the cells are infected by the viruses [55]. In china, the main reason for the cause of HCC is CHB, and recent study by Kouwaki T et al. demonstrated that the infected virus in HCC induces exosomal miRNA-21 and miRNA-29, inhibiting macrophages and dendritic cells and releasing IL-12. It is widely accepted that IL-12 activates natural killer (NK) cells, thereby undermining the innate immune responses [56]. This may in turn cause chronic infection of hepatitis B (Fig. 2a), and long history of CHB may bring the effect of Hepatic fibrosis. It is evident that the hepatic stellate cells (HSCs) transdifferentiates into myofibroblasts in response to certain stimuli, and this is associated with the pathogenesis of hepatic fibrosis and the development of HCC [57]. According to the research proposed by Angeles Duran et al.,p62/SQSTM1, a negative regulator of liver inflammation and fibrosis, promotes Vitamin D receptor (VDR) signaling activation in HSCs, and mediates retinoid X receptor,which in turn promotes heterodimerization during the inhibition of liver inflammation and fibrosis [58]. The above processes make a great difference in recruiting the target gene. Also, loss of p62 expression in HSCs increases myofibroblastic differentiation, while suppresses fibrosis and inflammation via VDR agonists in chemically-induced murine fibrosis and tumor models. Carcinoma-associated fibroblasts (CAFs) are most frequently observed in human carcinomas, and involve alpha-smooth muscle actin-positive myofibroblasts and actin-negative fibroblasts that promote the growth and progression of tumor [59]. Although the critical role of p62/SQSTM1 and CAFs in liver inflammation, fibrosis and tumor progressionhas been discovered previously, research on p62/SQSTM and CAFs in exosomes is still lacking. It is widely accepted that exosomes are used as carriers to block several signal transduction pathways [60], and hence much attention has been paid on the relationship between p62/SQSTM, CAFs and exosomes in order to explore whether they could prevent liver inflammation, fibrosis and tumor progressionthrough blocking signal transduction pathways. Chen L et al. have discovered that the exosomes can utilize miR-214 to interplay cellular shuttling in order to regulate the formation of connective tissue growth factor 2 (CCN2) [61]. Overexpression of CCN2 by exosomes in HSCs is found in the activation process of liver fibrosis [62, 63] (Fig. 2b). After several years, hepatic fibrosis proliferates into HCC. Emergingevidence proves that exosomes play a bigger role in this stage. The lipid components of exosomes not only participate in a variety of HCC biological processes, but also protect tumor-derived exosomes from enzymatic degradation [64]. The abundant protein Rab GTPase, annexin and similar exosomes could be used for HCC membrane transportation and fusion [65, 66]. Heat shock proteins such as HSP70, HSP60 and HSP90 could be used for early diagnosis and treatment of liver cancer [67]. Exosomal proteomics indicate that exosomal integrin αvβ5 showed close association with liver metastasis [68] (Fig. 2c). Tumor susceptibility gene 101 protein (TSG101), major histocompatibility complex (MHC) molecules and the ESCRT-III binding protein ALIX might be used as biomarkers for diagnosis [69, 70]. In addition to this, many nucleic acids, including messenger RNA (mRNA), microRNAs (miRNAs), long noncoding ribonucleic acid (LncRNA) and DNA, regulate cell genetics as well as epigenetics [71]. The process of CHB to HCC is displayed vividly in Fig. 2.

Emerging evidence indicates that exosomes play a significant role in the formation and metastasis of HCC and the substances in exosomes are also used for clinical diagnosis and treatment. There are many types of liver cells, and so the intercellular communication is indispensable for liver cells to regulate and associate with each other [72]. Exosomes are novel research hot spots that play a critical role in intercellular communication [73]. In the next part, a thorough introduction on how the exosomes exert their role in HCC and how clinical exosomes are used as diagnostic and therapeutic tools is discussed.

Due to spatial heterogeneity and temporal heterogeneity of tumor, traditional method of tissue specimen biopsy cannot obtain the full and dynamic information of tumor tissues. In recent years, a novel diagnostic technology named “liquid biopsy” has been emerged to overcome the shortcomings of traditional tissue biopsy [103, 104]. In the protection of phospholipid bilayer membrane, the exosomal substances cannot be degraded by any enzyme, and so exosomes are considered as an appropriate diagnostic tool.

