The formation of exosomes is a complex process, and numerous studies have described it in detail. The term exosome describes 40-100 nm-diameter vesicles that contain complex RNA and proteins. Exosomes form via the following axis: endosome-multivesicular body (MVB)-intraluminal vesicle (ILV). When MVBs fuse with the cell membrane, exosomes are released from parental cells (11). The formation of ILVs is the main process by which cargo, including proteins, lipids and nucleic acids, is loaded into vesicles. The endosomal sorting complex required for transport (ESCRT) machinery serves a critical role in the sorting of proteins into exosomes in parental cells (19). The RNA cargo of exosomes is enriched in small RNAs, especially microRNAs (miRNAs/miRs) (20). There are some other essential mechanisms of miRNA cargo loading in addition to the ESCRT machinery. Neutral sphingomyelinase 2 is the rate-limiting enzyme in the synthesis of ceramides, which could influence the loading of miRNAs into exosomes (21). Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of conserved nuclear proteins that bind to nascent RNA polymerase II transcripts to produce hnRNP granules. Several hnRNPs, especially hnRNP A2B1 and hnRNP Q, are implicated in miRNA packaging into exosomes (22). hnRNPA2B1 can recognize and bind to specific motifs in the 3′ untranslated regions (3′UTRs) of miRNAs and then transport miRNAs into exosomes (23).

The intracellular movement of MVBs involves the microtubule network, and localization of MVBs to the plasma membrane requires kinesin-dependent movement toward microtubule plus ends (24). The release of exosomes depends on the forward motion of MVBs to fuse with the plasma membrane (25). Rab GTPases, a subfamily of proteins in the Ras superfamily of GTPases (26), and soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor proteins can interact to induce exosome release (27).

Previous studies indicate that there are three main mechanisms underlying exosome uptake by recipient cells: i) Direct interaction (28); ii) fusion with the plasma membrane (29); and iii) internalization (30). In immune cells, major histocompatibility complex (MHC)-T cell receptor interactions can also facilitate the uptake of mutual exosomes (28). Once exosomes enter recipient cells, they trigger a series of biological effects through multiple pathways, including Erk1/2, Jak/STAT, NF-κB and PI3K/Akt signaling pathways (31).

The mechanisms of exosome formation are briefly summarized in Fig. 1. Notably, some molecules encoded by EBV can participate in the loading processes. In B cell-derived exosomes, the 3′ ends of miRNAs are uridylated, and miRNAs from the parental cells share adenylated 3′ ends, suggesting that 3′ end modification of miRNAs may be another mechanism for sorting miRNAs into exosomes (32). Nkosi et al (33) revealed that the viral protein latent membrane protein 1 (LMP1) can interact with the ESCRT pathway and associated proteins, including CD63, Syntenin-1, programmed cell death 6 interacting protein, tumor susceptibility 101, human growth factor-regulated tyrosine kinase substrate (Hrs) and charged multivesicular body proteins (CHMPs). Moreover, the study demonstrated that the LMP1-interacting proteins Hrs and Syntenin-1 serve major roles in directing LMP1 into EVs for packaging and secretion (33).

The TME is a complex interactome between tumor cells, adjacent cells (including adipocytes, fibroblasts, lymphocytes and dendritic cells) and the intercellular matrix. Cancer progression and metastasis are closely related to alterations in the TME; in particular, the characteristics of tumors, such as sustained proliferation, avoidance of immune surveillance, and activation of invasion and metastatic cascades, are influenced by the TME (34). In turn, cancer cells synthesize and secrete biomolecules to reprogram the surrounding cells and remodel the TME to be suitable for survival (35). The TME modulates numerous types of cell-cell communication through diverse signaling networks, including juxtacrine and paracrine interactions. Regarding paracrine signaling interactions, exosomes are an important and emerging mechanism of cell-cell communication (36). The TME also regulates the secretion of exosomes (37), and interactions of exosomes with the TME benefit the growth of the tumor. For example, exosomes derived from leukemia cells have been shown to accelerate cancer-associated fibroblast activation to remodel the TME to a more cancer-permissive state (38). Stress conditions, such as extracellular acidity and hypoxia, are common in the TME. On the one hand, the accumulation of lactic acid or H⁺ ions is a common characteristic of the TME, but TME acidity increases the release of tumor-derived exosomes (TEXs) (39). On the other hand, under hypoxic stress, tumor cells remodel the TME and facilitate angiogenesis by inducing the secretion of exosome-containing proteins associated with vascular endothelial growth factor (VEGF) signaling (40). Hypoxia can enhance miR-23a loading into lung cancer-derived exosomes. Endothelial cells take up exosomal miR-23a, which targets prolyl hydroxylase 1/2, resulting in the induction of hypoxia-inducible factor-1α (HIF1α) accumulation. Through this pathway, lung cancer cells remodel the TME and enhance tumor angiogenesis (41). Moreover, hypoxia-mediated enhancement of TEX release has also been observed in breast (42), bladder (43), prostate (44) and ovarian cancer (45) through multiple pathways, such as TGF-β2, TNF1α, IL-6, tumor susceptibility 101, Akt, integrin linked kinase 1 and β-catenin pathways.

