Tumor-Derived Extracellular Vesicles in Cancer Immunoediting and Their Potential as Oncoimmunotherapeutics

Simple Summary Tumor cell-derived extracellular vesicles (TEVs) are an important means of tumor communication with, and manipulation of, the patient’s physiology. TEVs influence the local tumor environment as well as the systemic conditions of the patient. Progressive changes in tumor interactions with the host immune system are defined as “immunoediting”. Here, we summarize TEV effects on the immune system during the stages of cancer immunoediting and outline the molecular and cellular characteristics of interactions that result in complete tumor regression versus tumor immune escape and progression. Generally, the cargo profile of TEVs naturally changes during immunoediting toward immunosuppression while different cell stress or treatment conditions can inhibit this process or even reverse it to immunostimulation by altering the TEVs cargos. Therefore, understanding potential immunotherapeutic properties and how they can be manipulated to treat cancer should be considered a new research approach in oncoimmunotherapy. Abstract The tumor microenvironment (TME) within and around a tumor is a complex interacting mixture of tumor cells with various stromal cells, including endothelial cells, fibroblasts, and immune cells. In the early steps of tumor formation, the local microenvironment tends to oppose carcinogenesis, while with cancer progression, the microenvironment skews into a protumoral TME and the tumor influences stromal cells to provide tumor-supporting functions. The creation and development of cancer are dependent on escape from immune recognition predominantly by influencing stromal cells, particularly immune cells, to suppress antitumor immunity. This overall process is generally called immunoediting and has been categorized into three phases; elimination, equilibrium, and escape. Interaction of tumor cells with stromal cells in the TME is mediated generally by cell-to-cell contact, cytokines, growth factors, and extracellular vesicles (EVs). The least well studied are EVs (especially exosomes), which are nanoparticle-sized bilayer membrane vesicles released by many cell types that participate in cell/cell communication. EVs carry various proteins, nucleic acids, lipids, and small molecules that influence cells that ingest the EVs. Tumor-derived extracellular vesicles (TEVs) play a significant role in every stage of immunoediting, and their cargoes change from immune-activating in the early stages of immunoediting into immunosuppressing in the escape phase. In addition, their cargos change with different treatments or stress conditions and can be influenced to be more immune stimulatory against cancer. This review focuses on the emerging understanding of how TEVs affect the differentiation and effector functions of stromal cells and their role in immunoediting, from the early stages of immunoediting to immune escape. Consideration of how TEVs can be therapeutically utilized includes different treatments that can modify TEV to support cancer immunotherapy.


