Tumor-Derived Exosome and Immune Modulation

Abstract

Tumor cells, like most other cells, release exosomes called tumor-derived exosomes (TEX) and are vital for intercellular communication. TEX are membrane-bound extracellular vesicles (EVs), containing unique cargo reminiscent of the parent tumor cells and possess immunomodulatory functions. TEX carries factors that directly promote immunosuppression in the tumor microenvironment and indirectly attract immunosuppressive T-regulatory (Treg) cells. The tumor-secreted exosomes can transfer their cargo by multiple mechanisms like fusion, phagocytosis, and receptor-mediated endocytosis, activating the recipient cells. TEX directly engages and releases cytokines, inactivating natural killer (NK) cells and T-cells and activating apoptosis. Tumor-derived exosomes also release soluble factors to suppress dendritic cell (DC) maturation while activating the expansion of immune-suppressive cells like Myeloid-derived suppressor cells (MDSCs) and Regulatory T (Treg) cells. Several studies have shown the relevance of TEX containing tumor-associated antigens (TAA) in reducing the efficacy of cancer immunotherapy and adoptive cell therapy. Hence understanding the basic biology and mechanism of TEX-mediated immunosuppression is critical in discovering cancer biomarkers and finding better immunotherapy and cell therapy approaches. In this chapter, we have discussed TEX biogenesis, TEX’s structural and molecular features, TEX-mediated immunosuppression, and its relation to immunotherapy.

1. Introduction

James E. Rothman, Randy W. Schekman, and Thomas C. Südhof pioneered and discovered the molecular principles regulating cellular cargo trafficking via extracellular vesicles and were jointly awarded the 2013 Nobel Prize in Physiology or Medicine. Since then extracellular vesicles (EVs)-mediated horizontally transport of cargo across donor to recipient cells, followed by phenotypic alterations in the latter, has aroused significant scientific attention. EVs are lipid bilayer-delimited particles spontaneously secreted practically from all kinds of cells. The EVs contain cargo, including proteins, nucleic acids, lipids, metabolites, and even organelles, representing the parent cell’s physiological state [1, 2, 3]. The terminology and classification of EVs are still emerging. Exosomes are a subgroup of EVs with a size ranging from 30 to 150 nm, produced via the parent cell’s endocytic pathway and engaged in intracellular communication. Exosomes are released from cells upon fusion of an intermediate endocytic compartment, multi-vesicular body (MVB), and plasma membrane. This process delivers intraluminal vesicles (ILVs) into the extracellular milieu and in circulation (Figure 1) [1, 2].

Figure 1.

Exosome biogenesis. The cytoplasmic outer layer protrudes to make up an initial secretory endosome (aka early endosome); intraluminal vesicles (ILVs) grow inwardly into the endosomal lumen constituting the multi-vesicular body (MVB). This process is known as the MVB biogenesis; these ILVs are secreted as exosomes when the MVB merges with the plasma membrane; but a few selected merges with the lysosome for degradation. The cargo of exosomes comprises of lipids, mRNA, miRNA, tRNA, lncRNA, DNA, proteins, adhesion molecules, receptors, and other functional compounds.

The conventional exosome secretion process involves a few key steps: ILVs formation and exosome biosynthesis within MVBs, MVB trafficking, and fusion with the parent cell’s plasma membrane followed by released via exocytosis (Figure 1). Once the exosomes reach a recipient cell, they either engage with the recipient cell’s surface molecules to promote juxtracrine downstream signaling, undergo fusion with the recipient cell’s membrane to deposit their payloads into the cytosol, or are taken by the recipient cells via processes like phagocytosis, macropinocytosis, and receptor-mediated endocytosis [1, 2, 4]. The fate of internalized EVs is still poorly understood and may be determined by exosomal heterogeneity and mode of cellular uptake. Internalized exosomes go to the early endocytic pathway after being endocytosed. Early endosomal membrane fusion may deliver the soluble cargo into the cytoplasm; in contrast, EV-associated membrane proteins undergo retrograde transport to the trans-Golgi network. Endosomal recycling may deliver them to the plasma membrane or be degraded in the lysosomes [5, 6, 7].

