The human umbilical cord is a promising source of MSCs (101). Different from bone marrow stem cells and adipose-derived stem cells (ADSCs), hucMSCs have been associated with painless collection, easy acquisition (102), more primitive cells, higher proliferative ability, less immune rejection (103), and ability for faster self-renewal (104) ( Figure 3 ). The hucMSCs differentiate into various cells in three germ layers (e.g., bone, cartilage, fat, skeletal muscle, myocardial cells, endothelial cells), and synthesize and secrete a group of trophic factors and cytokines (103). They also support the expansion and function of other cells (e.g., hematopoietic stem cells, embryonic stem cells, NK cells, islet-like cell clusters, neurons, and glial cells), and can migrate to and return to the pathological area (105). Evidence has shown that hucMSCs-derived exosomes (hucMSCs-Exo) have similar functions to hucMSCs, with low immunogenicity and non-tumorigenicity (102). As a new cell-free alternative therapy, hucMSCs-Exo have been widely used in regenerative medicine and cancer treatment (106, 107). Below, we introduce the application of hucMSCs-Exo in the treatment and diagnosis of cancer in various systems of the body.

BMMSCs are the first MSCs identified (84). They have been described as the progeny of fibroblasts possessing colony-forming ability and differentiation potential (170, 171). In addition to BMMSCs-specific markers (CD73, CD90, CD105) and negative surface markers (CD11b, CD14, CD19, CD34, CD45, CD79a), and human leukocyte antigen-DR (HLA-DR), human-derived BMMSCs also express other markers (CD10, CD29, CD44, CD133) (171). BMMSCs are multipotent cells that can differentiate into osteoblasts, chondrocytes, and adipocytes (172). They are widely used in the treatment of various diseases due to their self-regeneration, differentiation, and immune regulation (downregulation of T cells, B cells, NK cells, and antigen-presenting cells through various mechanisms) (173). Compared with hucMSCs and ADMSCs, BMMSCs have high potency for clinical use in the treatment of various diseases (e.g., bone and cartilage, immune system, nervous system, cardiovascular, viral/infectious, cancer, wounds and injuries). The exosomes produced by BMMSCs can also be utilized for this purpose (174, 175). Compared with BMMSCs, BMMSCs-derived exosome (BMMSCs-Exo) have smaller volume, are associated with less immune rejection, and can easily transport therapeutic agents, thus playing a great role in overcoming resistance to cancer treatment ( Figure 3 ). Consequently, BMMSCs-Exo show promise in the treatment of cancer.

ADSCs exhibit positivity for tumor susceptibility 101 (TSG101), CD63, CD9, CD13, CD29, CD44, CD73, CD90, and CD105 (216); in contrast, they show negativity for calnexin (CANX), CD31, and CD45 (217). Compared with BMMSCs, ADSCs have a longer life span, higher proliferative ability, shorter doubling time, and later senescence in vitro ( 218). Furthermore, the collection of ADSCs is more convenient and less invasive, while the yield is larger. Although ADSCs offer multiple advantages, their clinical application is limited due to the possible promotion of tumor development ( Figure 3 ). Subsequent research revealed that the promotive effect of ADSCs on cancer is attributed to the adipose tissue around the tumor and its progenitor cells (219). ADSCs exhibit selective tumor homing ability, rendering them a suitable vehicle for anti-cancer drug delivery (143). By improving the targeting ability of drugs, it is also possible to improve the treatment efficiency and safety of high-dose use (220). ADSCs are often used in regenerative medicine and autologous transplantation, and have great potential for tissue regeneration and wound repair. In recent years, with the continuous development of nanotechnology, ADSCs-Exo have attracted increasing attention. ADSCs-Exo possess many therapeutic bioactive factors unique to stem cells, which can accelerate wound healing and are essential for tissue repair (221). They also play a key role in enhancing cell regeneration (222), promoting angiogenesis (223), regulating inflammation, and remodeling the extracellular matrix. ADSCs-Exo are often used in tendon repair, corneal skin regeneration, treatment of diabetic skin injury, regulation of inflammation and angiogenesis, fracture healing, etc (224).