Introduction
Roughly 19.3 million new cancer cases are expected to be diagnosed in a year across the world. Before the recent establishment of immune-based cancer therapies, the immune system was generally considered to play a minor role in cancer biology. However, immunotherapies, specifically checkpoint blockade antibodies (CPB), are now established therapy for many cancers, with extensive ongoing research into new higher-impact immunotherapies [1,2]. It is now accepted that the immune system does recognize and attempts to eliminate cancer and an important question in oncology is: how do cancer cells evade the immune system? There is considerable information, but the process is complex and variable between tumor types and between patients, so the understanding is incomplete. The immune system acts as a double-edged that can control and eliminate tumors through or can help tumors progress through immunosuppression and support of angiogenesis [3,4].
The TME contains tumor cells, stromal cells including stromal fibroblasts, endothelial cells, and immune cells that can make up more than half of the overall tumor mass. These cells influence tumor cells and each other through complex interactions such as extracellular matrix (ECM) secretion, soluble factors, cell-cell communication, cytokines, chemokines, and inflammatory mediators [5][6][7]. Extracellular vesicles (EVs) are an important means of intercellular communication and include vesicles up to 1 µm with plasma membrane origin, and smaller lipid bilayer vesicles (30-100 nm), which according to the International Society for Extracellular Vesicles are called small extracellular vesicles (exosomes), that are cup-shaped or doughnut-shaped [8,9]. EVs carry and deliver membrane and cytosolic components, including proteins, lipids, and nucleic acids [10,11]. The physiological and pathological function of EVs depends on their contents and ability to deliver their cargoes. Like other secreted biologically active components, vesicular-based cell-to-cell communication does not require cell contact and can act over long distances [8]. Their internalization in target cells can be through direct fusion with the plasma membrane, endocytosis, phagocytosis, or through ligands on their surface binding to receptors on other cells [8]. Their compositions are associated with endosome biogenesis and parental cell type since EVs with different origins contain unique subsets of components with different cell type-associated functions [10].
The process of tumors interacting with the immune system has been characterized by three phases, elimination, equilibrium, and escape [12][13][14]. During the elimination phase, small tumors not clinically recognized are eliminated without awareness by anyone. During the equilibrium phase, tumors are held in check by immune pressure but not eliminated, and tumors in this stage are also generally not recognized clinically. Lastly, in the escape phase, tumors grow large and are clinically recognized as cancer [12]. It should be noted that most information about cancer comes from tumors in the escape phase, since before that they are small, not identified in humans, and generally hard to study in animal models.
During the elimination phase, the innate and adaptive immune systems work together to identify and eliminate transformed cells that have evaded genetic mechanisms that suppress malignancies. Tumor cells that survive the elimination phase replicate and enable the tumor to reach the equilibrium phase. Both innate and adaptive immunity appears to play a major role in limiting tumor progression in this phase and tumors are controlled, but not eliminated by the immune system. During equilibrium, the tumor is sufficiently immunosuppressive to avoid elimination but is unable to significantly expand. Ultimately in the escape phase, tumors with more robust immunosuppressive mechanisms overcome the immune system via different mechanisms, grow until they are clinically detectable, locally invade and generate metastases [12,13]. In each of the immunoediting phases, cancer-secreted factors interact with the immune system. Some of them can help the tumor grow by suppressing the functional immune cells, whereas others stimulate the immune cells to respond against cancer Figure 1 [12,13,15]. As noted below, EVs play a role in all three stages, and this role generally goes from an antitumor effect in the elimination phase to a protumor effect in the escape phase. cent studies show that cancer cells use EVs to communicate with one another and with stromal and normal cells [8,10]. The cancer-mediated immunoregulation mechanisms and factors they influence include the expression of surface molecules such as PD-L1 by cancer cells and the recruitment of immunosuppressive cell types such as Tregs and myeloidderived suppressor cells (MDSC). This review discusses the effects of TEV on the immune system in each phase of cancer immunoediting, their roles in immunoregulation in the TME, and their potential use in cancer immunotherapy. Figure 1. Cancer immunoediting and involved effector molecules and receptors: (a) During the elimination phase, the innate and adaptive immune systems work together to identify and eliminate transformed cells that have evaded genetic malignant cell suppression mechanisms. The generation of tumor antigens and extracellular vesicles by cancer cells trigger immunological responses against tumors that result in the detection and elimination of nascent cancer cells by the immune system (b) if they survive the elimination phase, tumors may reach the equilibrium phase. Adaptive and innate immunity limits tumor progression in this phase, therefore tumor growth is reduced, and surviving tumor cells are kept under control and possibly kept dormant by the immune system (c) Ultimately in the escape phase, tumors overcome the immune system by immune suppression through recruiting immunosuppressive cells, and suppressing effector immune cells via different mechanisms.

Tumor-Derived Extracellular Vesicles and Tumor-Supportive Cells
During the initial phases of tumor formation, the local microenvironment has mostly anticancer effects [14], but as tumors progress, the healthy microenvironment changes into the TME, immune protection is lost, tumor growth continues, and the stromal cells are influenced by cancer cells to support tumorigenic functions [19][20][21][22]. Along with tumor growth, immunoediting proceeds step by step with the formation of the TME. Tumors that successfully escape the equilibrium phase alter the surrounding healthy microenvironment by manipulating surrounding cancer-associated fibroblasts (CAFs), Tumor-associated macrophages (TAMs), MDSCs, and other immune cells and by recruiting new TME cells that further suppress the immune response via cytokines/chemokines, cell-to-cell contact, growth factors, and EVs [4,23]. EVs from malignant cells are important mediators of malignant cell communication in the TME and beyond and may support cancer metastasis, angiogenesis, therapy resistance, and immunoregulation leading to resistance to immune surveillance [16][17][18]. Recent studies show that cancer cells use EVs to communicate with one another and with stromal and normal cells [8,10]. The cancer-mediated immunoregulation mechanisms and factors they influence include the expression of surface molecules such as PD-L1 by cancer cells and the recruitment of immunosuppressive cell types such as Tregs and myeloid-derived suppressor cells (MDSC). This review discusses the effects of TEV on the immune system in each phase of cancer immunoediting, their roles in immunoregulation in the TME, and their potential use in cancer immunotherapy.

