An immune checkpoint protein called Programmed cell-death ligand 1 (PD-L1, B7-H1, CD274) has been found in various cells, including tumor cells and some immune cells (1, 2). In the immunosuppressive mechanisms, PD-L1/PD-1 plays a crucial role. When PD-L1 interacts with PD-1 on T cells, the intracellular protein tyrosine phosphatase SHP-2 activates the downstream signal pathway of PD-1, which impedes T-cell activation, that prevents T cells from killing cancer cells by preventing the proliferation and function of T-cells (3, 4). Currently, immune checkpoint inhibitors (PD-L1 or PD-1 monoclonal antibodies) have been utilized to treat various cancers (5). However, researchers are focusing on exosomes because they carry a variety of protein biomarkers linked with a particular disease and hence have the potential to function as early-stage disease diagnostic tools (6).

Exosomes are vesicles of about 40~160nm (on average 100nm) formed by endocytosis and can be secreted by variety of cells. Various molecules are contained within them, including DNA, RNA, protein, lipid and metabolites. Exosomes transport antigens and communicates between cells (7–9). It has been proven that exosomes are found in a wide range of biological fluids, including serum, plasma, breast milk, urine, cerebrospinal fluid, tears, bile and so forth (10, 11). Nevertheless, exosomes do not contain glycolytic enzymes and cytoskeleton proteins, and the release of active DNA does not rely on small extracellular vesicles as carriers. In addition, extracellular vesicles, including exosomes, do not transport double-stranded DNA and DNA-binding histones (12). The classification of exosomes is regulated by primitive cells, but the mechanism remains unclear. Their composition and surface markers reflect their origin. Therefore, in addition to the general exosomal markers that help distinguish exosomes from other extracellular vesicles (such as CD81, CD9, CD63, ALIX and TSG101), it can also detect exosomes based on special membrane surface markers from different cell sources. In addition, exosome lipid content plays a significant role in preserving the shapes of exosomes, participating in biogenesis and regulating the homeostasis of recipient cells (7, 12, 13). Exosomes are closely linked to the immune system’s activation, immunosuppression and immune escape (14). It was reported that PD-L1 exists in the exosomes of melanoma cell lines SK-MEL-28, B16F10, human lung cancer cell lines A549 and human embryonic lung fibroblast cell lineMRC-5 (15).

Exosomal PD-L1 (ExoPD-L1), like other forms of PD-L1 in cells, impedes immune cell function and promotes tumor development (16). ExoPD-L1 suppresses T-cell activities by attaching to PD-1 on the T-cell surface. PD-1 is a security checkpoint molecule that assists in preventing the immune system from attacking healthy cells and tissues. ExoPD-L1 attaches to PD-1 and sends a signal to T-cells, regulating their activity and suppressing the immunological response, leading to cancer cell proliferation. Conversely, PD-1 or PD-L1 inhibitors can be used to halt tumor growth by blocking their activity ( Figure 1 ). Thus it is crucial to understand how ExoPD-L1 contributes to cancer development and the different regulatory factors affecting the expression of ExoPD-L1. In this review, we highlight the role of ExoPD-L1 in immunosuppression, the factors that cause its upregulation, its utility as a marker for cancer diagnosis, detection methods for ExoPD-L1, and its potential as a target for combination therapy in cancer treatment.

Research has uncovered the presence of ExoPD-L1 in various cancer cell lines (7). Therefore, exosomal PD/PD-L1 is assumed to be an intersection of inflammation and tumor progression so, it is necessary to investigate the causing factors and mechanisms that lead to T-cell dysfunction.