The endosomal sorting complex required for transports (ESCRTs) machinery is pivotal for the biogenesis of MVBs and ILVs [2, 8]. Exosomes contain distinctive cargos, including DNA, messenger RNAs (mRNA), micro RNAs (miRNA), transfer RNAs (tRNA), long non-coding RNA (lncRNA), proteins, lipids, and metabolites, among other biologically active molecules. These payloads are carefully processed and packed into the exosomes. The contents vary with each type of cell and are influenced by different cellular phenotypes and metabolic states, thereby imparting differential biological functionality [7, 9]. Exosome protein composition analysis has indicated that certain proteins are exclusive to the cell and tissue of origin, while others are found in all exosomes. Among some of the reported, 9769 exosomal proteins (exocarta.org) are conserved, and some are cell type-specific, like the major histocompatibility complex (MHC) class-I and class-II, other cell surface receptors, and proteases. Exosomes include proteins associated with membrane transport and fusion (e.g., annexin, nuclear-related protein Rab family GTPase (Rab-GTPase), SNAREs, and heat shock proteins (HSPs). While exosomes also have membrane-associated proteins (Tetraspanins, ICAM, etc.), MVB-related proteins (ALIX and TSG101), and other proteins such as actin, myosin, and adhesion molecules such as integrins. Specific proteins are widely used as exosomal markers, including the tetraspanins (CD9, CD63, CD81, CD82, Tspan8, CD151), Alix, and Tsg101 [5, 8].

Exosomes also include cell-specific or conserved lipid content (like cholesterol, sphingomyelin, phosphatidylserine, and saturated fatty acids). Lipids are involved in exosome biosynthesis as well as maintaining homeostasis in recipient cells, in addition to safeguarding exosome structure. Additionally, exosomes contain a variety of RNAs that are active and can influence the transcriptome of recipient cells [10]. Exosomes contribute to maintaining cellular homeostasis and cell-to-cell communications and are secreted by cells in normal physiological and pathological settings [5, 11].

There is an unprecedented need to study the role of exosomes and TEX to understand tumor progression that can aid in cancer diagnosis, prognosis, and therapeutic interventions. Tumor cells are reported to secrete more exosomes than healthy cells, thereby inhibition of exosome production, release, and reduction of circulating level may be an effective cancer therapy approach [12, 13]. Understanding the interaction of cancer cells with the body’s immune system is key to cancer immunology and immunotherapy success. Immune suppressive features exist in the tumor microenvironment, limiting responses to immune-regulated assaults on the tumor [14]. Several immune cell types get functionally specialized and activated to fight and neutralize tumor cells and tumorigenesis. On the contrary, the tumor cells either evade immune identification, or induce an immunosuppressive tumor microenvironment (TME) to thwart the immunological onslaught. The tumor cells strategically use TEX-driven immunosuppression in the TME and within the tumor [15].

Recent studies show that tumor-derived exosomes are widely generated and contain a wide range of immunosuppressive chemicals. Here the role of TEX is extremely significant in intercellular communication and TME remodeling. This remodeling has the potential to aid cancer cells in avoiding detection by the immune system. According to several studies, TEX released by tumor cells modulates tumorigenesis, metastasis, and angiogenesis and facilitates drug resistance [14, 15, 16]. For example, when tumor cells are subjected to hypoxia, they release exosomes with increased angiogenic and metastatic potential, supporting the concept that tumor cells respond to a hypoxic milieu by releasing exosomes to promote angiogenesis or allow progression of the tumor cells to a more suitable habitat [16, 17]. TEXs are implicated in regulating the bioactivities of their target cells via the transmission of their oncogenic cargo. EVs/exosomes have also been termed “oncosomes” in these circumstances, and they transport active proteins, lipids, and nucleic acids to recipient target cells and control gene expression, therefore regulating their function. These exosomes may potentially aid in the establishment of metastatic niches. The molecular basis driving immune avoidance and the development of metastatic niches is still ambiguous [9, 15, 18]. In the tumor microenvironment, TEXs are copiously secreted and transport a range of immunosuppressive molecules. Associations between TEXs and immune cells can suppress immune cell function and the anti-tumor immune system, both directly and indirectly. Thus, TEXs are currently being investigated as prospective candidates for cancer immunotherapy due to these features and the discovery that large quantities of these TEXs in cancer patients correspond with tumor load and progression of the disease [15, 18].