Tumor-Derived Extracellular Vesicles and Tumor-Supportive Cells
During the initial phases of tumor formation, the local microenvironment has mostly anticancer effects [14], but as tumors progress, the healthy microenvironment changes into the TME, immune protection is lost, tumor growth continues, and the stromal cells are influenced by cancer cells to support tumorigenic functions [19][20][21][22]. Along with tumor growth, immunoediting proceeds step by step with the formation of the TME. Tumors that successfully escape the equilibrium phase alter the surrounding healthy microenvironment by manipulating surrounding cancer-associated fibroblasts (CAFs), Tumor-associated macrophages (TAMs), MDSCs, and other immune cells and by recruiting new TME cells that further suppress the immune response via cytokines/chemokines, cell-to-cell contact, growth factors, and EVs [4,23].

TEVs Modulate Macrophage Activity in TME
Macrophages are complex and plastic cells that adopt a range of phenotypes from strongly immune suppressive to strongly immune stimulatory depending on the environmental signals they receive. While there are no clear distinctions on this continuum, for convenience they are often divided into M1 subtypes with pro-inflammatory properties that express cytokines such as IFN-γ, TNF-α, CXCL-10, IL-12, and high amounts of nitric oxide (NOS), and M2 subtypes with an anti-inflammatory function that release anti-inflammatory cytokines such as IL-4, IL-10, IL-13 and express high amounts of arginase-1(ARG1), scavenger receptors, and mannose receptor [24]. TAMs, begin to shift phenotypic from M1 to M2 through macrophage polarization with exposure to tumor-derived factors and TEVs in the TME and hypoxic conditions and act as a bridge between the adaptive and innate immune systems [25,26]. The M2 cells manifest local supportive functions for the tumor [26,27]. Cancer-derived extracellular vesicles increase M2 polarization by activating signaling pathways such as STAT3, p38MAPK, NF-κB, ERK1/2, and PI3K/AKT [28][29][30] and reprogram M1 tending cells into the cancer-promoting M2 end of the macrophage phenotype spectrum. Table 1 outlines a variety of data on specific activities of TEV in mediating immunosuppression.

TEVs Modulate Fibroblast Activity in TME
In the normal state, fibroblasts are activated during wound healing to help in the process by creating an extracellular matrix (ECM), which serves as a scaffold for other cells [51]. Cancer-associated fibroblasts (CAFs) resemble myofibroblasts and often make up the majority of the cancer stroma [51]. Unlike normal fibroblasts (NFs), CAFs create an excessive ECM and secrete pro-invasive molecules such as ECM-degrading proteases. Hence, CAFs support ECM remodeling, and invasion by producing various kinds of cytokines, growth factors, chemokines, and matrix-degradable enzymes [52][53][54]. The molecular mechanisms that convert normal fibroblasts (NFs) to CAFs in TME are not fully understood. MiRNAs have a major function in the transition and activation of fibroblasts, as evidenced by the fact that dysregulation and disruption of miR-1, 206, 31,214, 155, and 31 secretion leads to the differentiation of NFs to CAFs through modulating FOXO3a, vascular endothelial growth factor (VEGF), and CCL2 signaling [55,56]. Recent studies show that crucial miRNAs in TEVs promote the differentiation of NFs into CAFs [21,[57][58][59] (Table 2). Ovarian cancer vesicular miR-630 transformed NFs into CAFs by activating the NF-κB and inhibiting the KLF6 pathway [60]. In another study, lung cancer vesicular miR-210 activated the JAK2/STAT3 pathway, and ten-eleven translocation 2 (TET2) promoted the transformation of NFs into CAF [61]. The JAK2/STAT3 pathway activated by miR-210 resulted in increased expression of some pro-angiogenic factors such as FGF2, MMP9, and VEGF. In addition, breast cancer vesicular proteins such as survivin and ITGB4 converted NFs into myofibroblasts by increasing superoxide dismutase 1 (SOD1) and lactate in CAFs in a BNIP3L-dependent manner [62]. In bladder cancer, the vesicular TGF-β protein activates the TGF-β pathway and triggers CAF differentiation by SMAD pathway activation [19,63].

