As endogenous cellular components, exosomes are vesicles (30–100 nm) derived from the endosomal compartments called multivesicular bodies (Fig. 1) that are secreted by various cells into the extracellular microenvironment. When exosomes are isolated by density gradient centrifugation or ultracentrifugation, they appear as round vesicles in solution. The ultrastructure is resolved by dehydration, where it appears cup-shaped under an electron microscope (10). Exosomes are double-membraned organelles formed by periodic endocytosis of intracellular fluid throughout the life cycle of eukaryotic cells. As early endosomes mature and develop into late endosomes, the inner membrane sprouts inward to form intraluminal vesicles (ILVs), which contain randomly engulfed parts of the cytoplasmic content, rich with mRNAs, microRNAs (miRNAs/miRs), proteins and lipids. ILVs that are released to the extracellular environment are called exosomes (11). Studies show that exosomes can be found in various extracellular fluids, such as blood, urine, ascites, semen and cerebrospinal fluid.

Exosomes can be used as delivery vehicles for a variety of bioactive molecules, for example proteins, lipids, mRNA, miRNA, long non-coding RNA (lncRNA), genomic DNA and cDNA. This unique composition is also occasionally used as an identifier for a particular exosome (12). Previous research indicates that the common proteins in exosomes include tetraspanins, co-stimulatory molecule CD86 and adhesion molecules, such as integrins, ICAM1, CD166 and CD146. Besides specialized proteins, exosomes may also carry common proteins, such as HSP-70, HSP84 and HSP90 (11). Exosomes can accelerate peptide loading onto MHC I and II, thereby mediating a rapid immune response. Exosomes also carry signal transduction proteins, for example, receptor tyrosine kinases and membrane transport and fusion proteins, such as the GTPases Rab5 and Rab7 (11). Studies have also reported that nucleic acids are carried by exosomes, comprising of a diverse mix of DNA (13,14), RNA (15), lncRNA (16) and miRNA (17,18) molecules. Exosomal miRs (miR-15, −16, 151 and −375) can promote angiogenesis and tumor progression in the TME (11). The bioactive cargo carried by exosomes can participate in the modification of immune response of an ovarian cancer microenvironment (19).

In infectious and non-infectious pathological conditions, both non-tumorous and tumorous cells tend to release exosomes more actively, whereas the number of exosomes quantified from the blood of patients with ovarian cancer is 3–4 times higher compared with in healthy individuals (11). The exosomes extracted from two human ovarian carcinoma cell lines OVCAR-3 and IGROV1 have a density ranging from 1.09–1.15 g/ml, while ~2,230 proteins are detected in the exosomal cargo, including other significant exosomal protein markers (20). Andre et al (6) in 2002 detected human epidermal growth factor (EGF) receptor (Her2/neu gene) signaling in exosomes of patients with ovarian cancer via western blotting. Activated matrix metalloproteinase (MMP)-2, MMP-9 and urokinase plasminogen activator are found in exosomes derived from the ascites of patients with ovarian cancer, which promotes protease activation to increase degradation of the extracellular matrices (ECMs) and tumor cell invasion and metastasis (21).