2. Structural and molecular features of TEX

Electron microscopic (EM) evaluation suggests that the structural features of tumor-derived exosomes are comparable to that of most other exosomes [10]. Like many other cells, tumor cells release TEX, which are spherical membrane-bound vesicles that frequently have a diameter ranging from 30 to 150 nm, forming an aggregation of varying sizes. Considering that exosomes carry cell-type-specific molecules, it has been proposed that TEXs vary from normal healthy exosomes in regards to their structural, molecular, and biomechanical features [19]. TEX have lipid bilayer membrane structures that include transmembrane proteins and receptors. Numerous proteins, nucleic acids, and a diverse range of compounds can be discovered within the exosomal cavity, specific to the cancer cell type. The nucleic acid constituents have a role in intracellular transmission, chemotherapy resilience, micro-angiogenesis, tumor microenvironment alteration, immune response modulation, and tumor invasion and metastatic stimulation [16].

According to several proteomics studies, TEX contain membrane proteins, Rab family proteins, annexin, proteins associated with the Endosomal Sorting Complexes Required for Transport (ESCRT) complex like Alix TSG101, MHC molecules, heat shock proteins, and tetraspanins (CD63, etc.), all of which are endosomal pathway proteins. TEXs have also been reported to include tumor antigens such as Mart1, gp100, TRP, and Her2-neu, TGF-β, FasL, TRAIL (TNF-related apoptosis-inducing ligand), and beta-glycan [10, 19, 20]. TEX carry molecular cargo (Table 1) that comprises of specific lipids, MHC components, tumor-associated antigens, and other proteins derived in part from the surface of parent tumor cells [10, 15, 16, 19, 20]. These suggest that they can either activate or repress the immune system, although several studies reveal that TEXs enhance immunosuppression in the tumor microenvironment. Exosomes secreted by primary tumor cells can be transmitted to distant metastatic organs before tumor cells arrive at their final destination, according to growing data. This procedure allows for the formation of an accommodating pre-metastatic niche that promotes the proliferation of disseminated tumors. TEXs have immunosuppressive and immunostimulatory effects that are not limited to the tumor microenvironment [15, 16].

TEX Cargo	Types of molecules	Functional activity
Protein	Heat shock proteins; Membrane transport and fusion related proteins; Enzymes; Tetraspanins; ICAM; MVB related proteins; Cell adhesion; Proteins; Receptor proteins	Regulation of proliferation, growth, metastasis, migration, angiogenesis, adhesion, immunological suppression, and a variety of other physiological processes related to the tumor cells; also modulate immunotherapy effects
Lipids	Phosphatidylserine; Lactosylceramide	Can be potentially used as biomarkers in many cancers
Nucleic acids	DNA; RNA miRNA mRNA; lncRNA circRNA	Enhancing proliferation and growth of tumors, promoting angiogenesis, inhibiting immunological activities, and increasing metastasis all contribute to cancer and influence immune response stimulation

Table 1.

Cargo of TEX with their molecular features and functions.

Ubiquitination is a feature of normal exosomal/TEX proteins that permits them to be identified by ESCRT-0, whereas deubiquitination is critical in sorting them into ILVs. Membrane transport and fusion proteins such as annexin, Rab-GTPase, and HSPs such as Hsp60, Hsp70, and Hsp90; Tetraspanins such as CD9, CD63, CD81, CD82, CD106, Tspan8, intercellular adhesion molecules (ICAM); MVBs associated protein such as ALIX and TSG101; and certain other proteins such as integrins, cytoskeletal construction proteins like actin and myosin are content of TEX [6, 15, 20]. These proteins are critical for exosome functioning. Since tumor cells are always under stressful conditions such as hypoxia, acidosis, nutrient shortage, etc., Hsp90 expression is high in numerous cancer cells. Hsp90 is linked to poor tumor prognosis and tumor development in breast cancer, pancreatic cancer, and leukemia. A study suggested that certain TEX’s expressed surface TGF-β and beta-glycan, which can stimulate the SMAD signaling and govern fibroblast development into myofibroblasts [19, 21].

Exosome-bound miRNAs may also aid tumor growth in a number of ways.