TEVs Effect on MDSC Formation in TME
MDSCs normally protect the host from the damaging consequences of excessive immune activation in pathological conditions such as wound healing, but in the TME, MDSCs promote angiogenesis, invasion, and metastasis, as well as block antitumor immunity [68]. MDSCs can generate robust immunosuppressive responses via numerous pathways such as the release of reactive oxygen species (ROS), NO via iNOS, arginine depletion by arginase, secretion of immunosuppressive cytokines such as IL-10 and TGF-β, and stimulation of apoptosis of immune effector cells via the Fas ligand pathway [68][69][70][71]. Therefore, MDSCs are critical mediators in helping cancers evade the immune system. Immature myeloid cells (IMCs) can fail to differentiate under several pathologic conditions (infection, inflammation, and cancer) and develop the features of dysfunctional myeloid cells that include MDSCs through a variety of mechanisms involving numerous substances that accumulate in the TME. Several growth factors and interleukins such as GM-CSF and interleukin IL-6 promote the differentiation of IMCs into MDSCs via activating the STAT-3 signaling pathway [72,73]. MDSCs are a diverse category of IMCs with immunosuppressive characteristics and activities [69]. It also was reported that immature natural killer (NK) cells, can be converted to MDSC [74]. Various tumor-derived factors within or on TEVs surface induce MDSCs in vitro, including IL-1β, IL-6, IL-10, prostaglandin E2 (PGE2), TGF-β, stem cell factor (SCF), and VEGF [72,75] (Table 3). The most important vesicular mediators involved in the differentiation of IMCs into MDSCs include PD-L1, PGE2, TGF-β, and HSP70 [75][76][77][78]. STAT pathways participate since various TEV can differentiate bone marrow myeloid cells into MDSCs by activating STATs [79,80].

TEVs-Mediated Communication between Tumor and Immune Cells
TEV early in tumor development can stimulate antitumor immunity. The interaction between immune cells and cancer in TME is categorized into seven potential steps ( Figure 2) [4] which is called the "cancer-immunity cycle". In the right conditions, TEV from tumor cells can also support antitumor immunity. TEVs contain and transfer TAs and damage-associated molecular patterns (DAMPs) to innate immune cells, especially dendritic cells (Step 1) [85,86]. Tumor-derived EVs are a source of shared TAs for CTL cross-priming.

TEVs-Mediated Communication between Tumor and Immune Cells
TEV early in tumor development can stimulate antitumor immunity. The interaction between immune cells and cancer in TME is categorized into seven potential steps ( Figure  2) [4] which is called the "cancer-immunity cycle". In the right conditions, TEV from tumor cells can also support antitumor immunity. TEVs contain and transfer TAs and damage-associated molecular patterns (DAMPs) to innate immune cells, especially dendritic cells (Step 1) [85,86]. Tumor-derived EVs are a source of shared TAs for CTL cross-priming.  . One or more of these stages may be disrupted in many cancer patients, resulting in ineffective immune responses to cancer. Disruption at any stage of this cycle is caused by cancer cells and their secreted factors, including via TEVs [86,87]. This disruption and immune system suppression block antitumor immunity and support cancer progression.