ADSCs-Exo are an ideal potential drug delivery carrier with broad application prospects in tumor therapy (225). Lou et al. reported that ADSCs-Exo can be used as an effective carrier for the delivery of miR-199a. They can effectively improve the sensitivity of HCC cells to doxorubicin by targeting the mechanistic target of rapamycin kinase (mTOR) pathway (226). Studies have also shown that the delivery of miR-122 through ADSCs-Exo provides a new idea for improving the sensitivity of HCC to chemotherapy (227). Rezaeian et al. showed that ADSCs-Exo could affect prostate cancer, bladder cancer, and renal cancer cell lines. The 5637 cell line of primary bladder tumor, ACHN cell line of metastatic renal adenocarcinoma, LNCaP cell line of metastatic prostate cancer, and the prostate adenocarcinoma PC3 cell line were used. It was found that ADSCs-Exo exert a synergistic apoptotic effect on LNCaP, PC3, and 5637 cells, but not on ACHN cells. This difference was attributed to the increase in tumor protein 53 (TP53) expression and decrease in BCL2 gene expression in the PC3, 5637, and LNCaP cancer cell lines treated with exosomes (228). Liu et al. used ADSCs-Exo as a vector to deliver tumor suppressor miR-138–5p for the treatment of bladder cancer. The results showed that ADSC-Exo-miR-138–5p could inhibit the proliferation, migration, and invasion of bladder cancer cells in vitro and in vivo, This evidence indicated that ADSCs-Exo-miR-138–5p is a promising therapeutic agent for bladder cancer (213). Shojaei et al. used ADSCs-Exo to deliver the tumor suppressor miR-218 (downregulation was associated with EMT and angiogenesis) to breast cancer cells; the purpose of that study was to evaluate the tumor suppressor properties of miR-218 in vitro. The results demonstrated that ADSCs-Exo could effectively restore the levels of miR-218 in breast cancer cells and significantly reduce the expression of miR-218 target genes (RUNX family transcription factor 2 [RUNX2] and RPTOR independent companion of MTOR complex 2 [RICTOR]) in breast cancer cells (MDA-MB-231). The findings also indicate that miR-218 can prevent breast cancer progression by simultaneously targeting angiogenesis and EMT (229). Shojaei et al. also studied the usefulness of ADSCs-Exo as a carrier of miRNA-381. The treatment significantly downregulated the expression of genes and proteins related to EMT and inhibited the progression of triple-negative breast cancer in vitro ( 230).

MSCs are the most widely used cells in cell therapy (231). Importantly, cell therapy is also associated with some significant risks. In some cases, ADSCs-Exo may also promote tumor progression. For example, Qu et al. found that ADSCs-Exo secreted into ascites activated the mitogen-activated protein kinase (MAPK) signaling pathway through forkhead box M1 (FOXM1) thereby regulating the ADSCs-Exo-mediated progression of ovarian cancer and promoting peritoneal metastasis of epithelial ovarian cancer (232). Wang et al. reported that ADSCs-Exo could promote the invasion, migration, and proliferation of osteosarcoma cells, as well as increase the expression of collagen β(1-O)galactosyltransferase 2 (COLGALT2) (233). Lin et al. also reported that ADSCs-Exo could activate the Wnt/β-catenin signaling pathway, thus promoting the migration and proliferation of breast cancer cell line MCF7 (234) ( Figure 4 ).

In addition to mesenchymal stem cells (MSCs) derived from umbilical cord, bone marrow and adipose tissue, exosomes derived from dental pulp, peripheral blood and placenta have great potential in disease treatment and biomarker diagnosis, and are expected to become a reserve force for treatment and diagnosis (235, 236). Dental pulp stem cell-derived exosomes (DPSC-Exos) have similar biological characteristics with bone marrow blasts and are closely related to tissue regeneration (237). Qiao et al. found that DPSC-Exos can inhibit periodontitis and promote epithelial healing in rats with periodontitis, and its mechanism is to regulate inflammation by inhibiting the IL-6/JAK2/STAT3 signaling pathway (238). The advantages of clinical transfusion and easy availability of peripheral-blood-derived exosomes have broadened the scope of their clinical application. Kang et al. prepared a mouse model of myocardial infarction and found that exosomes loaded with miR-21 mimics enhanced fibrosis, while exosomes loaded with miR-21 inhibitors reduced fibrosis, Human peripheral blood-derived exosomes loaded with miRNA can be used as a therapeutic tool for heart diseases (239). Placental mesenchymal stem cell-derived exosomes (Pd-MSC-Exos) can be detected in maternal blood as early as 6 weeks after conception (240), and their levels increase with gestational age (241). Zheng et al. analyzed the mechanism of Pd-MSC-EVs affecting liver fibrosis and found that Pd-MSC-EVs may inhibit the activation of hepatic stellate cells (HSC) through the miR-378c/SKP2 pathway. Thus, Pd-MSC-EVs are expected to become effective drug candidates for the treatment of liver fibrosis (242).