By decreasing the expression of E-cadherin in normal fibroblasts, miR-9 in exosomes originating from triple-negative breast cancer cells (TNBC) might increase tumor cell migration and enhance the transition of fibroblasts into cancer-associated fibroblasts. Exosomal miRNAs of mesenchymal stromal cells have also been demonstrated to be transported directly to tumor cells, promoting cancer growth and inducing treatment resistance in multiple myeloma, colorectal, and gastric cancer cells [6, 21]. lncRNA is a newly discovered regulatory RNA that can be packed into exosomes and functions as a messenger in intercellular communication to control tumor development and other related processes while also reshaping the tumor microenvironment. lncRNAs-ATB, for example, a new cancer-associated lncRNA that was anomalously exhibited in many cancers, is known for enhancing tumor progression and growth primarily by competitively anchoring miRNA to stimulate epithelial-mesenchymal transition (EMT) [21].

Although prevailing data indicates that TEX may have different immune-triggering/immunosuppressive activities based on the cargo they transmit and the functional capacity of immune cells in the tumor microenvironment, reconciling these two opposing factors of exosome features has proven to be challenging. TEX can potentially manipulate a wide variety of functions in target cells simultaneously, and the consequence is determined by the content of their cargo and the target cell’s potential to accept or decline the conveyed signals.

3. TEX and immune modulation

The TEX are responsible for the tumor proliferation, metastasis, and antitumor response through immune and non-immune pathways. On the one hand, TEX interacts with immune cells and conveys negative signals, thereby interfering with their antitumor functions, while on the other hand, TEX promotes tumor development and facilitates tumor escape by reducing immune effector cell activity [20]. TEX transport immunoinhibitory, immunosuppressive mediators, and costimulatory molecules (like cytokines, MHC I and II, etc.) that directly or indirectly regulate immune cell formation, maturation, and antitumor activity [15, 20]. These molecules with unique cell surface motifs assist TEX in modulating the immune response, which works by the coordinated action and networking of different components. Human cancer cell exosomes may induce inter-and intracellular signals to the tumor microenvironment modulating immune cell infiltration (Figure 2). Recent studies show that it can suppress the immune response both in antigen-specific and non-antigen-specific fashion. For example, TEX can induce apoptosis by the transfer of FasL and TRAIL to activated T cells [22].

Figure 2.

Role of TEX in cancer progression. TEX are responsible for transferring oncogenic proteins and nucleic acid. It can promote angiogenesis and thrombosis by activating the endothelial cells. Also, it helps in the conversion of MSCs and fibroblast to myofibroblast to promote angiogenesis and metastasis. Further, it impairs the immune response by directing apoptosis in T cells and NK cells, promoting Treg cell activity, expanding MDSC, and inhibiting DC differentiation and function. Also, it assists in developing drug resistance by delivering multidrug resistance proteins and miRNA. Moreover, it helps in neutralizing antibodies and expelling the anticancer drugs. DC: dendritic cell; MDSC: myeloid-derived suppressor cell; Treg: regulatory T cell; Breg: regulatory B cells; M2 Mϕ: M2 macrophage; NKT: natural killer T cells; NK: natural killer cells; MHC: major histocompatibility complex; TEX: tumor-derived exosomes; TGF-β: transforming growth factor-beta; IL-10: interleukin 10; TNFα: tumor necrosis factor-alpha; TNFR1: tumor necrosis factor receptor 1; TRAIL: TNF-related apoptosis-inducing ligand; TRAIL R2: TRAIL receptor 2; PGE₂: prostaglandin E2; HSP: heat shock protein.

TEX include membrane-bound NKG2D ligands including MICA, MICB, or ULBP1-6, which may directly suppress NK and CD8+ T cells [23]. Also, TEX is known to suppress the expression of CD3- ζ chains in T cells to prevent their activation, and NKG2D inhibition in natural killer (NK) cells prevents NK cell-dependent toxicity [24, 25]. Tumor-derived exosomes may also suppress the anti-tumor immune response by producing prostaglandin E2 (PGE₂). In the presence of TGF-β, PGE₂ promotes the growth of myeloid-derived suppressor cells (MDSCs) and their suppressive function. PGE₂ also inhibits NK cell cytolysis and IFN-γ production, as well as T cell IL-2 production and responsiveness [26]. TEX can also modulate the antigen-presenting cells; for example, TEX miRNA may bind to TLRs, triggering an inflammatory response. For example, miR21 and -29a secreted from exosomes of lung cancer cells bind to the human and murine TLRs and stimulate the secretion of pro-inflammatory cytokines like IL-6 and TNF-α [27]. TEX can also disrupt the differentiation of peripheral blood monocytes into functional dendritic cells. For example, TEX-released by colorectal and melanoma cells, for example, was shown to impede CD14+ monocyte differentiation into dendritic cells instead of causing them to highly immunosuppressive MDSCs (Figure 2) [25]. Tumor-derived exosomes have emerged as an important factor in the loss of antigen presenting cell function and decreased anti-tumor immune responses in patients with cancer [28].