It is believed that multiple mechanisms are involved in regulating PD-L1 expression in cells. It includes the following aspects: various oncogenic pathways of the tumor microenvironment, transcription factors and regulation of PD-L1 at post-transcriptional regulation level (different miRNAs) (44–47). The type 3 transmembrane protein CMTM 6 is widely expressed in organisms. Through co-localizing with intracellular PD-L1, CMTM6 reduced PD-L1 ubiquitination to reduce PD-L1 degradation by lysosomes, thereby enhancing PD-L1 inhibitory ability on T cells (48, 49). Another member of the CMTM family, CMTM4, also contributes to PD-L1 expression. It was found that PD-L1 was more effectively suppressed by inhibiting both CMTM4 and CMTM6 simultaneously (49). In recent years, research has focused on the RNA binding protein (RBP) in order to understand PD-L1 regulation. Human antigen R as an RBP binds to the 3’UTR of CMTM6 mRNA that is highly enriched in AU elements (AREs), resulting in more stable CMTM6 mRNA, thereby inducing PD-L1 expression (50). Tu et al. developed a PD-L1 antibody called H1A. Compared to earlier PD-L1 antibodies, it has a distinct mechanism. H1A mainly destroys the binding of CMTM6 and PD-L1, making PD-L1 more susceptible to lysosomal degradation (1). Tristetraprolin, as RBP, is phosphorylated upon oncogenic RAS signaling, reducing its ability to bind to mRNA, thereby reducing its inhibitory effect on PD-L1 (51). Apart from PD-L1 inhibiting immune function by connecting to PD-1, PD-L1 can also be used as an RBP to compete with SC10 and SC4 components of RNA exosome for DNA repair elements NBS1 and BRCA1 mRNA, protecting target RNA from degradation and enhancing resistance to DNA damage. PD-L1 is up-regulated after DNA damage, which is effective for the immune escape of tumor cells and resistance to DNA damage treatment.

The ExoPD-L1 expression has been found to strongly correlate with transforming growth factor (TGF-β) in the tumor microenvironment in breast cancer cells. They revealed that TGF-β increased the ExoPD-L1, which impaired CD8⁺ T cell function. Furthermore, combining exosome loss and TGF-β inhibition enhanced T cell function (52). According to reports, when T cells are activated, they secrete IFN-γ, which could up-regulate ExoPD-L1 and then ExoPD-L1 inhibits T cell’s function, promoting tumor growth. However, it was found that ExoPD-L1 expression is significantly linked with IFN-γ in patients with metastatic melanoma, and ExoPD-L1 inhibits CD8⁺ T cell functions. They also observed that ExoPD-L1 levels were positively linked with IFN-γ and tumor load in patients and that ExoPD-L1 concentration varied during anti-PD-1 treatment (43). In human papillomavirus positive cervical squamous cell carcinoma cell lines SiHa and CaSki showed overexpression of CXCL10 binds to CXCR3, which is highly expressed in human foreskin fibroblasts activates JAK-STAT pathway, which up-regulates PD-L1 and ExoPD-L1 (53). Another study also showed that the level of ExoPD-L1 in metastatic melanoma was higher than that in healthy people and primary melanoma, but the number of exosomes was not notably different (43).

5-Fluorouracil (5-FU) down-regulated the expression of cytotoxic factors IFN-γ, TNF-α, IL-2, IL-6 and GM-CSF in gastric cancer and up-regulated the expression of PD-L1 through the miR-940/Cbl-b/STAT5A axis. At the same time, it significantly up-regulated the expression of ExoPD-L1 (54). Sulfasalazine (SAS) is an FDA-approved cystine/glutamate antiporter xCT inhibitor. The study found that the level of glutamate in the plasma of patients with melanoma increased significantly, which was conducive to tumor growth. SAS inhibited xCT by up-regulating reactive oxygen species, thereby inhibiting tumor progression. However, when combined with xCT inhibition and anti-PD-1/PD-L1 antibody, SAS enhanced the binding of transcription factors IRF4 and EGR1 to PD-L1 promoter, increased PD-L1 mRNA and protein levels, increased tumor ExoPD-L1 level, and reduced CD8⁺ T cell activity (47).

Due to their characteristics and composition, exosomes are being increasingly used as diagnostic tools. Exosomes reflect their parent cell’s characteristics through their distinctive contents. Exosomes from cancer cells possess distinctive genetic and epigenetic makeup which, makes them tumorigenic (55, 56).