As the exosomes have many unique substances that can be expressed by tumor cells, we utilized them for the early detection of tumor. miRNAs are a class of conserved RNAs in the evolutionary history of humans and also participate in the development of liver cancer. According to the latest research, miRNA is considered as a potentially new and ideal marker [105]. Won Sohn et al. found that the levels of miR-18a, miR-221, miR-222 and miR-224 in exosomes of patients with HCC were lower than those in patients with HBV, indicating that these 3 miRNAs could be used as novel serum markers for detecting HCC [106]. Apart from miRNAs, lncRNAs are also used as biomarkers for clinical diagnosis of HCC [107]. Xiang Ma et al. discovered in an experiment that exosomes mediate the regulation of lncRNAs X-inactive-specific transcript in the expression of blood cells, and indicate that Xist expressed by mononuclear cells and granulocytes might act as valuable biomarkers in the diagnosis of female HCC patients [108].

Another study also reported that miR-30d, miR-140 and miR-29b showed significance in the survival of patients with liver cancer. Therefore, these exosomal miRNAs act as prognostic biomarkers for liver cancer and guide in the treatment of advanced liver cancer [109]. For proving the relationship of circulating exosomal noncoding RNAs (ncRNAs) in tumors, Lee YRet al. have conducted a series of experiments, which eventually showesa relationship of ncRNAs (miRNA-21 and lncRNA-ATB) with TNM staging and prognosis of HCC [110]. Xu Het al. indicates that serum exosomallncRNAs ENSG00000258332.1 and LINC00635 combined with serum AFP might be a promising method for diagnosis and prognosis of HCC [111]. Other experiment also supported that exosomal lncRNA acts as a prognostic factor in HCC, and lncRNA (LINC00161) significantly upregulates in HCC patients when compared to normal patients, which is well stabilized and specific [112]. In addition to lncRNA, circPTGR1, a circRNA, is particularly expressed in exosomes of 97 L and LM3 cells, and is increased in the serum exosomes of HCC patients. Wang G and his collaborator indicated that it could be used for clinical staging and prognosis [113].

HCC is not a sensitive disease to common chemotherapy. miR-122 could promote the sensitivity of HCC cells to chemotherapeutic drugs, and exosomes can be used as a biological carrier of miRNAs. These findings indicates that exosomes from self-body cells have less immunogenicity when compared with other vehicles [114]. Emerging evidenceshows the feasibility of applying exosomes as nanocarriers, for example, low immunogenicity, high biocompatibility, less toxicity and so on [115, 116]. At the same time, MSCs could secrete large amounts of exosomes. Researchers have found that transfection of miRNA-122 into adipose derived mesenchymal stem cells formed by MSCs could produce exocrine bodies containing miRNA-122, improving the miR-122-target gene expression and promoting sensitivity of cancer cells to chemotherapy [117]. Fang Wang and other researchers have found that stellate cell-derived EVs could load miR-335-5p. What makes us exhilarated is that miR-335-5 could be introduced into HCC cells to inhibit tumor growth and metastasis, which thereby provides a new treatment strategy for liver cancer [118]. Another research by Kenji Takahashiet al. have found that during the process of mediating chemotherapeutic stress response, RNAi-mediated knockdown of EVs (exosomes) lncRNAs could reduce the function and progression of tumor cells in HCC, which promotes the treatment of HCC [119].

Exosomes are involved in the occurrence, development, and metastasis of tumors, providing new clues for the treatment of HCC. We also found that many substances in exosomes including miRNAs serve as new biomarkers. It is also of great significance in improving the early diagnosis of HCC. Therefore, exosomes have become a hot research topic currently. This review has first introduced the role of exosomes in the development of CHB to HCC. Secondly, the mechanism of exosomes in tumor growth and metastasis is also discussed. The last but not the least, it is elucidated that exosomes could be used for clinical diagnosis and treatment. The function of the substance in HCC exosomes are conclued (Table 1). Although several studies have been put forwarded in investigating the relationship of exosomes and liver cancer, research on the formation mechanism of liver cancer by exosomes is still not deep enough. This is because of effective separation and specific detection of circulating exosomes in cancer cells [24, 125]. Meanwhile, the use of exosomes in studying the 4 serum markers of liver cancer (AFP, AFP-L3, GP73, and GPC3) has rarely been reported. Different researchers have drawn different views regarding the same exosomes. The major reason for this might be due to individual differences. So, environment, aging, gender, reason for HCC, and multi-center should be combined to produce more accurate results. For over the past several years, exosomes are used in immune therapeutic method only in 3 phase I clinical trials [126]. With more research conducted on exosomes, it is believed that exosomes could be successfully used in clinical diagnosis of early stage HCC in near future.