Mesenchymal stem cells are currently known to be the only cells that can be prepared on a large scale and have the ability to prepare exosomes on a large scale (243). Therefore, compared with exosomes from other sources, exosomes derived from mesenchymal stem cells have the following advantages: easy access, low immunogenicity, inhibition of the function of various immune effector cell types, promotion of immune regulation, anti-inflammatory, anti-aging and wound healing (191). Therefore, mesenchymal stem cells have become an ideal cell source in regenerative medicine and immunotherapy (244). MSCs-Exo can also be used for the treatment of skin lesions caused by chronic diabetes, Yang et al. combined hucMSCs-Exo and Pluronic F-127 (PF-127) hydrogel in diabetic rats and found that it could significantly accelerate the speed of wound healing, promote granulation tissue regeneration by increasing the expression of Ki67 and CD31. The expression of vascular endothelial growth factor (VEGF) and transforming growth factor beta-1 (TGFβ-1) was up-regulated to promote wound healing (245). Song et al. constructed ECM hydrogel loaded with ADSCs-Exo, and once injected into the wound site, ECM-Exo formed the hydrogel at a physiological temperature of nearly 37°C. ADSCs-Exo can be released slowly and continuously from the hydrogel to maintain a high concentration at the wound site. The ECM hydrogel gradually degrades in vivo. It can effectively reduce inflammation and promote angiogenesis, collagen deposition, cell proliferation and migration to accelerate wound healing (246), Wang et al. mixed collagen (COL-I) and platelet-rich plasma (PRP) and added thrombin to prepare a biological carrier, and delivered ADSCs-Exo in the carrier. The study found that the scaffold released a large number of growth factors, such as TGF-β, PDGF, FGF, HGF, and VEGF. These growth factors play a key role in wound healing and angiogenesis. Meanwhile, ADSCs-Exo also plays an important role in promoting tissue repair, regeneration and angiogenesis. In vitro experiments proved that the ADSCs- Exo based stents can induce angiogenesis, accelerate the healing process (247). The application of MSCs-Exo and its carrier provides a new idea for the treatment of skin injury. In addition, Hu et al. compared human amniotic mesenchymal stem cells (hAMSCs) and Schwann cell-like cells (SCLCs) derived exosomes in the treatment of peripheral nerve injury (PNI). The results showed that SCLCs-Exo enhanced the recovery of motor function in the rat model, alleviated gastrocnemial-muscle atrophy, promoted axon regeneration, myelination and angiogenesis, and up-regulated the expression of glial cell-derived neurotrophic factor, myelin positive regulator and myelin protein in Schwann cells. SCLCs-Exo is a potential new treatment for PNI (248).

DCs are antigen-presenting cells (APCs) with the unique ability to induce primary and secondary immune responses. They also play an important role in tumor immunotherapy, and are involved in anti-tumor immunity, activating tumor-specific T cells to eliminate tumor cells. DCs are often used in the preparation of vaccines (272, 273); however, their use is limited due to the high manufacturing cost, time constraint, difficult preservation of living cells, and possible functional and phenotypic changes after injection (274). DEX have also attracted attention as immune cell-derived exosomes. They are characterized by the expression of tumor antigens, MHC class I (MHC-I), class II (MHC-II), and T cell costimulatory molecules on their surface. After capturing and internalizing the antigen-MHC complex, these antigen-MHC complexes are presented to T cells via APC, thereby triggering the release of antigen-specific CD4⁺ and CD8⁺ T cells (156). DEX are nanovesicles containing functional MHC-peptide complexes that promote T cell-dependent tumor rejection. There are three mechanisms through which DEX stimulate T cell production. Firstly, DEX directly stimulate T cells to exert their effect; however, direct T cell stimulation appears to be inefficient in priming naive T cells. Secondly, the antigenic peptide-MHC complex is transferred to the APC for more efficient stimulation of T cell responses by APC presentation. Thirdly, T cells may be indirectly activated through tumorigenesis (275). Through research on mice, Viaud et al. also found that DEX could promote the proliferation and activation of NK cells by promoting IL-15Rα and NKG2D, thereby producing an anti-metastatic effect mediated by NK1.1 cells (276). DEX carry numerous molecules related to the immune function of DCs; these molecules are bound by tumor cells, transforming them into immunogenic targets. Compared with exosomes from immature DCs, those derived from mature DCs have less loss after endocytosis and greater ability to stimulate T cells (277, 278). These observations provide a good basis for targeted therapy of tumors. DEX can be used to load neoantigens, which is not susceptible to environmental influences and can retain function and phenotype as a new nanovaccine, which can more easily transport antigens to lymph nodes and trigger a strong immune response (268, 274). Lu et al. studied exosomes derived from HCC antigen-expressing DCs in three different HCC mouse models. They demonstrated that α-fetoprotein-rich DEX could trigger effective antigen-specific anti-tumor immune responses and reshape the tumor microenvironment (TME) in HCC mice, thus providing a cell-free vaccine option for HCC immunotherapy (279). Zhong et al. used microwave ablation combined with DEX to treat mice with HCC. They found that the number of CD8⁺T cells at the tumor site and the plasma IFN-γ concentration were increased, whereas the number of regulatory T cells and the IL-10 concentration were decreased. The results showed that the combination of microwave ablation with DEX can significantly inhibit tumor growth and improve the immune microenvironment, thereby providing a new direction for the development of vaccines based on DCs and DEX (280). The membrane structure of DEX avoids high degradation, while ensuring good biocompatibility and in vivo safety. Compared with DC-based vaccines, DEX have higher immunogenicity and stronger resistance to immunosuppression, and have shown better anti-tumor effects in preclinical studies. Hao et al. reported that intravenous injection of an exosome vaccine is superior to subcutaneous injection, inducing stronger anti-tumor immunity. A Phase I study of DEX failed to demonstrate its immune competence (281). Therefore, Viaud et al. developed second-generation DEX with enhanced immunostimulatory properties. The clinical grade process of the IFN-γ-DEX vaccine and its quality control parameters currently used in phase II trials were studied. IFN-γ is a key cytokine that regulates the expression of CD40, CD80, CD86, and CD54 induced by DCs on DEX, leading to direct and effective peptide-dependent CD8⁺T cell weight gain potential in vitro and in vivo ( 282).