Macrophages are the first line of defense against foreign pathogens and key effectors of innate immunity, they are the key cells to bridge innate and adaptive immunities (73). According to their activation pathways, macrophages can be divided into two types: Classically-activated macrophages (named M1) and alternatively-activated macrophages (named M2) (74). Macrophages are usually polarized into M1 phenotype after being induced by IFN-γ, TNF-α, IL-6 and lipopolysaccharides. Their surface highly expresses MHC II and co-stimulatory proteins, such as CD80 and CD86 (75). M1 macrophages are generally considered pro-inflammatory and release IL-6, IL-12, TNF-α and reactive oxygen species (ROS), which are considered intermediates that are associated with cytotoxicity and anti-tumorigenic properties (76,77). M2 macrophages are polarized by IL-4, IL-13, IL-10 and IL-33 (56). The markers on the surface of M2 macrophages include found in inflammatory zone 1, mannose receptor 1 and MHC II (74). M2 macrophages have the capacity to secrete TGF-β, IL-6 and arginase-1 to facilitate neovascularization, inhibit the adaptive immune response, ensure tumor cell survival and remodel the ECM, which are all generally considered as tumor-promoting functions (78,79). In addition, TAMs are the main cells in the tumor immune microenvironment that have two phenotypes: M1-Like TAMs and M2-like TAMs (80). In the immune microenvironment of ovarian cancer TAMs usually manifest as the M2-like phenotype. The variety of biomarkers on the surface are scavenging receptor B (CD163), mannose receptor (CD204), IL-10 and chemotactic factor ligands CCL18 and CCL22 (81,82). IL-10 secreted by TAMs can activate Treg cells and promote tumor progression (83). miR-29a-3p and −21-5p, which are abundant in TAM-derived exosomes, can be transferred to CD4⁺T cells; thereby inhibiting STAT3 from regulating the ratio of Treg:Th17 and creating an immunosuppressive microenvironment that is necessary for the ovarian tumor to evade an active immune response, thus helping in tumor progression (84).

Tumor-derived exosomes can induce macrophages to differentiate into the M2 phenotype and TAMs, which have been confirmed in extracts from various organs, including: Ovaries, colorectal regions, endometrium, pancreas, melanoma, liver, breast and lung cancer cells (85–91). In a hypoxic microenvironment, high expression of miR-940 in exosomes has been derived from epithelial ovarian cancer cells, which induces macrophages to differentiate into the M2 phenotype, thus promoting proliferation and metastasis of epithelial ovarian cancer (92). Similar research shows that under hypoxic conditions, exosomes derived from epithelial ovarian cancer activate hypoxia-inducible factors that induce macrophages to highly express miR-21-3p, −125b-5p and −181d-5p and promotes their polarization to the M2 phenotype through the cytokine signal transduction 4/5/STAT3 signaling pathway, which was also verified in vivo. The JAK-STAT pathway mediates inflammatory immune response by converting cytokine signals, and SOCS is the key regulator of the pathway (76). Through microarray analyses, some researchers found that the expression of miR-221-3p was upregulated in exosomes that were derived from M2 macrophages. Additionally, miR-221-3p can target the inhibition of cyclin-dependent kinase inhibitor 1B, thus promoting the proliferation of ovarian cancer cells via transition from G₁ to S (93). In addition, epithelial ovarian cancer-derived exosomes overexpress miR-222-3p and transfer it to macrophages to induce them into M2-like polarization by the SOCS3/STAT3 pathway. miR-222-3p targets downregulation of SOCS3 gene expression and activates STAT3 expression (94). This transfer of miR-222-3p can facilitate the progression of ovarian cancer (94). Wu et al (95) have shown that TAM-derived exosomes inhibit endothelial cell migration by targeting the miR-146b-5p/TRAF6/NF-kB/MMP2 pathway, whereas ovarian cancer-derived exosomes can reverse the role of TAMs in endothelial cells by transferring lncRNAs.

In order for macrophages to differentiate into TAMs, macrophage-derived exosomes are important components involved in the antitumor immune response (84,93,96). The TNF-related weak inducer of apoptosis (TNSFS12 or TWEAK)-stimulated macrophage-derived exosomes can be internalized by the tumor cells, which can inhibit ovarian cancer metastasis. A study revealed that TWEAK-stimulation increased the expression of miR-7 (a tumor suppressor) in exosomes released by macrophages, which downregulates the activity of the EGFR/AKT/ERK1/2 signaling pathway and inhibits ovarian cancer metastasis (96). This was performed in mouse models where TWEAK-stimulated macrophage-derived exosomes blocked the metastasis of epithelial ovarian cancer (96). In addition, Baj-Krzyworzeka et al (97) have shown that tumor-derived exosomes can activate monocytes by increasing HLA-DR expression, upregulating reactive oxygen intermediates and TNF and by accumulating and secreting IL-10 and IL-12 mRNA. Exosomes activate monocytes and induce them to differentiate into macrophages (97–99). At present, there are few studies on macrophage-derived exosomes in ovarian cancer, which is an area that needs to be explored further. Exosomes derived from breast cancer cells have upregulated levels of miR-130 and miR-33, which can alter the polarization of macrophages from M2 to M1 phenotype and inhibit tumor progression (100). The exosomes derived from TAMs of progranulin (PGRN)-negative tumor tissues have upregulated expression of miR-5100, which inhibits the invasion, migration and EMT of breast cancer cells by targeting the CXCL12/CXCR4 axis (101). Although a similar study in ovarian cancer has not been performed, studies have shown that expression of PGRN protein relates to poor ovarian cancer prognosis (102,103). Moreover, high expression of PGRN can induce EMT in ovarian cancer cells (104). This suggests that exosomes are a potential therapeutic target for ovarian cancer.