Elimination Phase TEV Involvement
This phase has not been directly detected in vivo in humans since it occurs with very small tumors. The innate and adaptive immune systems collaborate to identify and eliminate tumors that have evaded intrinsic tumor suppressor mechanisms in developing tumors [14]. Cancer immunosurveillance is proposed to remove newly generated neoplastic cells that have the potential to develop tumors. The processes of how the immune system is alerted of the presence of primary tumor cells remain unknown. Among the possibilities, the generation of neoantigens by abnormal cells within the created inflammatory environment (via immune cell subsets, recognition molecules, and effector cytokines) results in the detection of nascent cancers, and the traditional warning signals such as IFNs are likely involved [88][89][90]. T cells are the primary immune cells that identify and eliminate tumor cells [88,91]. However, B cells and their antibodies also seem to play a role in recognizing and removing these cells [92]. IFN-γ has a direct anti-proliferative impact on tumors through the STAT1 pathway and causes the release of cytokines such as CXCL9, 10, and 11 that increase immune activation by recruiting effector T cells [93,94]. IFN alpha and beta (type I IFNs) also play an important role in activating CD103 + DCs to cross-present tumor antigens [15,95].
Physical characteristics of the tumor environment such as hypoxia can cause tumor cell death, potentially resulting in the release of DAMPs such as Heat shock proteins (HSPs) and high mobility group box 1 (HMGB1), which act as ligands for Toll-like receptors on innate immune cells [85]. EVs can carry TAs, interferon, and DAMPs that stimulate immunological responses against tumors [96,97]. EVs carried CEA and HER2 TAAs that triggered immune responses and improved anti-tumor responses in vivo [98]. The release of tumor antigens and EVs can be altered under various TME situations. For example, an acidic microenvironment, quite common for tumors, increased the number of secreted EVs [99].
TEVs can play a key role in NK cell activation, DC maturation, and CD8 + effector T-cell development [100,101]. TEVs may also carry surface proteins derived from cancer cells which promote the uptake of TEVs by DCs. There are reports supporting LFA-1/CD54 and mannose-rich C-type lectin receptor interactions as enabling TEV uptake by DCs [102,103]. Uptake of TEVs by DCs enhanced DC expression of co-stimulatory receptors such as CD80, CD86, and also MHC II expression and boosted interferon and cytokine production along with DC maturation [104][105][106]. Breast cancer cells generated EVs that convey dsDNA to DCs, causing IFN alpha and beta expression in a STING-dependent manner and elevation of costimulatory molecules in DCs [107].
Furthermore, TEVs carry molecules that promoted CD8 + T-cell activation and enhanced tumor cytotoxic T lymphocyte (CTL) responses in vivo in mice [105,106,108]. EVs generated from brain tumors were delivered to mice on days 7 and 14 post-tumor inoculation, stimulating antibody production and T-cell activation. Antitumor antibodies and T cells present at the time of tumor inoculation appear to have caused enough tumor cell death to generate further T-cell antitumor response [109].

Equilibrium Phase TEV Involvement
Molecular processes that initiate immune-mediated cancer dormancy/control, i.e., the equilibrium phase (EqP), are not well understood in part because this phase is hard to model and has been minimally characterized in humans [130]. Not surprisingly, when overall mechanisms are poorly understood, there is not much known about the involvement of EVs in the equilibrium phase. In the equilibrium phase, the adaptive effector functions and the resistance of the tumor are in a dynamic balance. There are clear indications that tumors in the escape phase having metastasized, can return to equilibrium following chemotherapy and be dormant for many years before relapse. This occurs in particular with metastatic breast tumors where metastatic cells stop proliferating but survive in a quiescent state [131]. The role, if any that the immune system plays in maintaining this dormancy is not clear.
In the EqP, TEVs may suppress different adaptive immune cell types through various mechanisms such as inhibiting effector cells such as CD8 + T cells and NK cells, suppressing DC maturation and activation, increasing M2 and TAM immune suppressive polarization, and stimulating CAF differentiation [64,132,133]. However, as we noted previously, TEV can also mediate tumor-suppressing signals. TEVs containing miR-23b derived from mesenchymal bone marrow cancer stem cells (CSC) can induce cancer dormancy via downregulation of the MARCKS gene that mediates breast cancer cells' differentiation into CSCs through the Wnt-β-catenin pathway [134,135].
Considering PD-L1 and IFN-γ in the EqP of tumors is of interest for understanding the involvement of TEV and highlighting the complexity of molecular interactions. While IFN-γ supports CD8 T-cell effector function, IFN-γ stimulation also increases the quantity of PD-L1 on melanoma-released EVs that in turn suppressed the effector function of CD8 + T cells [136]. IFN-γ induced tumor dormancy when the interferon-gamma receptor 1 (IFNGR1) expression level was low but resulted in tumor elimination when it was high [137]. GW4869 treatment or Rab27a knockdown can inhibit vesicular-PD-L1 secretion, and significantly augment anti-PD-L1 therapeutic efficacy in 4T1 tumor growth [138]. Animal studies have shown that TEVs can also impair the production of interferons as well as decrease innate immune activity via EGFR-and MEKK2-dependent pathways [139].