Once EBV infection occurs, the viral genome is disassembled and integrated into the host genome. Thus, even in latency, EBV⁺ B cells could persistently express viral proteins and nucleic acids (64). LMP1 is a major oncoprotein of EBV, and numerous studies have demonstrated that it can be loaded on the membrane of exosomes (14,33,65,66). LMP1 also exists in exosomes derived from LMP1-transfected DG75 cells (a Burkitt lymphoma cell line). DG75 exosomes can be taken up by isolated B cells within the peripheral blood mononuclear cell population, which leads to enhanced proliferation and induces B cell differentiation toward a plasmablast-like phenotype via induction of activation-induced cytidine deaminase and the production of circle and germline transcripts for IgG1 in B cells (62). In vitro, after infection of B cells, LMP1 induces immortalization and aberrant proliferation, leading to the development of lymphoblastoid cell lines (LCLs) (67). The N-terminus and transmembrane domain 1 are sufficient for targeting LMP1 to extracellular vesicles (EVs) (68). Kobayashi et al (14) reported that ubiquitin C-terminal hydrolase-L1 and C-terminal farnesylation, a post-translational lipid modification, contribute to the direction of LMP1 to exosomes. Moreover, Rialland et al (69) found that the B cell receptor can modulate the protein content of exosomes upon stimulation and target its bound antigen to these vesicles. On the other hand, exosomes derived from LCLs carry an EBV glycoprotein, gp350, and preferentially target B cells via the interaction of this glycoprotein with its ligand, CD21 (70). In addition, these exosomes contain high levels of MHC-II molecules, which induce homogeneous antigen-specific T cell responses (66). The transmembrane freedom of exosomes is important for cellular interactions and this property might become the basis of exosome-targeted therapy.

Exosomes released from EBV⁺ B cells are internalized by recipient cells primarily via caveolin-dependent endocytosis (71). When exosomes derived from EBV⁺ B cells enter the TME, they can create an immunosuppressive microenvironment that affects T cell immune responses to ensure the proliferation of tumor cells. Cells with latent EBV infection can continuously produce EBV-encoded RNAs (EBERs), which elicit proinflammatory responses after sensing by pathogen recognition receptors (72,73). Lupus antigen (La) is an abundant RNA binding protein in the nucleus of latently infected B cells that binds nascent viral Pol III transcripts, protecting the 3′ ends from degradation by exonucleases (74). EBERs are transported from the nucleus to the cytoplasm and shed in exosomes by binding to La (16). Based on these studies, Baglio et al (75) proposed that by interacting with La and loading into exosomes, EBV nuclear 5′pppEBER1 escapes cytosolic detection in cells with established latent infection. Then, the viral cargo loads are internalized by plasmacytoid dendritic cells (pDCs), triggering antiviral immunity through exosomes. Another study showed that exosomes secreted from P3HR1 cells (an EBV⁺ Burkitt lymphoma B cell line) can increase the production of indoleamine 2,3-dioxygenase (IDO), TNF-α and interleukin (IL)-6 in human monocyte-derived macrophages (MDMs) via the retinoic acid-inducible gene I pathway. Moreover, EBER-1-activated IDO in MDMs suppresses the proliferation of T lymphocytes and diminishes the cytolytic activity of CD8⁺ T cells (76). Thus, EBER1⁺ exosomes derived from EBV⁺ B cells might promote tumorigenesis by inhibiting cellular immunity in the TME.