In a tumor environment, NK cells are the first line of defense within the immune system. NK cells mainly kill target cells in four ways: i) Antibody-dependent cell-mediated cytotoxicity via the Fas/FasL pathway (105), ii) the perforin-granzyme pathway (106), iii) binding to target cells through adhesion molecules (107) and iv) releasing cytokines to attack target cells (108,109). NK cells are important effectors in the cancer immune surveillance (110,111). Upon activation, they secrete pro-inflammatory factors and chemokines, for example IFN-γ, TNF, IL-6, GM-CSF and chemotactic cytokine ligand 5 to mediate antitumor immune responses, affect antitumor activity and promote formation of the tumor microenvironment (112). IL-15 enhances the antitumor activity of NK cell-derived exosomes and has been validated in mouse models (113). Exosomes derived from NK cells express the killer protein (CD56), FasL, perforin, granulysin, and granzyme A and B, which show antitumor activity and play a role in immune surveillance (114). Killer proteins expressed by NK cell-derived exosomes can participate in NK cell-mediated cytotoxic killing effects (115), and the expressed DNAX accessory molecule 1 (DNAM-1/CD226) receptor can bind to DNAM-1 ligands on the cell membrane of tumor cells to exert a cytotoxic tumor cell killing effect (116). This is an important role of NK cells in cancer immune surveillance. NK cell-derived exosomes express FasL and perforin and exert cytotoxic effects in melanoma (117). NK cell-derived exosomes carry miR-186, which can inhibit neuroblastoma growth (118). The antitumor effect of NK cell-derived exosomes on invasive melanoma and neuroblastoma has been confirmed, exosomes derived from NK cells can potentially be used in cancer treatment (117,118).

Although NK cells are a part of the innate immune system and are capable of killing tumor cells, tumor microenvironment also affects the cytotoxicity of NK cells (119). Exosomes released by ovarian cancer cells highly express KLRK1/ natural killer group 2 (NKG2D) ligands in the manner of MHC I chain-related protein A and B and UL16-binding protein. This downregulates the expression of NKG2D receptors on peripheral blood mononuclear cells, affecting the activation of NK cells and suppressing their natural killing effect (120).

Adaptive immune effector cells include CD8⁺, CTL and CD4⁺Th. In acute infection, CTL is the main effector cell and has a specific killing effect on tumors through the perforin-granzyme, Fas/FasL and TNF pathways (121). CD4⁺Th cells play an auxiliary role in activating CTL (122). In contrast, during chronic infections and cancer, T cell dysfunction occurs due to continuous stimulation of antigens (123). In addition, the cytokines produced by CD4⁺Th cells can indirectly participate in antitumor immune effects. In theory, dendritic cell-derived exosomes can stimulate T cells through three different mechanisms: i) Direct presentation of exosomal MHC I and II to T cells, ii) indirect stimulation via exosomal MHC presentation to APCs and iii) degradation and recycling of the exosomal MHC/peptide complex in the microenvironment (124).

Research shows that T cell-derived exosomes can regulate gene expression and extracellular signal transduction of DCs by carrying miRNA and T cell receptor protein (TCR)-rich vesicles (125). MHC I, MHC II and T cell co-stimulatory molecules on the surface of B lymphocyte-derived exosomes can stimulate the proliferation of T lymphocytes and thus prevent tumor progression (126). T follicular helper cell-derived exosomes facilitate the proliferation of B lymphocytes (127). CD4⁺T cell-derived exosomes regulate the gene expression of B lymphocytes by transporting their miRNA cargo to B lymphocytes, which therefore decreases the level of antibodies produced by B cells (128).