Zhu et al. developed an anti-tumor vaccine candidate by coupling mucin 1 (MUC1) glycopeptide antigen to DEX. They found that MUC1-DEX induced high MUC1-specific immunoglobulin G antibody titers with strong binding affinity to MUC1-positive tumor cells in vivo. This treatment enhanced cytotoxicity of CD8⁺T cells from immunized mice against MUC1-positive tumor cells. It also inhibited tumor growth and prolonged the survival time of mice in preventive and therapeutic tumor-bearing mouse models (283).

Silva et al. reported that DEX play a role in tissue regeneration. DEX are naturally loaded with chemoattractants, which can promote cell recruitment. Osteopontin and MMP9 have been confirmed in EVs (284). Triptolide has beneficial effects in the treatment of cancer (e.g., gastric carcinoma, lung cancer), but causes multi-organ toxicity. DCs are the main targets of triptolide, inducing immunosuppression. Rao et al. packaged triptolide in DEX for targeted delivery to reduce toxicity. They reported that triptolide with DEX could play a role by reducing CD4⁺ T cells and increasing regulatory T cells in vivo to reshape the immune environment (285).

Barnwal et al. reported that myeloid-derived DEX were obtained from bone marrow in the presence of tumor antigen. Studies have demonstrated that colony-stimulating factor 1 receptor (CSF-1R) inhibitor (PLX-3397) targeting the colony-stimulating factor 1/CSF1R (CSF1/CSF1R) signaling pathway can deplete tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells responsible for an immunosuppressive TME. In a B16-F10 mouse model of melanoma, DEX combined with PLX-3397 regulated the TME by transferring Th1/Th2 to dominant Th1 population and depleting TAMs and myeloid-derived suppressor cells. These findings also provide a new strategy for the treatment of melanoma (286). DEX carry many molecules associated with the immune function of DCs, and their incorporation into tumor cells can transform them into immunogenic targets. Romagnoli et al. treated breast cancer cell line SK-BR-3 with DEX, and subsequently used these DEX to stimulate SK-BR-3 cells sensitized with CD3⁺ T cells. The investigators generated DEX-SK-BR-3-trimer CD3⁺ T cells, and revealed that the sensitizing T cells cultured from tumor cells treated with DEX had a stronger ability to secrete IFN-γ compared with non-DEX-treated cells. These data suggest that incorporation of DEX into tumor cells enhances the activation of T cells, thus potentially producing a more effective response. Collectively, these findings imply that DEX may become an important tool in cancer immunotherapy (287) ( Figure 5 ).