The miRNA, HSP 70, prostaglandin E2, and TGF-β are found in the TEX and play an essential role in the differentiation of the monocytes [29]. It has been reported that the above factors can also be transported distantly by the TEX towards altering the function and differentiation of myeloid cells for favoring the MDSCs at the metastatic sites [30]. After that, MDSC induces the regulatory T cells (Treg), which play a crucial role in the tumor-suppressive microenvironment. The CD4+ T cells are directed towards the Th2 and Treg due to the expression of cytokines, TGF-β, MMPs, and growth factors in MDSCs [31]. TEX has been shown to convert the CD4+FoxP3+ T cells into Tregs via IL-10 and TGF, which are very suppressive and resistant to apoptosis [32]. Also, it has been reported that CD11b+ TEX in the tumor-bearing mice can suppress the specific response to tumor antigens via the MHC class-I independent and MHC class II-dependent pathways [33]. It suggests that TEX first stimulates the antigen-presenting cells containing CD11 in the tumor microenvironment, which then secretes the CD11b and MHC class II immunosuppressive vesicles in the circulation. Adenosine synthesis in T cells was reported to be increased by Treg coincubated with TEX, which have CD39 and CD73 ectonucleotidases [34, 35]. TEX-mediated adenosine production is implicated in suppressing activated B cells and may in-turn activate B cells into regulatory B cells (Figure 2).

Exosomes may promote innate and adaptive immunity, as seen in infected macrophages, which produce exosomes containing bacterial cell wall components that activate uninfected macrophages [36]. Among the adjuvants found in tumor exosomes is heat shock protein 70 (hsp70), which may stimulate anti-cancer immune responses. Researchers discovered that Hsp70/Bag-4-positive human pancreatic and colon cancer cells secrete exosomes that promote the migration and cytolytic activity of NK cells [37]. They also showed that Hsp70-positive exosomes operate in macrophage activation, as measured by TNF production [38]. Biomolecular cargo found in exosomes from DCs might facilitate the development of cell-free DC-based cancer vaccines [36, 39]. Exosomes from other cellular sources may also activate an immune response. When human alveolar epithelial cells were treated with TNF-α+ mature DC exosomes, they in turn produced inflammatory mediators such as IL-8, MCP-1, MIP-1, RANTES, and TNF-α as a result [39]. Advanced stage NSCLC or metastatic melanoma patients who received DC exosomes in phase I clinical trials showed enhanced NK cells activity [40, 41]. Injection of DC exosomes restored NKG2D levels in patients with metastatic melanoma, and tumor regression in mice was encouraged [42]. Though exosomes and TEX might induce immune activation or immunosuppression in the tumor microenvironment, but most reports for TEX suggest an immunosuppressive mode of action.

4. Mechanism of TEX-mediated immune suppression

Immunostimulatory TEX from tumors may spread to distant tissues and organs, impairing systemic anti-tumor immune responses. Signaling molecules in the tumor microenvironment aid in tumor development and inhibits the immune response, with T cells being highly vulnerable to TEX-driven negative messages [7, 43]. Unlike other leukocytes which engulf other cells, T cells interact with the TEX using the ligands and surface receptors. It leads to the signal-driven influx of Ca²⁺ in the cells and activation and suppression of pathways along with downstream modified responses. Two major receptors on the T cells that are negatively regulated by the TEX are the interleukin 2 receptor (IL-2R) and T cell receptor (TCR) [44]. The TCR zeta chain is downregulated consistently on the incubation of TEX with the T cell. In addition, JAK expression and phosphorylation are diminished, which is responsible for the production of IL-2, IL-7, and IL-15, all of which are deleterious to T cell proliferation. Further, TEX downregulates the proliferation of CD4+ T cells but upregulates the expansion of CD8+ T cells [45]. However, in the case of normal cell-derived exosomes, the proliferation of all T cells is experienced [46]. Also, TEX regulates the STAT5 function in the T cells, as STAT5 phosphorylation in the case of CD4+ T cells increases while the phosphorylation of STAT5 in CD8+ T cells decreases [47]. The level of CD69 on the surface of CD4+ T cells is also reduced due to TEX immunosuppressive function.