However, a previous study confirmed that tumor cell-derived exosomes recruit lymphocytes to suppress the antitumor immune response, promoting tumor progression by increasing tumor invasion, angiogenesis and upregulating proinflammatory cytokines, such as IL-6 and VEGFA (129). Exosomes can induce T cell apoptosis and weaken the immune response to achieve immune escape. FasL is a transmembrane protein in the TNF family of proteins that is increased in malignant tumors. FasL is overexpressed in tumor-derived exosomes (11,130). FasL recognizes the Fas receptor on the surface of target cells and triggers intrinsic apoptosis of target cells via the FADD-procaspase-8 pathway, to induce T cell apoptosis and suppress the immune response (11). In addition, Fas can also mediate the apoptosis of lymphocytes through antibody-dependent direct cytotoxicity. A previous study has showed 41 kDa FasL and HLA class I antigens are upregulated in ovarian cancer-derived exosomes and inhibit T cell antitumor immune function through FasL/CD3-ζ ligand-receptor binding (131). This inhibitory function has also been observed in other tumors, such as exosomes derived from human prostate cancer cells that induce CD8⁺T cell apoptosis by FasL/Fas (132). FasL is also found in exosomes from melanoma and colorectal cancer, which is considered a new immune escape pathway (133). The expression of circ-0001068 (a novel biomarker for ovarian cancer and inducer of PD1 expression in T cells) in exosomes derived from ovarian cancer cells is upregulated and can be delivered to T cells as well. Once in T cells, circ-0001068 downregulates the expression level of miR-28-5p and thus induces the expression of PD1 (134). Exosomes released by ovarian cancer cells can carry plasma gelsolin and induce CD8⁺ T cell apoptosis, which weakens immune surveillance (135).

Exosomes from the ascites of patients with ovarian cancer can inhibit T cell signaling molecules, inhibit the expression of CD3-ζ and JAK 3, and induce T cell apoptosis (136). In the ascites of patients with ovarian cancer, the ganglioside GD3 expressed on the surface of exosomes binds with the TCR and actively arrest T cell function, which greatly reduces the antitumorigenic effect of T cells (137,138). Moreover, a previous study showed that this inhibitory effect mediated by GD3 and TCR is related to the sialic acid group in exosomes (137). When the sialic acid group is hydrolyzed, this inhibitory effect disappears, which indicates that GD3 can be a new target for ovarian cancer immunotherapy (137). In addition, the ascites-derived exosomes of patients with ovarian cancer can effectively block T cells in a reversible manner due to the early translocation of NF-kB and later functional activation of IFN-γ. Researchers have demonstrated that this can be reversed within 24–48 h by the removal of exosomes. Therefore, targeted removal of exosomes will increase the antitumorigenic effect of the host T cells (139).

Tumor-derived exosomes can also inhibit T cell activation to cause immunosuppression. Functional CD39 and CD73 expressed by exosomes can dephosphorylate exogenous ATP and cAMP to form adenosine, and inhibit T cell activation through the adenosine A2A receptor. Therefore, exosomes increase the production of extracellular adenosine to regulate the antitumor immune effect of the T cells (140). In addition, phosphatidylserine (PS)-positive exosomes derived from the ascites of patients with ovarian cancer block the NF-kB and NFAT pathway signaling cascade in T cells, and reversibly inhibit T cell activation (141). Thus, depletion of anti-PS antibodies or blocking PS can notably eliminate the inhibition of T cells, which could be another new treatment method for patients with ovarian cancer (142).