Escape Phase TEV Involvement
Clinically recognized tumors have generally moved from equilibrium to escape. In the equilibrium phase, genome instability and accumulation of mutations in cancer cells over time leads to selection for low immunogenicity, expression of immune suppressive ligands, and escape from the immune system [140]. Tumors can eventually overcome antitumor immunity through mechanisms already mentioned, including tumor antigen editing, loss of MHC I expression, and expression of immune inhibitors such as PD-L1 [141][142][143] or suppressive mediators such as IL-10 [144], TGF-β [145], and TRAIL decoy receptors [146,147]. Recruitment and activation of immune-suppressing cells such as Tregs also contribute to escape [148].

Effect of TEVs on Dendritic Cells
Maturation of DCs requires inflammation-related stimuli which stimulate the expression of co-stimulatory molecules such as CD86, CD80, and CD40. TEVs can modify or block the differentiation of immature myeloid cells (IMC) to DC or divert the DCs maturation from IMC to MDSC or M2 macrophage (Tables 2 and 3) by interacting with bone marrow IMC and inducing the production of IL-6, and decreasing expression of CD83 and CD86, as reported for breast cancer, murine mammary adenocarcinoma, and melanoma [149,150]. TEVs also can disrupt DC maturation and T-cell immune response with HLA-G-associated mechanisms in renal cancer [133] (Table 4). Some vesicular proteins such as MALAT1 directly interact with DCs and induce DC autophagy, which decreases DC-mediated T-cell activation [151]. Furthermore, TEV-treated DCs were ineffective at inducing CD4 + T-cell proliferation and activation but promoted differentiation into Treg [152]. TEVs fatty acids can create immunologically dysfunctional DCs by increasing intracellular lipid content by activating the peroxisome proliferator-activated receptor (PPAR) resulting in extra fatty acid oxidation (FAO) which shifts the DCs' metabolism toward oxidative phosphorylation of mitochondria and the disruption of the function of DCs [153][154][155]. It was reported that human prostate cancer-derived extracellular vesicles purified from cultured cells contained PGE2 and triggered the expression of CD73 and CD39 on DCs in vitro, resulting in the generation of adenosine from ATP and inhibition of TNF-α and IL-12-production which reduced T-cell activation [156]. Inhibited antigen recognition, processing, and presentation by interacting with TIM-3 on DCs [142] Ascites of glioma patients PD-L1 Impaired DCs maturation via formation of immunosuppressive monocytes [77] Blood samples from glioma patients GSC20 GSC267 GSC17 MEC-1

LLC PD-L1
Myeloid precursor cells were unable to differentiate into CD11c+ DCs in the presence of vesicular PD-L1 and resulted in DCs death [152] LLC A549 MALAT1 Inhibited DC function and T-cell proliferation and increased DC autophagy via AKT/mTOR Pathway [151] Breast cancer

MDA-MB-231 TS/A Vesicular cargo
Inhibited the development of myeloid precursor cells into DCs by increasing IL-6 production and reducing CD83 and CD86 expression [149] 4T1 PD-L1 Myeloid precursor cells were unable to differentiate into CD11c+ DCs in the presence of vesicular PD-L1 and resulted in DC death [152] Blood samples from melanoma patients 4T1 HSP72 and HSP105 Promoted DCs to IL-6 secretion in a TLR2-and TLR4-dependent manner which activated STAT3-dependent MMP 9 activity [161] Cancers 2023, 15, 82 13 of 26 HSP72 and HSP105 on the membrane of TEVs interact with TLR2 and TLR4 on DCs which induced IL-6 secretion by DCs that increased STAT3-dependent MMP-9 transcription activity in cancer cells resulting in tumor invasion [161]. Galectin-9 on glioblastoma-derived EVs binds to the TIM3 DCs receptor and inhibits antigen presentation by DCs, leading to disrupted antitumor immune responses of cytotoxic T cells [142]. Important DC receptors such as Tim-3 and galectin-9 [157] and SIRPα as the ligand for CD47 were up-regulated on the tumor cells' membranes and derived TEV [143,164]. TLR4 on the DCs decreased after treatment with pancreatic cancer-derived vesicular miR-203 resulting in reduced expression of cytokines such as TNF-α and IL-12, subsequently reducing DC maturation and Th1 differentiation [125]. Besides the vesicular proteins, vesicular miRs also affect DC's function. For example, miR-212-3p transferred to DCs by pancreatic cancer-derived extracellular vesicles suppressed regulatory factor X-associated protein (RFXAP), decreased MHC II expression, and reduced antigen presentation by DCs [165]. Table 4 summarizes reports of TEV impacts on DC.