T cell inhibitory and apoptotic receptors are directly engaged by tumor-derived exosomes (TEX). To enhance T cell death, TEX expresses Fas ligand (FasL), TNF-alpha, TRAIL, and Galectin-9 that interacts with counterpart T cell receptors like FAS, TNF R1, TRAIL R2, and Tim3 [48]. The TEX is also found to inhibit the antigen-specific T cell, as in the case of human melanoma, wherein specific T cells are generated via the melanoma-specific pulsing of the DC cells. Also, evidence suggests the presence of membrane-bound FasL and PD-L1 on TEX [49]. TEX-mediated apoptosis of CD8+ T cells is associated with canonical features like the caspase 3 cleavage, annexin V binding, loss of mitochondria membrane potential, DNA fragmentation, and cytochrome C release [45]. It suggests the involvement of extrinsic and an intrinsic mechanism for the cascade of apoptosis of the CD8+ T cells. Also, the PI3K/AKT pathways are the main target for TEX-driven apoptosis in CD8+ T cells: AKT dephosphorylation in a time-dependent manner decreases the expression of BCL-xL, BCL-2, and MCL-1 along with the increase in BAX was observed when TEX was incubated with CD8+ T cells [50]. All these data indicate that TEX may not be internalized by the T cells; instead, the negative signaling by the surface receptors modulates the function of T cell response. Also, TEX-driven transcription changes are regulated by the type of recipient cell, activation status, and presence or absence of exosomes. TEX is also responsible for the apoptosis of CD8+ T cells with the help of death ligands and interaction via the Fas/FasL pathway [51]. Also, it has been shown in the lymphoma animal model that TEX protects cancer cells from complement-dependent cytolysis by binding to the complements [52].

Cancer cells upregulate PD-L1 to avoid immune identification by causing anergy in PD-1+ T cells. Although immune checkpoint inhibitors have shown extraordinary effectiveness, most patients do not react to PD-1/PD-L1 inhibition. Paracrine immunosuppression may now include recently discovered exosomal-PD-L1 as well as cell-to-cell interaction [53]. By both direct and indirect means, exosomes seem to be capable of immunomodulating PD-L1 (Figure 3). Recent research shows that PD-L1 is active on exosome membranes and may promote tumor development by inhibiting CD8+ T cell proliferation and cytotoxicity [53]. Exosomes from human breast cancer cells contain PD-L1, while PD-L1 knockout (KO) cells do not [54]. In a unique experiment, two groups of mice were treated with exosome-expressing PD-L1^FLAG and exosome-expressing no PD-L1 (PD-L1-KO). Animals treated with exosome-expressing PD-L1^FLAG showed considerably larger tumor volumes than mice treated with exosome-expressing no PD-L1, demonstrating that exosomal PD-L1 stimulates breast cancer tumor development. Exosomes expressing PD-L1 dramatically reduced T cell death, demonstrating that PD-L1 signaling may limit T cell killing of cancer cells [55]. Exosomes isolated from the WM9 and WM164 human melanoma cell lines express PD-L1, and may function as an anti-PD-L1 antibody sink [34, 38]. Chen et al., also reported exosomal PD-L1 in human non–small cell lung cancer (NSCLC) and breast cancer cell lines. Recent findings suggest that PD-L1 exosome’s immunosuppressive effects are not limited to the tumor microenvironment, and that exosomes might cause systemic alterations in adaptive immune components (Figure 3) [49]. Functional modifiers that stimulate PD-L1 expression in target cells might be delivered via tumor-derived exosomes. T cell dysfunction may be caused directly by PD-L1+ monocytes or macrophages as a result of a PD-L1-PD-1 interaction [56]. Further investigation is needed regarding exosomal PD-L1 and exosome-induced PD-L1 in immune modulation and cancer. Exosomal-PD-L1-based paracrine immune regulation may help create novel therapeutic options.

Figure 3.

TEX and PD-L1: By activating the PD-L1/PD1 axis, cancer cells adapt and exploit the immune system to elude immune monitoring. a. Exosomal-PD-L1 interacts with T-cells through paracrine signaling, inactivating T cell effector activity. b. Induction of functional PD-L1 in target cells by exosome cargo (miRNA). The PD-L1 on the surface then interacts with T cells, inactivating effector activity.