DCs are unique, in that they can activate T cells. They can also activate immune responses or induce immune tolerance (143). Mast cell-derived exosomes contain HSP60 and HSP70, which can promote DC maturation and exert antitumorigenic immune effects in a mouse model (144). The ovarian cancer microenvironment is rich in cytokines and angiogenic factors, which can change the phenotype and function of DCs. Most studies corroborate that the ability of exosomes to stimulate T cells can be enhanced through the interaction of exosomes with DCs (145–147). Exosomes derived from the ascites of patients with ovarian cancer present tumor antigens and can induce differentiation of dendritic cells and tumor-specific cytotoxic T lymphocytes (6). Exosomes isolated from the ascites of patients with ovarian cancer express MHC I molecules, HSP70 and HSP90. DCs treated with these exosomes can promote T cell activation and produce cytotoxicity (148). DC-derived exosomes present antigens to DCs, and then these DCs can activate T cells. These results suggest that the exosome is a potential safe and feasible immunotherapy for advanced tumors (149). At present, the antitumor immunotherapy of exosomes derived from DCs is in phase II clinical trials of advanced malignant tumors.

Although DCs have antitumorigenic activity, their function may be inhibited in tumor immune microenvironment. The ovarian cancer microenvironment is rich in factors that inhibit monocyte differentiation into DCs. Ascites-derived exosomes from patients with ovarian cancer induce apoptosis in DCs by activation of the Fas/FasL pathway and mediating TRAIL apoptosis-inducing signal molecules in mature DC precursors. In one investigation, ovarian cancer-derived exosomes were cultured with dendritic precursor cells for 48 h. Exosome co-cultured DCs had an apoptotic rate of 12.6% while the control group had an apoptotic rate of 8.6% (21). Overall, exosomes may induce apoptosis of DCs and stimulate precursors of mature DCs.

MSCs are an important member of the stem cell family, which play an important role in cancer progression. They are present, albeit in small numbers, in a variety of tissues (bone marrow, umbilical cord blood, umbilical cord, placenta and adipose) and are reported to have multidirectional differentiation and regeneration properties (150). In addition, MSCs also have the capability of immune modulation, which in a number of cases can cause immunosuppressive effects. De Miguel et al (151) demonstrated in vitro that MSCs can inhibit the proliferation of immune cells (lymphocytes, NK cells and DCs) and inhibit secretion of cytokines, thereby inhibiting the cytotoxic effect of T and NK cells via indoleamine 2,3-dioxygenase, while also activating and inducing the maturation of DCs. A co-culture of ovarian cancer cell lines (SKOV-3 and OVCAR-3) and MSCs demonstrates that adhesion, migration, invasion, proliferation and chemical resistance of ovarian cancer cells is enhanced, leading to accelerated tumorigenicity (152). In the tumor microenvironment, exosomes derived from MSCs also play an immunoregulatory role. Bone marrow MSC-derived exosomes have anti-inflammatory, anti-apoptotic, pro-angiogenic and immune-regulating effects (153). Bone marrow MSC-derived exosomes inhibit the proliferation of T and B cells and affect mRNA function; downregulating the expression of CXCL8 and marginal zone B and B1 cell-specific protein the level of IgM to affect the anti-tumorigenic function of B cells (154). MSC-derived exosomes upregulate MMP-2 and activate ecto-5′-nucleases, causing tumor cells to become more malignant and thus altering the tumor microenvironment, as well as enhancing tumor heterogeneity (155). In addition, cancer cell-derived exosomes affect the tumorigenicity of MSCs. In vitro analysis demonstrates that SKOV-3 and OVCAR-3 cell line-derived exosomes can enhance the migration capacity of MSCs (156). In the microenvironment of ovarian cancer, cancer stem cells are associated with creating drug resistance and making the tumor mass refractory to a specific drug (147,157). Vera et al (158) have revealed that upon treatment with cisplatin, exosomes released by ovarian cancer that are rich in cancer stem cells can upregulate IL-6, IL-8 and VEGFA, increasing the migration capacity of cancer cells. In addition, factors secreted by MSCs can induce endothelial cell angiogenesis and accelerate the migration of low-invasive ovarian cancer cells. Exosomes have also been shown to enhance the oncogenicity of MSCs, leading to drug resistance and tumor progression (158). The expression of miR-146a in MSC-derived exosomes is upregulated, which targets laminin γ-2 to regulate the phosphoinositide 3-kinase (PI3K/Akt) signaling pathway. This subsequently inhibits the proliferation of ovarian cancer cells and induces chemotherapy resistance (159).

