Exosome-mediated communication in the tumor microenvironment contributes to hepatocellular carcinoma development and progression

The tumor microenvironment (TME) is the cellular environment in which the tumor develops. Apart from the tumor cells, the TME includes various cell types, extracellular matrix (ECM), growth factors, proteolytic enzymes and their inhibitors, and signaling molecules [1, 2]. TME influences tumor growth, metastasis, and ultimately prognosis. Therefore, the fundamental role of TME is to dynamically interact with malignant cells [3]. The TME contributes significantly to the pathogenesis of hepatocellular carcinoma (HCC). Indeed, by offering, inhibiting, or stimulating growth signals, this TME is an essential modulator of HCC development and progression and a source for identifying targets for potential therapeutic agents [4].

The interactions of HCC cells with the surrounding TME are based on complex systemic networks. In addition to direct cell-to-cell contact, intercellular communication through secreted factors plays a key role in intercellular signaling. Among these secreted factors, exosomes are the major components of extracellular vesicles (EVs), which range in size from 30 to 150 nm. EVs originate from multivesicular bodies (MVBs) and are generated by all cell types [5, 6]. Upon early to late endosome maturation, biomolecules are endocytosed and transported into early endosomes. In late endosomes, intraluminal vesicles (ILVs) are formed by inward budding of the endosomal membrane and result in a large MVB. MVBs can fuse with the plasma membrane, and the ILVs released into the extracellular space are referred to as “exosomes” [7, 8]. However, studies on the genesis and release of exosomes have revealed that apart from the sorting of cargo molecules, the procedure is tightly associated with energy mediators, such as SNAREs, Rabs, and Ras GTPases [9]. Exosomes are generated in the form of endocytosis, exocytosis, protein transport, and protein sorting. During this process, exosomes are packed with lipids, proteins, DNA, mRNA, miRNA, and other ncRNAs [5, 10], which are horizontally transferred from donor to recipient cells. Exosomes can carry biomolecules from tissues to body fluids [11‐15]. These properties contribute to the role of exosomes in intercellular communication, i.e., shuttling of signaling molecules between nearby and remote cells [16‐18]. The surface of the exosome contains a large number of molecules related to antigen presentation. In vivo and in vitro, exosomes have similar effects as antigen-presenting cells, which can induce and enhance immune responses. Exosomes have widely dissimilar sizes and contents and are heterogeneous in biological effects and targeting specificities. Thus, exosomes have attracted attention as important vehicles for specific signals in tumor progression, metastasis, immune modulation, angiogenesis, and tissue regeneration [19].

In the liver, exosomes are secreted by three main cell types: liver epithelia (i.e., hepatocytes and cholangiocytes), immune cells (i.e., T and B cells, dendritic cells, and NK cells), and nonparenchymal liver cells (e.g., liver stellate cells) [20‐22]. Further evidence suggests a role for exosomes derived from different liver cells in the intracellular communication for the coordination of cell behaviors proper functioning. For example, exosomes derived from hepatocytes and cholangiocytes are important mediators of proliferation processes [20, 23]. T cell- and B cell-derived exosomes are involved in inflammation [24]. Exosomes derived from hepatic stellate cells (HSCs) may be involved in the pathogenesis of liver fibrosis [25]. Furthermore, primary hepatocyte-derived exosomes promote the activation of stellate cells, which in turn participate in liver disease progression [26]. Moreover, lipid-induced EVs derived from hepatocytes also cause an inflammatory macrophage phenotype [27]. Exosomes derived from the cells of other organs and tissues are involved in various types of liver disease [21]. For example, exosomes are involved in the progression of viral infections, including viral transmission, immune response, and antiviral effect [28, 29]; several studies have suggested that EVs increase with alcoholic hepatitis and show upregulation, even with excessive alcohol consumption [30, 31]. The role of exosomes in liver fibrosis by regulating connective tissue growth factor 2-dependent fibrogenesis in HSCs has also been reported [32].

