Exosomes in the tumor microenvironment of cholangiocarcinoma: current status and future perspectives

Abstract

Cholangiocarcinoma (CCA) refers to an aggressive malignancy with a high fatality rate and poor prognosis. Globally, the morbidity of CCA is increasing for the past few decades, which has progressed into a disease that gravely endangers human health. Exosomes belong to a class of extracellular vesicles (EVs) with diameters ranging from 40 to 150 nm that can be discharged by all living cells. As communication messengers of the intercellular network, exosomes carry a diverse range of cargoes such as proteins, nucleic acids, lipids, and metabolic substances, which are capable of conveying biological information across different cell types to mediate various physiological activities or pathological changes. Increasing studies have demonstrated that exosomes in the tumor microenvironment participate in regulating tumorigenesis and progression via multiple approaches in the tumor microenvironment. Here, we reviewed the current research progress of exosomes in the context of cancer and particularly highlighted their functions in modulating the development of CCA. Furthermore, the potential values of exosomes as diagnostic and therapeutic targets in CCA were overviewed as well.

Background

CCA remains a highly lethal malignancy of the biliary system. It can be classified into three major groups based on their lesion locations: intrahepatic, perihilar, and distal CCA. Globally, the proportion of CCA is second only to hepatocellular carcinoma (HCC) among all primary liver tumors, accounting for roughly 15%, and accounts for 3% of all gastrointestinal malignant tumors [1, 2]. Despite advancements advances in CCA cognition, diagnosis, and treatment for the past few years, due to its high malignant degree, strong invasiveness, and the occult in initial, most patients are first diagnosed already in the late-stage that severely constrains therapeutic options. Although curative surgery is a preferred selection for some early-stage patients, the fact of tumor recurrence or metastasis after resection remains frustrating. Meanwhile, on account of the high heterogeneity of CCAs, the systemic therapeutics bring little effect for advanced patients who are not possible for radical surgery, leading to a very poor prognosis that only 7%-20% of patients can reach the five-year survival [3, 4]. Nevertheless, with the advancements in genetic profiling of CCAs, emerging treatments such as targeted or immunological therapeutics may be able to assist patients with this deadly cancer to get better outcomes. Considering the current situation of CCAs, exploring new diagnostic and therapeutic strategies remains a matter of priority.

EVs are enveloped by a lipid bilayer that can be discharged by numerous cell sorts. According to the size and formation pathway of vesicles, it can be classified into two major subsets approximately, called ectosomes and exosomes [5]. The former are vesicles with a diameter of 50 nm ~ 1 μm via plasma membrane budding outward directly, while exosomes are EVs ranging from 40 ~ 150 nm in diameter generated in the opposite way, which involves plasma membrane invagination and endosomal formation [5]. Exosomes contain multiple substances and are broadly distributed in different body fluids like plasma, urine, bile, and cerebrospinal fluid (CSF), which play important roles in a variety of normal or abnormal biological behaviors [6,7,8]. Recently, researches about cancer exosomes have received tremendous attention. Intercellular communication in the microenvironment plays a significant role in regulating tumor development, where exosomes are key messengers that mediate this cell-to-cell communication [5, 9]. Previous researches have illustrated that exosomes participate in tumorigenesis or metastasis in multiple ways, their potential usages in cancer diagnosis and prognosis have also been deeply explored [9]. Although certain studies have reviewed the roles of EVs in the progression of CCA [10], a more comprehensive summarization of exosomes in CCA remains insufficient up to now.

In this article, we systematically summarized the research status of exosomes in the tumor fields. Based on the existing researches of exosomes in CCA, we specifically emphasized their significant roles in regulating tumor development and potential values in diagnosis and treatment.

Research status of exosomes

Biogenesis, secretion and internalization

As a type of EVs, the synthesis progress of exosomes involves three major phases: 1) plasma membrane invagination and early endosomes formation. 2) intraluminal vesicles (ILVs) and intracellular multivesicular bodies (MVBs) generation. 3) the fusion of MVBs and plasma membrane leads to exosomes secretion [9]. Generally, the biogenesis of MVBs mainly depends on the following two pathways: endosomal sorting complexes required for transport (ESCRT)-dependent or ESCRT-independent mechanisms, and the former is the most classic pathway [6]. Once mature, MVBs can integrate with autophagosomes then degrade through the lysosomal pathway or secrete into extracellular space as exosomes by fusing with the plasma membrane [11]. In this biogenesis and secretion process, other components such as tumor susceptibility gene 101 (TSG101), Rab family of GTPases (like Rab27A and Rab27B), soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, apoptosis-linked gene 2-interacting protein X (Alix), ceramide, tetraspanins (CD63, CD9, CD81), and phospholipids are also getting involved [5, 12, 13].