Previous studies have confirmed the antitumor effect of MSC-derived exosomes. Human adipose MSC-derived exosomes can induce apoptotic signals by upregulating pro-apoptotic signaling via BAX, CASP9, CASP3 and downregulating the anti-apoptotic protein BCL2 to inhibit A2780 and SKOV-3 cell proliferation, wound-repair and colony-forming ability (160). In mouse models of ovarian cancer, paclitaxel-loaded MSC-derived exosomes have strong antitumor effects, which suggests that they can be used as drug carriers to target ovarian cancer (161).

Tregs negatively regulate the antitumor response in both a direct and indirect manner while also playing a key role in immune escape (162). The increase of Tregs in the tumor microenvironment of patients is related to poor prognosis and shortened overall survival (OS) time (55). Treg-derived exosomes can exert immunosuppressive effects by expressing CD73 and inhibiting the proliferation of CD4⁺T cells (107). Curiel et al (163) in 2004 analyzed 104 specimens of epithelial ovarian cancer and found that CD4⁺CD25⁺FOXP3⁺ Tregs inhibited T cells in vivo and promoted tumor development. After tumor-derived exosomes activate Tregs, the expression levels of STAT3/SMAD2/3/IL-10/TGF-B increase and the expression of granzyme B, perforin and FasL are upregulated, thereby reducing the antitumor immune response. In addition, exosomes act on the SMAD2/3 and STAT3 signaling pathways to convert CD4⁺CD25T cells into CD4⁺CD25⁺FOXP3⁺ Tregs, which upregulates their immunosuppressive function and anti-apoptotic potential (164).

MDSC is an immunosuppressive cell of marrow-derived cells, which are induced to differentiate into DCs, macrophages and granulocytes. These MDSCs can depress the activity of T and NK cells, which can significantly suppress immune cell response (165). IL-6 is produced by autocrine activation in a Toll-like receptor 2 (TLR2)/myeloid differentiation primary response 88-dependent manner, triggering STAT3 activation and promoting the immunosuppressive function of MDSCs (166). A similar study found that HSP70 is highly expressed on the surface of ovarian cancer-derived exosomes, which fuses with and activates MDSCs by binding TLR2 to promote cancer progression (166,167). Notably, the study found that the A8 peptide blocked this HSP70/TLR2 binding to weaken the ability of tumor-derived exosomes to activate MDSCs in a mouse model. Drugs, such as cisplatin and 5-fluorouracil, cause tumor cells to release more exosomes with HSP70 surface expression to activate MDSCs. When cisplatin or 5-fluorouracil is used in combination with A8 peptide, it can effectively antagonize the activation of MDSCs caused by cisplatin or 5-fluorouracil, which greatly enhances the antitumor effect of these drugs. Overall, this study has notable implications for novel ovarian cancer therapies (168).

As an important element of the tumor microenvironment, CAFs secrete a variety of growth factors and pro-inflammatory cytokines (TGF-β, VEGF, IL-6 and CXCL12) which promotes angiogenesis and recruits immunosuppressive cells into the tumor microenvironment to assist in immune evasion (169). Exosomes derived from epithelial ovarian cancer induce adipose tissue-derived mesenchymal stem cells to differentiate into tumor-associated myofibroblasts and upregulate tumorigenic factors such as stromal cell-derived factor 1 and TGF-β (170). TGF-β receptor and SMAD signaling can regulate the expression of multifunctional proteoglycan VERISCAN protein, encoded by the VCAN gene. Upregulating VCAN activates the NF-kB signal pathway, which upregulates the expression of CD44, MMP-9 and hyaluronic acid-mediated motor receptors that collectively promote the migration and invasion of ovarian cancer cells (171). Further the CAF-derived exosomes can be internalized by SKOV-3 and CAOV-3 cell lines leading to a more aggressive tumorous phenotype, promoting the EMT of ovarian tumors. This evidence suggests that CAF-derived exosomes have the potential to provide a breakthrough in the treatment of ovarian cancer (172).