However, what is the role of exosomes in HCC, and how is TME remolded by exosomes? With these considerations, we will focus here on the role of exosomes in setting up and modifying the HCC microenvironment that encourages tumor development and progression. We will discuss the specific mechanisms by which exosome-based communication regulates the immune response and benefits tumor survival, growth, angiogenesis, and metastasis (Fig. 1). We will also summarize the potential clinical applications of exosomes as therapeutic modalities.

Despite advances in prevention, screening, and novel diagnostic and therapeutic technologies, HCC therapy has encountered a bottleneck. The 5-year survival rate of patients with HCC is still < 20% [33, 34], which indicates that HCC remains a highly lethal disease. An emerging concept is that the TME plays an important role in initiating and maintaining carcinogenesis [35]. The TME is a varying environment that describes the behavior of cancer. The TME is the cellular environment that tumor cells need for survival, growth, proliferation, and metastasis [36]. It is a complex and heterogeneous system that is orchestrated by two major groups of cellular and noncellular elements.

The cellular elements in the TME, such as HSCs, fibroblasts (cancer-associated fibroblasts or CAFs), immune cells (T lymphocytes, B lymphocytes, NK cells, natural killer T cells, and tumor-associated macrophages or TAMs), and endothelial cells (ECs), play a critical role in tumor-stromal interactions that can modulate biological activities of HCC. A study has shown that activated HSCs infiltrate the stroma of HCC and are associated with tumor proliferation [37]. One of the mechanisms is that HSCs reduce HCC central necrosis [38]. CAFs are the best studied stromal cells and are defined as loose fibroblasts found within the tumor mass. CAFs maintain the stem cell-like properties of HCC cells by secreting IL-6 [39] and promote tumor invasion and metastasis by secreting matrix metalloproteinase-9 (MMP-9) [40]. Immune cells are commonly described to restrain tumor development; however, when they intimately interact with transformed cells in the TME, these cells can alternatively propagate tumor progression [41, 42]. For example, the deficiencies and malfunction of immune cells can introduce an immunosuppressive microenvironment for tumor cells to achieve immune tolerance and evasion. ECs in TME are important suppliers of nutrients, oxygen, and various growth factors for HCC development.

The noncellular elements include growth factors, such as transforming growth factor-β (TGF-β), insulin-like growth factor (IGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF). The remaining noncellular elements also include proteolytic enzymes, ECM, and inflammatory cytokines. These factors can provide a pliable environment that encourages further HCC growth and propagation. TGF-β is an essential component of liver disease by participating in responses to surroundings and acting as a continual target for tumor-derived signals [43]. IGFs are mainly synthesized in the liver and are functionally related to HCC proliferation [44, 45]. Another important growth factor, FGF, is characterized by multiple functions that can mediate cell proliferation, angiogenesis, wound healing, and tissue repair [46]. HGF in the TME can promote malignant cell growth and survival [47]. Abnormal ECM in the TME can play a critical role in promoting angiogenesis and inflammation [48]. Many inflammatory cytokines, such as IL-6 and IL-8, as well as MMP-2 and MMP-9, participate in regulating the inflammatory microenvironment and contribute to the increased migratory and invasive potential of hepatocytes, thus facilitating cancer metastasis [49, 50].

Together, these data suggest that TME has a determinate protumorigenic effect and works as a booster in HCC progression and metastasis. TME has been considered to modulate the survival and growth of cell lines by providing inhibitory or stimulatory signals [4]. However, how are signals transmitted to and received from the TME? In recent years, growing evidence indicates that exosomes are important vehicles of specific signals in physiological scenarios [5]. In addition, exosomes have been increasingly studied as novel intercellular communication mediators for tumor onset and progression in TME [19].