EBV-miRNAs are loaded into exosomes to induce a series of downstream effects (77). Higuchi et al (78) showed that exosomes derived from EBV⁺ lymphoma cells can regulate the activity of macrophages and induce the immunoregulatory phenotype in vitro. In this process, the expression levels of TNF-α, IL-10 and ARG1 are partially regulated by EBV-BamHI A rightward transcripts (BART)-miRNAs. Ito et al (63) observed that a phosphatidylserine-exposing subset of EVs secreted from lymphoma cells transformed with the EBV strain Akata converted surrounding phagocytes into tumor-associated macrophages (TAMs) by inducing an inflammatory response partially mediated by EBV-miRNAs. Using mass spectrometric analysis, the study indicated that several immunomodulatory proteins, especially integrin αLβ2 and fibroblast growth factor 2 (FGF2), are key factors in the TAM-inducing ability of EVs. Moreover, in the clinic, higher loads of BART miRNAs correlate with worse outcomes in elderly patients with EBV⁺ DLBCL. Furthermore, EBV-BART-miRNAs might be the link between EBV⁺ B cells and uninfected T cells or NK cells. Haneklaus et al (79) showed that exosomal EBV-miR-BART15 released from EBV⁺ B cells can enter uninfected T cells, targeting the miR-223 binding site in the NLR family pyrin domain containing 3 3′UTR to inhibit inflammasome-mediated IL-1β production, which was consistent with previous interpretations (80,81). T cells can be suppressed by LMP1⁺ exosomes (66); thus, EBV⁺ exosomes display immunosuppressive effects. Extranodal NK/T cell lymphoma, nasal type (ENKTCL) is a rare EBV-associated non-Hodgkin lymphoma (82). However, the mechanism of EBV entry into NK cells remains unknown. Lee et al (83) reported that EBV mRNAs and CD21 RNA can be transferred into NK cells from B cells by exosomes. However, this transfer is not sufficient to maintain EBV persistence or allow EBV entry into NK cells. Therefore, whether EBV genomic components can affect NK/T cells via exosomes requires further investigation.

These studies indicate that exosomes derived from EBV⁺ B cells can affect uninfected cells, and highlight the immunomodulatory function and oncogenic effect of exosomes. EBV hijacks host cells to build an immunosuppressive TME by secreting exosomes. Moreover, Ahmed et al (84) reported that exosomes derived from cells with both type I and III latent EBV infection induce apoptosis in B cells, T cells and epithelial cells via the Fas cell surface death receptor (Fas)/Fas ligand (FasL) pathway. As the majority of studies of exosomes derived from EBV⁺ B cells have been conducted in vitro, further studies, especially in vivo studies, are needed to clarify the complete roles of exosomes.

NPC is a malignant tumor derived from nasopharyngeal epithelial cells that has a high incidence in southern China, Mediterranean Africa and some regions of the Middle East (85). The role of EBV in NPC has been studied for decades. Virtually all NPCs are EBV⁺ (64); however, the complete mechanisms of EBV in the development and progression of NPC remain unclear. Viral proteins or nucleic acids might be involved in these processes. In recent years, researchers have focused on the roles of exosomes, and these studies are summarized to highlight the role of EBV in NPC.

Although the oncogenicity of LMP1 is known, there are no effective strategies to target this oncoprotein. With further studies, researchers have begun to focus on EBV⁺ exosomes. In 2006, Keryer-Bibens et al (86) first demonstrated that NPC cells can release HLA class II⁺ exosomes containing galectin 9 and/or LMP1. Galectin-9 is a ligand of the membrane receptor T cell immunoglobulin and mucin domain-containing protein 3, which is able to induce apoptosis in mature Th1 lymphocytes, weakening immunological surveillance (87). A previous study demonstrated that LMP1 can target the epidermal growth factor receptor (EGFR), inducing cell proliferation (88). Moreover, LMP1 increases the loading of EGFR into exosomes secreted by NPC cells, and exosomes containing LMP1 and EGFR are taken up by epithelial cells, endothelial cells and fibroblasts, which activates the ERK and PI3K/Akt pathways to affect cell proliferation (89). On the other hand, HIF1α regulates numerous key aspects of tumor development and progression by promoting increases in proliferation, invasiveness and neoangiogenesis (90). Aga et al (91) demonstrated that LMP1⁺ exosomes secreted by NPC cell lines contain HIF1α. Furthermore, LMP1⁺ exosomes and HIF1α can counterbalance the levels of E-cadherin and N-cadherin in recipient cells, consistent with EMT-associated changes. FGF-2, a member of the FGF family, is active in embryogenesis and morphogenesis, and serves a key role as an angiogenic factor involved in tumor progression and invasion (92). Ceccarelli et al (93) demonstrated that FGF-2 is packaged into exosomes and LMP1 selectively promotes this secretory process. LMP1 can interact with nucleic acids in addition to human proteins. For instance, miR-203 functions as a tumor suppressor in NPC and can be downregulated by LMP1 (94). A further study demonstrated that aspirin can reverse EMT and exosomal LMP1 might serve a pivotal role in this process. Exosomal LMP1 exhibited potential EMT-inducing ability, aspirin suppressed exosomal LMP1 secretion from EBV⁺ cells by influencing NF-κB, and a decrease in exosomal LMP1 uptake alleviated the inhibition of miR-203 by LMP1. The aforementioned results were further confirmed in an in vivo study; thus, aspirin could inhibit NPC lung metastasis by reversing the LMP1/NF-kB/exosomal LMP1/miR-203 axis in nude mice (17).