Considering the difference in the origin and microenvironment, exosomes have a strong heterogeneity, which is mainly reflected in the regulation of the target cell functions [5]. Once exosomes are secreted by the host cells, they can be absorbed by target cells through various approaches like endocytosis, plasma membrane integration, and specific protein interactions [14]. Among these internalization ways, endocytosis is the most widely studied pattern. According to the characteristics of components involved in endocytosis, several subtypes are broadly divided up, including phagocytosis, macro-pinocytosis, clathrin-mediated endocytosis (CME), and caveolin-dependent endocytosis (CDE), as well as lipid raft-mediated internalization [15]. Moreover, several proteins such as tetraspanins, integrins, proteoglycans, and lectins, also participate in the internalization of exosomes by the unique ligand-receptor interactions[9]. However, on account of the heterogeneity of exosomes, whether or not exosomes uptake is specific remains controversial(15). Therefore, it is essential to further investigate the detailed routes of exosomes uptake (Fig. 1).

Fig. 1

Biogenesis, secretion, and internalization of exosomes. The formation of exosomes initially depends on the invagination of the plasma membrane, followed by the generation of ILVs and MVBs. Once mature, MVBs can fuse with lysosomes and be degraded, or integrate with the plasma membrane and finally get released, i.e., exosomes. During this process of synthesis and secretion, ESCRT-dependent and ESCRT-independent mechanisms are two common approaches, other components like the Rab family of GTPases, SNARE, ceramide, and tetraspanins are also involved. Exosomes can be uptake by receptor cells to perform specific functions through various mechanisms, such as phagocytosis, macro-pinocytosis, ligand-receptor interaction, CME, and CDE. As plasma membrane-derived vesicles with lipid bilayer structure, exosomes carry a variety of components, including RNAs (mRNA, MiRNA, LncRNA, and CircRNA), proteins (TSG101, Alix, HSP, CD9, CD63, and CD81) and metabolites, etc.

Isolation and identification

Currently, frequently-used isolation strategies include centrifugation (differential or density gradient centrifugation), particle size separation, size-exclusion chromatography, microfluidic technique, and immunoaffinity capture [16]. Until now, the most common method is still differential centrifugation due to its high exosome yields and relatively cheap price. However, it also has some deficiencies like complicated procedures, low separation efficiency, and susceptibility to contamination by soluble substances in cell culture medium or other body fluids [17]. Other isolation methods like size-exclusion chromatography, with a relatively high yield but difficult to achieve mass production, and immunoaffinity capture, advanced in specific separation yet costly with low yields [16, 17]. So far there is not a standardized method that can achieve both economic and high purity at the same time. Therefore, the exploration of better purification methods remains a major challenge in the exosome-related fields.

In terms of identification, the International Society of Extracellular Vesicles proposed to identify exosomes mainly from the following three aspects: 1) Exosomal morphology identification, 2) Exosomal size detection, 3) Exosomal biomarkers identification [18, 19]. Among them, transmission electron microscopy (TEM), cryo-electron microscopy (Cryo-EM), and atomic force microscopy (AFM) are the most direct methods for visual observation of exosomes [20]. Real-time nanoparticle tracking technology based on the principle of Brownian motion can be used to obtain the size distribution of exosomes [21]. In addition, enzyme-linked immunosorbent assay (ELISA), flow cytometry (FCM), and western blotting (WB) are available means to detect the specific proteins or other markers expressed on exosomes [20, 22]. Reportedly, several transmembrane proteins like CD9, CD63, and CD81 are considered to be representative hallmarks, however, a recent study suggested that compared with other tetraspanins, CD63 is the unique biomarker, while CD9 and CD81 are not specific for exosomes [23]. Moreover, other components related to the formation of exosomes such as Alix, TSG101, and Heat shock proteins (HSP) can also serve as classical hallmarks [5].

The roles of exosomes in malignancies

Since exosomes have played an essential role in multiple pathological changes through mediating intercellular communication, it has also received enormous concerns in the cancer area over the past few years [9]. Related studies have pointed out that cancer-cell-derived exosomes can modulate tumor progression through a variety of mechanisms [9, 24]. Besides, as mentioned above, exosomes contain complex cargoes that are widespread in various body fluids, which also partly represent the heterogeneity of their parental cells, making them available for cancer diagnosis and prognostic by serving as novel biomarkers [25]. Moreover, recent studies have focused more on the tumor microenvironment (TME), where the signal interaction mediated by exosomes also makes a difference in tumor development [5, 26].

Exosomes induce or accelerate tumorigenesis. Exosomes secreted by HCC cancer cells promoted tumorigenesis through the Hedgehog pathway [27]. Mirna-224-5p-enriched exosomes secreted by non-small cell lung cancer (NSCLC) cells accelerated neoplasia by directly binding with the androgen receptor (AR) [28]. On the contrary, exosomes distributed in the plasma of patients with medulloblastoma inhibited tumorigenesis by targeting FOXP4 (forkhead box protein 4) and EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) directly through their miRNA cargoes [

Availability of data and materials

Not applicable.