Effect of TEVs on T Cells
TEVs have a broad array of mechanisms by which they impact T cells. TEVs modify antitumor response by reducing T-cell viability, proliferation, and effector activities [166][167][168]. TEVs can disrupt T-cell effector function indirectly by blocking APC maturation [142,151,152] or directly by inhibiting activated CD8 + T-cell function, inducing CD8 + T-cell death through pro-apoptotic molecules (galectin-group proteins and FasL), promoting Treg expansion, and inducing T-cell exhaustion [169,170]. PD-L1 enriched glioblastoma-derived EVs perhaps surprisingly suppress monocytes rather than T-cells [77]. Nasopharyngeal carcinoma-derived vesicular galectin-9 induced apoptosis in CD4 + T cells via interaction with Tim-3 [171], as well as impairing T-cell function by interaction with TIM3 receptor on DCs in glioblastoma [142]. TEVs can carry pro-apoptotic Bax that induces apoptosis in CD8 + T cells [172] and downregulates JAK3 expression which blocks CD8 + T-cell activation [167,173]. In Treg cell activation, both CD45 negative and positive EVs derived from plasma in head and neck cancer induced Treg differentiation of CD4 cells, but CD45(-) EVs also reduced CD8+ T-cell activation due to their higher adenosine concentrations [174]. EVs generated from multiple myeloma reduced the viability of CD4 + T cells and boosted the proliferation of Treg cells [175].
Pancreatic cancer cell EVs can stimulate p38 MAP kinase signaling in T lymphocytes that causes ER stress, which triggers the PERK-eIF2-ATF4-CHOP signaling cascade resulting in T-cell death [181]. Vesicular microRNAs in the serum of patients with nasopharyngeal carcinoma influenced T-cell differentiation and activation through suppression of the MAPK1 signaling pathway [182], while EVs with a high amount of miR-24-3 reduced CD4 + and CD8 + T-cell proliferation by targeting FGF11 [183]. In addition, mesothelioma cells' EVs carrying TGF-β decreased proliferative response to IL-2 in T effector cells, but not in T-reg cells [184].

HSP70
Altered development and cytotoxicity of T cells in an HSP70-dependent way via miR-21/PTEN/PD-L1 regulatory pathway [170] Blood samples from OSCC patients

PCI-13
FasL Induced apoptotic pathways in T cells through triggering caspase-3 cleavage, the release of cytochrome c that led to disrupting mitochondrial membrane, and decreased TCR-ζ chain production [172] Breast cancer MCF7 CD73, CD39 Inhibited T cells via the adenosine A2A receptor [192] BT-474 MDA-MB-231