Weakening or evading the immune surveillance function of lymphocytes is beneficial to the proliferation of tumor cells. Mrizak et al (95) showed that NPC cell-derived exosomes (NPC-Exos) express CCL20 on their surface, which prioritizes the infiltration of regulatory T cells (Tregs) into the tumor. In addition, NPC-Exos recruit CD4⁺ T cells and induce their conversion into suppressive Tregs. Under induction by NPC-Exos, Tregs change their phenotype and their immunosuppressive ability is enhanced. These findings show that EBV⁺ exosomes can exert immunosuppressive effects not only by affecting pDCs, MDMs and CD8⁺ T cells, but also by recruiting Tregs.

EBV-BART-miRNAs have been found in exosomes secreted from infected epithelial cells, and these miRNAs can affect mitochondrial respiration in exosome recipient cells to modify the TME to support the growth of infected cells, thereby contributing to viral fitness (96). In another report, Meckes et al (89) showed that EBV-BART-miRNAs (including miR-BART-1, 4, 5, 7, 9, 11, 12, 13, and 16) are also selectively loaded into exosomes secreted from cultured EBV⁺ NPC cells. These viral miRNAs have been found to have biological functions and to be involved in molecular mechanisms associated with tumor immune evasion, proliferation, apoptosis resistance, invasion and metastasis (97). EBV-miR-BART3 can target the tumor suppressor integrator complex subunit 6 to promote cell proliferation and transformation in NPC (98). In a nude mouse model, EBV-BART3-3p was found to directly target the tumor suppressor gene tumor protein 53, which led to inhibition of senescence in GC cells, suppression of NK cells and macrophage infiltration into tumors (99), which are post-transcriptional regulation processes independent of genetic mutations. However, few studies have addressed whether EBV-miRNAs can mediate these changes through exosomes, and the specific mechanism remains unclear. Tumor occurrence results from the accumulation of a number of different factors. As the functions of EBV⁺ exosomes in NPC have been gradually explored, it can be speculated that viral oncogenic cargo might be transferred to uninfected cells through exosomes, resulting in the occurrence or progression of NPC.

Similarly, exosomes derived from NPC cells share analogous immunosuppressive functions like those of exosomes from EBV⁺ B cells. Importantly, exosomes from NPC cells could also induce the angiogenesis, EMT and metastasis of tumors (17,95). Therefore, we speculate that targeting EBV⁺ exosomes is of significance for the treatment of NPC, and might be able to reverse the progression of tumors by blocking tumor cell immune escape and inhibiting tumor angiogenesis and EMT at the same time.

EBVaGC accounts for ~10% of GC cases worldwide, with variable frequencies among geographic regions (100). In 1990, a possible association with EBV was first reported in a case of gastric carcinoma (101). To date, studies investigating EBV⁺ exosomes in EBVaGC are limited. In 2013, Choi et al (102) reported that miR-BART15-3p is enriched in exosomes derived from EBVaGC cells and can induce apoptosis partially by inhibiting the translation of the apoptosis inhibitor baculovirus inhibitor of apoptosis repeat-containing ubiquitin-conjugating enzyme. However, DCs are pivotal to tumor immunity (103), and Hinata et al (104) found that the maturation of DCs is suppressed by exosomes derived from EBV⁺ epithelial cells, which weakens tumor immunity. The aforementioned studies show the paradoxical functions of EBV⁺ exosomes, including inhibition of host immunity while promoting EBV⁺ cell apoptosis, which might explain why the prognosis of EBVaGC is favorable compared with that of other types of GC (105).

Although there are a number of different kinds of EBV-associated tumors, they primarily include B cell lymphoma, NPC and EBVaGC. These three different tumors have different TMEs but share an analogous immunosuppressive characteristic, which might be induced by EBV⁺ exosomes (63,95,104). The known mechanisms of different EBV⁺ tumor cell exosomes acting on various target cells are summarized in Fig. 2.