Currently, immunotherapy for blocking PD-L1/PD-1 has made remarkable achievements (75). Tumor-derived exosomes are a significant source of PD-L1, and the ExoPD-L1 have a potential role in PD-L1/PD-1 antibody resistance (76). Liu et al. found that PD-L1 expression in melanoma cells could be increased by inhibiting xCT via IRF4/EGR1, thus promoting the secretion of exosome PD-L1. This leads to M2 macrophage polarization and ultimately to the induction of anti-PD-1/PD-L1 therapy resistance (47). Gliblastoma (GBM)-derived stem cells (GSCs) can be enriched by continuous low-dose temozolomide (TMZ) stimulation treatment in GBM. GSCs produced exosomes containing high PD-L1 and interacted with TMZ-sensitive GBM, activated AMPK/ULK1 pathway to promote cell protective autophagy, inhibit cell apoptosis and enhance the TMZ resistance of GBM (33).

Therapies that block the PD-L1/PD-1 pathway have experienced great success in the past, and immunotherapy targeting this pathway has achieved unprecedented, durable response rates in a various cancers (77). Various PD-L1/PD -1 immune checkpoint inhibitors are available in human clinical trials ( Table 2 ) for treating multiple tumor types. PD-L1/PD-1 blocking therapy is primarily mediated by tumor-infiltrating T cells (90, 91). However, sometimes using a single method for tumor treatment will only show a weak effect. In addition, researchers found that some PD-L1 tumor patients still responded to PD-L1 monoclonal antibody (mAb) checkpoint inhibitors while receiving anti-PD-L1 mAb treatment. And this is critical for cancer patients treated with immunotherapy (92). Whether PD-L1 positive or negative solid tumor patients, PD-L1/PD-1 inhibitors or antibodies can inhibit tumor growth and prolong the OS of patients. Therefore, the PD-L1 level is not the only basis for selecting inhibitors (37, 93). Researchers have found that combination therapy usually achieves better results. For example, researchers found that the loss of ExoPD-L1 alone has no obvious inhibitory effect on the tumor. Still when the exosome secretion inhibitor GW4869 and ferroptosis inducer (Fe3⁺) were assembled into nano units and combined with an anti-PD-L1 antibody, the results were significant in enhancing T cell activation, proliferation, and in inhibiting melanoma cell metastasis (94). Yang et al. found that when combined with exosome inhibitor GW4869 or knockout Rab27a and PD-L1 antibody, the therapeutic effect of anti-PD-L1 antibody can be enhanced in a PD-L1-dependent manner and much stronger tumor suppression (26). When mice were injected with TRAMP-C2 cells lacking Rab27a and PD-L1, PD-1⁺ CD8⁺ T and CD4⁺ T cells decreased. Therefore, even a brief deletion of ExoPD-L1 will lead to long-term immunity to tumor cells (21). Moreover, in recent years, bispecific antibodies have also been shown to have great advantages in the treatment process. For instance, a bispecific antibody LY3434172, targeting both PD-1 and PD-L1 at the same time, has a stronger activation effect on T cells than using anti-PD-L1 or PD-1 antibody alone or both antibodies at the same time (95). In another study, researchers developed a bispecific antibody, YM101, that binds both TGF-β (a negative regulator of anti-tumor immunity) and PD-L1. This bispecific antibody simultaneously inhibits the biological effects of TGF-β and PD-L1/PD-1 pathways and has more potent anti-tumor effects than anti-TGF-β and anti-PD-L1 monotherapy (96). However, some researchers believe that more attention should be paid to adaptive immune resistance in subsets of cancer patients to better guide cancer immunotherapy, rather than the endless pursuit of combination clinical trials (97). Therefore, either monotherapy or combination clinical trials should benefit patients.