Potentials of EVs in Cancer Therapy
TEVs have both immunostimulation and immunosuppression effects [96,97,161], but the potential has not yet been clinically utilized. Admittedly, TEV-focused therapy will be challenging. One challenge to using EVs in cancer immunotherapy is developing a system that provides uniform reagents for clinical use. However, a variety of preclinical studies illustrate the therapeutic potential of TEVs.
TEVs from tumors in the elimination phase stimulated immune cell responses against cancer development [101,104,128], while, not surprisingly, by the time tumor progression occurs, TEVs tend to suppress immune cells and support tumor immune escape. Three general cancer immunotherapy approaches involving TEV can be conceived: I) inhibition of TEV secretion II) increasing the immunostimulatory factors on TEVs' surfaces, and III) using EVs as carriers in cancer vaccines.
Cells under stress produce more immunostimulatory molecules on TEVs and secrete more EVs, which can be caused by treatment-induced stress [210]. Thus some cancer therapies can increase the production of immunostimulatory vesicular factors and this may help to disrupt and even overcome the process of usual tumor immunoediting [211,212]. Opposing increased immunostimulation from tumor EVs, treating with immune checkpoint inhibitors can boost the secretion of immunosuppressive EVs [136].
Hyperthermia is a useful cancer treatment and heat or other stress can modulate TEVs. Heat-stressed B lymphoma cells' EVs possess more IL-6 and IL-17 stimulating molecules such as HSP90, HSP60, HSP70, CD40, and CD86, which can turn Tregs into Th 17 cells [211,213]. Heat stress boosts MHC-I expression on tumor cells [106] and generates TEVs equipped with chemokines such as CCL2,3,4,5, and CCL20 that functionally activate DC and T cells more strongly against tumors [214] thus stimulating "self-vaccination" [215]. Irradiated mouse breast cancer cells' TEVs transmit dsDNA to DCs and induce DC to overexpress costimulatory molecules as well as STING-dependent type I IFN [107] and irradiated melanoma cells' TEVs contained DC activation DAMPs such as HSP70, HMGB, and other stress-related proteins [124]. EVs from Melphalan (a genotoxic drug) treated myeloma cells can boost NK cell IFN-γ production via activating the NF-κB pathway in a TLR2/Hsp70-dependent manner [123]. IFN-γ treated cancer cells secreted a high amount of immune stimulatory TEVs that enhance the number of M1 macrophages by improving their capacity to ingest TEVs and promoting antibody production against cancer cells [216] as well as reducing Tregs and suppressing the expression of PD-L1, VEGF receptor 2, and IDO-1 [217]. In another strategy, modification of the vesicular contents by non-stress methods such as a lentiviral vector encoding two B7 costimulatory molecules (CD80, CD86) increased CD86 and CD80 expression in DCs and induced proliferation of CD4 + T cells, Th1 cytokine secretion, and CTL response [218].
TEVs have been studied as vaccine carriers and employed as immunogens for DC loading. Their immunogenicity in boosting DC-driven anti-tumor immunity was greater than tumor lysate, and they increased splenocyte proliferation and IL-2 release in mouse leukemia and melanoma cancer models [219,220]. In different syngeneic mouse models with large tumors, TEVs equipped with HMGB1 augment DC immunogenicity and elicit long-lasting antitumor immunity and tumor suppression [221]. Overall, EVs from a variety of cell types, including immune cells such as DCs and cancer cells, have the potential as a cancer vaccine and cancer immunotherapeutic [222] such as for colon cancer [2].
TEVs have potential use as drug carriers since they have an affinity for ingestion by cancer cells, are biocompatible and non-toxic with long half-lives in circulation, and their potential has been evaluated [223][224][225]. Some studies carried doxorubicin and in comparison to free doxorubicin, they boosted the therapeutic efficacy [224,225]. Additionally, TEVs carrying doxorubicin and paclitaxel crossed the blood-brain barrier (BBB) as part of in vivo studies [223]. This strategy of using TEVs to carry chemotherapy drugs as a cancer treatment has clinical potential and needs further study.

Conclusions
TEVs cargos are not static during cancer development; they change as tumors evolve and are stressed for various reasons. TEVs modulate immunostimulating or immunosuppressing effects against cancer cells by modifying immune cells during the tumor immunoediting phases [12]. TEVs play an immunostimulatory role in the early stages of immunoediting [8,12,96,101], are more immunosuppressive in the escape phase, and finally, at the late stages, they are more uniformly immune suppressive and play a major role in cancer immune escape [19,156,168]. Normally. the cargo profile of TEVs naturally changes in tumor immunoediting toward immunosuppression, while different cell stress or treatment conditions can inhibit this process or even reverse it to immunostimulation by altering the TEVs cargos profile. Therefore, to benefit from the therapeutic effects of EVs, the secretion of immunosuppression TEVs could be inhibited by disrupting the normal process of immunoediting. Generating EVs to apply therapeutically could be achieved by stimulating the release of immune-activating vesicular cargoes in vitro by using suitable treatment methods and subsequently using EVs in vivo as adjuvant therapy. Since TEV's very diverse cargo profiles depend on the cancer cell condition, understanding the immunotherapeutic properties of TEVs to utilize against cancer should be considered a new research line in oncoimmunotherapy.