The immune microenvironment is shaped by intricate interactions between tumor cells and the host immune response. The evolution of cancer is strongly related to the tumor immune microenvironment. HCC shows a high degree of malignancy, and its poor overall survival outcome is due to the collapse of immune surveillance, which is closely associated with the suppression of host immune responses. Mounting evidence indicates that exosome-derived interactions are involved in the modulation of the microenvironment with immunosuppressive and tolerogenic characteristics. By regulating immune responses, tumors can evade immune destruction, and progression is ensured in this protumorigenic immune environment [51‐54]. Pioneering work by Wang et al. [55] demonstrated that T cells could swallow HCC-derived exosomes containing 14-3-3ζ. The expression of 14-3-3ζ was upregulated in HCC cells and in tumor-infiltrating T lymphocytes (TILs). This process was related to impaired activation (CD69 expression), proliferation (Ki67 expression), and antitumor functions. In addition, 14-3-3ζ overexpression inhibits the vitality and proliferation of peripheral blood CD3+ T cells, promoting the differentiation of naive T cells from effector T cells to regulatory T cells. These findings suggested that HCC-derived exosomes containing 14-3-3ζ could inhibit the antitumor functions of TILs in the HCC microenvironment. NKG2D is a C-type lectin-like activating receptor expressed on all NK cells. Evidence has shown that NKG2D binding to its ligands mediates immune system activation and plays an important role in cancer immune surveillance [56, 57]. In fact, exosomes secreted by liver tumor cells induce NKG2D downregulation on NK cell surfaces, which leads to impaired cytotoxic function and is beneficial for HCC immune escape and progression [58]. Rao et al. demonstrated that HCC-derived exosomes can trigger DC-mediated immune responses by serving as vectors for a variety of antigens and remold the HCC tumor immune microenvironment [59]. Within the TME, tumor-derived exosomes play a key role in the polarization status of macrophages. In this setting, macrophages are observed to shift to a protumorigenic macrophage phenotype (M2 polarization), leading to tumor angiogenesis and metastasis [60, 61]. Melatonin is a hormone with certain cytotoxic and immunomodulatory effects, and this hormone can inhibit the functions of tumors. A recent study found that after treatment of hepatocellular carcinoma cells with melatonin, HCC-derived exosomes can alter the immunosuppressive status through the STAT3 pathway in macrophages [62].

HCC-derived exosomes can modulate the phenotype and function of immunocytes that can be beneficial for tumor cells to escape immune destruction. In turn, exosomes secreted by immunocytes in TME can affect antitumor immune responses. Mast cells can be stimulated by hepatitis C virus E2 envelope glycoprotein (HCV-E2) to secrete a large number of exosomes rich in miR-490. These exosomes are transferred into HCC cells and inhibit the metastasis of tumor cells via inhibiting the ERK1/2 pathway [63]. Exosomes derived from TAMs indisputably play a key role in immune suppression, angiogenesis, and tumor progression. For example, human macrophages can transfer exosomes with miR-142 and miR-223 to liver cells and inhibit the proliferation or growth of tumor cells [64]. In conclusion, HCC is characterized by the ability to employ different strategies to evade host immune surveillance, and exosomes are important promoters to alternatively propagate tumor progression by immune regulation.

As exosome-based communication in the HCC microenvironment can function as a determinant factor to promote the malignant behavior of cancer cells, exosomes in the HCC microenvironment may provide an opportunity for targeted therapeutic intervention to eventually prevent HCC and prolong the duration of survival for HCC patients. Because exosome-mediated immune regulation in TME plays an important role in the occurrence and development of HCC, it is reasonable to expect that the exosome-based communication system is beneficial to stimulate the immune response and to mediate HCC immunotherapy. In addition, increasing interest is focused on the utility of exosomes as therapeutic tools for drug and biological molecule transfer.

TME is the cellular environment that malignant cells need for survival, growth, proliferation, and metastasis. Exosomes, as a novel intercellular communication mediator in the TME, have attracted attention for promoting tumor onset and progression. The present review summarized the role of exosomes in setting up and modifying the TME for the development and progression of HCC. Here, we revealed the specific mechanisms through which exosome-based communication within the HCC microenvironment promoted the malignant behavior of cancer cells. It is possible that exosome-mediated therapy in HCC patients could involve the multifaceted roles of exosomes in the TME. Future work is needed to illuminate the potential mechanisms by which exosomes have effector functions in the TME to provide an opportunity for targeted therapeutic intervention based on exosome communication systems.