Another interesting aspect of TEX-mediated immunomodulation involves the anti-phagocytic surface proteins called ‘do not eat me’ signals, such as the cluster of differentiation (CD) 47 help healthy and normal cells evade macrophage-mediated phagocytosis, while a loss of ‘do not eat me’ signals in apoptotic or senescent cells leads to their systemic clearance [57]. Cancer cells use a similar technique to avoid macrophage-mediated clearance by overexpressing ‘do not eat me’ signals on their surface. TEX overexpressing CD47 renders phagocytic inactivation by interacting with SIRPα of immune cells and thereby enjoys prolonged circulation [58]. Downregulation of CD47 expression or inhibition of CD47-SIRPα can be an interesting approach to activate cancer cell/TEX phagocytosis [59].

5. TEX and cancer immunotherapy

There is a controversy regarding the role of exosomes in immunotherapy due to their immunostimulatory and immunoinhibitory action. However, it is imperative that TEX is responsible for the impairment of immune response and take part in the active progression of cancer as it contains several immunoinhibitory, tumor antigens, and invasive molecules to directly or indirectly suppress the immune system. It suppresses the proliferation and differentiation of the immune cells, including the remodeling of genetic materials. For example, tumor-associated antigens (TAA) bind to the antibodies produced against the cancer cells, thus reducing its efficacy in reaching the primary cancer tissue [18]. The efficacy of trastuzumab is severely affected by the TAA cargo of TEX [3]. Also, TEX has been found to play a role in inhibiting antibody-dependent cell-mediated toxicity (ADCC), which is majorly responsible for cancer prevention and the primary activity of humanized antibodies [60]. Evidence suggests that TEX also neutralizes the beneficial effect of immunotherapies. For example, TEX carrying HER2 or other TAA lessen the potential of antibody-driven immunotherapy. The TEX are present in all body fluids, which can neutralize the therapeutic antibodies, thus blocking access to the tumor. Also, the engineered T and NK cells are susceptive to the TEX carrying immunoinhibitory ligands like FasL, which is majorly responsible for the apoptosis of the adapted T cells [22]. The relapsed case of myelogenous leukemia showed the enriched presence of exosomes with immunosuppressive cargo in the plasma. These patients also responded poorly to the adoptive NK92 therapy [61]. It was speculated that a negative immunosuppressive exosome-based signaling was behind the failure of natural killer-based adoptive cell therapy. This was later confirmed by co incubating the patient-derived exosomes with NK-92 cells, wherein function and antileukemia activity of NK-92 cells were severely affected, while the vice-versa was experienced on the blocking of immunosuppressive exosomes in ex-vivo studies. Thus, it was established that TEX interferes with the immune cells and limits the therapy’s therapeutic efficacy. The new target of checkpoint inhibitors is the TEX PD-L1 to mitigate the resistance faced by the current antibody approaches [62]. It could also be used as a predictor for anti-PDL1 therapy.

Also, the Tim-3 and Galectin-9 is the upcoming checkpoint inhibitor that negatively regulates the antitumor immune response [63]. The TEX are also responsible for the variations in the PD-1/PD-L1 treatment in different patients. The low surface expressivity of PD-L1 may be the reason for the low response or resistance to immunotherapy [64]. Also, there is less information on whether the exosomal PD-L1 function varies with cancer type or not. The role of exosomal PD-L1 is to be elucidated further, which may help understand the early diagnosis and better therapeutic outcomes. Exosomes usually confer resistance by manipulating the cell-cell communication in the tumor microenvironment [10]. For example, the exosomes emanating from the macrophages are responsible for the drug resistance in ovarian cancer treatment in hypoxic conditions.

Further, HER2 overexpressing exosomes were found to neutralize the effect of trastuzumab [3]. In gastric cancer cells, the miRNA-21 containing exosomes secreted by the M2 macrophages were found to inhibit the action of cisplatin [65]. The miRNAs containing exosomes are responsible for converting the monocytes to MDSCs. It has also been shown in the breast cancer model of animals that exosomes release is responsible for developing the premetastatic niche in the lungs under the influence of chemotherapy [66, 67]. Thus, the role of TEX in the development of protumor immunity and progression of metastases is involved. Also, the immunosuppressive molecules affect the antitumor activities and maturation of immune cells. Although the dual role of TEX is studied like one is the development of metastatic niche and increasing the invasiveness of cancer cells while on the other side is helping in inducing the tumor-specific immune response for the cell lysis. Thus, the role of TEX in controlling the effectiveness of immunotherapy is an interesting area to be explored.