Evidence has shown ExoPD-L1 promotes tumor progression and metastasis in varieties of cancer cells by suppressing T-cell functions. Moreover, ExoPD-L1 elevations are associated with advanced cancer stages, larger tumor sizes, positive lymph node status, and distant metastasis in NSCLC patients whereas sPD-L1 shows no significant correlation (98). Melanoma patients with ExoPD-L1 have also been shown to have a significant association with tumor response and progression of the disease (15). Multiple variable can up-regulate ExoPD-L1 expression. The IFN-γ and ExoPD-L1 concentrations are correlated positively. When IFN-γ increases ExoPD-L1, it suppresses T-cell activity in metastatic melanoma and glioblastoma (23, 43). In addition, Rab27a and nSMase2 are also responsible for the upregulation of ExoPD-L1 in prostate cancer (21). Therefore, it is essential to identify what triggers the overexpression of PD-L1 in exosomes. Consequently, inhibiting the variables can be beneficial in cancer treatment. Furthermore, it is also crucial to understand the precise molecular mechanism by which ExoPD-L1 suppresses T-cell activity and promotes immunosuppression in cancer cell development. Several inhibitory roles of ExoPD-L1 have been addressed above, the molecular mechanisms involved in ExoPD-L1 mediated immunosuppression are mostly unknown. Therefore, further research is needed to explore the precise mechanism by which ExoPD-L1 suppresses the immune system and promotes tumor growth might be helpful in cancer treatment.

Besides ExoPD-L1, tumor-derived exosomes contain diverse immunomodulators that contribute to tumor progression by impairing immune cell function. However, these molecules can also be noninvasive biomarkers for tracking and treating cancer (36). Exosomal lncRNA RPPH1 enhances colorectal cancer (CRC) metastasis by inducing epithelial-mesenchymal transition (EMT) via interaction with β-tubulin III (TUBB3), blocking its ubiquitination. Exosomal lncRNA RPPH1 levels are closely linked with disease status (99). In a study on breast cancer, TEX containing miR-105 promoted metastasis by disrupting tight junctions in endothelial monolayers. Notably, inhibiting the overexpression of miR-105 in intensely metastatic tumors mitigates these effects (100). TEX-expressing Fas ligand (FasL) suppresses immune function in human and mouse cancer cell lines. They also play a critical role in CD8⁺ T-cell apoptosis; conversely, apoptosis can be encountered by anti-FasL antibodies (101, 102). Tumor-derived extracellular vesicles transfer active TGF-β type II receptors (TβRII) to CD8⁺ T cells, which trigger the activation of SMAD3. This activated SMAD3 associates and cooperated with the TCF1 transcription factor, resulting in CD8⁺ T cell exhaustion, ultimately contributing to immunotherapy failure (103).

In our investigation, we sought a reliable prognostic marker that would enable us to track the course of cancer. The ExoPD-L1 levels are significantly related to the development and response of tumors as well as the survival of patients with metastatic cancer (15, 28, 43). ExoPD-L1 levels tend to rise with tumor progression and are highly connected with overall survival; they can be used as a predictive factor to monitor cancer patients. Moreover, circulating exosomes are more reliable than biopsies and plasma for identifying and quantifying PD-L1. Nevertheless, the size of exosomes and the variability of PD-L1 make it challenging to quantify ExoPD-L1. Thus, it is pivotal to find a precise and simple way to diagnose ExoPD-L1. However, above mentioned effective ExoPD-L1 extraction method ( Figure 2 ) can be utilized to diagnose ExoPD-L1. The association of higher ExoPD-L1 expression with cancer suggests that it could also be considered in the cancer treatment by employing PD-1 or PL-L1 inhibitors ( Figure 1 ). More importantly, exosome loss should also be considered in developing a combination therapy because generally, the immunosuppression of immune cells by exosomes is PD-L1-dependent.

In a nutshell, the existence of ExoPD-L1 in cancer cells is a hotly debated research area that may have implications for the diagnosis and treatment of cancer. Currently, the research on ExoPD-L1 is still in the infant stage; therefore, further studies are required to clarify the role of ExoPD-L1 in cancer and its potential therapeutic application.

Study concept and design: RH and LT. Drafting of the manuscript: RH and MJ. Critical revision of the manuscript for important intellectual content: LT and MJ. Obtained funding: RH and LT. All authors contributed to the article and approved the submitted version.