6. TEX and cancer vaccine

The use of exosomes in cancer treatment is made possible by the fact that exosomes can serve as delivery vehicles for genes and biological therapeutics. Tumor cells must be targeted explicitly while limiting adverse effects on healthy tissues to treat cancer effectively. Due to their ability to deliver their contents inside cancer cells, exosomes play a significant role in improving the therapeutic index of cancer treatment [68]. Exosomes exhibit these abilities because of the multivalent display of their surface fractions derived from cells, and it is impossible to recreate this complexity in synthetic nanoparticles [68]. Researchers have researched the development of therapeutic cancer vaccines, also known as active specific immunotherapy, and discovered that exosomes offer a great deal of promise for cancer immunotherapy and therapeutic cancer vaccines [69]. Exosomes from different cell types, such as immune cells, cancer cells, and normal cells, are compared for their effectiveness of exosome-based cancer immunotherapy. B cells secrete exosomes harboring MHC class II peptides that aid in antigen presentation to CD4+ T lymphocytes. Dendritic cell-derived exosomes (DEXs) play a critical role in anti-tumor immune response and may trigger a particular Cytotoxic T lymphocytes (CTLs) response and activate a T cell-dependent anti-tumor response [13].

TEXs, are modified for cancer vaccine development as they are a natural source of tumor antigens and can activate APCs to display them effectively [68]. Several studies have reported positive outcomes with TEX-based vaccination and observed activation of T-cell mediated antitumor immune reactions and tumor reduction [70, 71]. Researchers found that TEX immunization not only protected against tumor development and stimulated Th1 immune responses in melanoma animal models but also might limit lung metastasis [72]. In another study, immunization of syngeneic mice with exosomes generated by L1210 leukemia cells reduced tumor growth and provided resistance against subsequent tumor challenges [73]. In another study, both T lymphocyte proliferation and specific CTL activity were all stimulated when treated with HeLa cell-derived TEX [74]. Though many studies support the use of TEX-based vaccination, it is unlikely that TEXs alone can initiate acceptable levels of anti-tumor immunity due to their role in immunosuppression and the low immunogenicity of their components. Multiple strategies are devised to maximize antigen immunogenicity for effective tumor vaccination. TEXs might be modified by the following approaches (a) incorporating electroporated siRNA, (b) engineered to express tumor-associated and pathogenic antigens concomitantly, (c) tagging known immune boosters like CpG DNA and TLR ligands, (d) Direct fusion of TEXs with antigens, and (e) using external stimulus to increase TEX release [68, 75]. Innovative approaches are needed to make TEX-based cancer vaccines a reality.

7. Conclusion

Exosomes play a crucial role in facilitating the cell-cell environment under normal and pathological conditions. TEXs are emerging as the key immunoregulatory players for cancer cell-to-tumor microenvironment communication, influencing tumor development and metastasis. It’s exciting and intriguing that the TEX can train immune effector cells to suppress or stimulate the immune system. TEX-driven interactions may be direct or indirect, and the presence or absence of immune recipient cells in the TME may alter the outcome. Oncotransducers, which generate juxtracrine or paracrine signals, may alter suppressive pathways formed in immune recipient cells and resulting in faster tumor growth. Immunotherapies are unlikely to function at its full potential due to TEX -based antibody sequestration. Reports also suggest that TEX-induced immunostimulatory signals can modify the TME to enhance immune activation rather than tumor progression. Hence more investigations are warranted. TEX-engineering to activate the anti-tumor potentials may lead to future therapeutics. Presently, the key challenge of the tumor biomarker identification and validation procedure is dependent on decoding the messages in TEX cargo and correlating them to clinical data. The practical usage of TEX may revolutionize cancer detection, diagnosis and therapy.

Acknowledgments

M.K.P. acknowledges S. Dubinett, B. Gomperts, and V. Hartenstein for providing constant support and mentoring.

Conflict of interest

The authors declare no conflict of interest.