Macrophages develop from hematopoietic stem cells, namely monocytes in the bone marrow. They play roles in phagocytosis (a major mechanisms of innate immunity) and antigen presentation. Five activated macrophage phenotypes have been identified, namely M1 macrophages, M2 macrophages, CD169⁺ macrophages, TCR⁺ macrophages, and TAMs; inactivated macrophages (termed M0) have also been identified. Among these phenotypes, M1 and M2 have been primarily studied (201, 288). These two phenotypes were distinguished according to their function and the level of inflammatory factor secretion. M1 macrophages exhibit an anti-tumor and pro-inflammatory phenotype, and can release pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), C-C motif chemokine ligand 2 (CCL2), IL-6, inducible nitric oxide synthase (iNOS), IL-1α, IL-1β, IL-12, IL-23, IL-18, type I IFN (-α and -β), C-X-C motif chemokine ligand 1–3 (CXCL1–3), CXCL5, and CXCL8–10. In contrast, M2 macrophages show a pro-tumor and anti-inflammatory profile (289). Arabpour et al. reported that MSC-Exo reduced inflammation by promoting M1 to M2 polarization and increasing anti-inflammatory cytokines and chemokines (290). Pritchard et al. reported that lung tumor-derived exosomes can also promote the polarization of M2 macrophages (290). Inactivated M0 can be induced to M1 under the action of lipopolysaccharide, TNF-α, and IFN-γ, while cytokines (e.g., IL-4 and IL-13) are required to induce M0 to M2 (291, 292). M1 macrophage exosomes have the ability to target lymph nodes and can be absorbed by local macrophages and DCs. Macrophage-derived exosomes are involved in immune activation and regulation, and serve as anti-cancer drug carriers.

Rayamajhi et al. designed hybrid exosomes by hybridizing small EVs from mouse macrophages with synthetic liposomes. The hybrid exosomes were loaded with water-soluble doxorubicin. The toxicity of the exosome-doxorubicin hybrid to cancer cells and drug release were enhanced under acidic conditions; this finding indicates the possibility for drug delivery to the acidic cancer environment (293). Li et al. developed a macrophage-derived exosome-coated polylactic acid-glycolic acid nanoplatform for targeted chemotherapy of triple-negative breast cancer. To further improve tumor targeting, the surface of exosomes was modified with peptides (253). The results showed that the engineered exosome-coated nanoparticles significantly improved the cellular uptake efficiency of doxorubicin and anti-tumor effect in vivo and in vitro, and induced the apoptosis of tumor cells.

Despite the availability of many options for the treatment of pancreatic cancer, chemotherapy is currently the main therapeutic modality (294). Chemotherapy drugs play a major role in the treatment of cancer; however, the development of chemotherapy resistance limits its efficacy. Therefore, it is necessary to develop more effective treatments. Zhao et al. have shown that it is possible to develop a specific M1 macrophage-derived exosome-gemcitabine delivery system and load it with noracilor (DFX). DFX is designed to deplete iron, thereby inhibiting the expression of the ribonucleotide reductase regulatory subunit M2 (RRM2), This approach improved the efficacy of gemcitabine. This delivery system can inhibit tumor cell proliferation, attachment, and migration, reverse the chemoresistance of tumor cells to gemcitabine, and significantly enhance the efficacy of gemcitabine. Therefore, this system provides a new strategy for the treatment of pancreatic cancer (295) ( Figure 6 ).

Neutrophils are the most abundant type of innate immune cells in the human body with a tendency to inflammation (296). Neutrophil-derived exosomes have the same effect to achieve tumor targeting. Surgical resection is commonly used for the treatment of glioma. However, surgery is often accompanied by infection and metastasis, while chemotherapy after surgical resection has a poor effect due to the existence of the BBB. Wang et al. developed a new pro-inflammatory drug delivery system to overcome the risk of inflammation and metastasis after surgical resection of glioma using neutrophil-derived exosomes as a carrier of doxorubicin. Through in vivo and in vitro experiments, it was confirmed that neutrophil-derived exosomes had inflammatory tendency and could target the inflammatory site of tumor to improve the therapeutic effect and the overall survival of patients with glioma (297). Neutrophil-derived exosomes can also act as carriers by delivering cytotoxic proteins and activating caspase signaling pathways (298). Vargas et al. found that neutrophil-derived exosomes could be internalized by airway smooth muscle and alter its proliferative properties. These exosomes play an important role in asthma progression, promoting airway remodeling in patients with severe and corticosteroid-insensitive asthma (299). Ou et al. found that senescent neutrophil-derived exosome piRNA-17560 enhanced cellulite and obesity-associated protein (FTO) expression in breast cancer cells, as well as chemotherapy resistance. Senescent neutrophils may be a therapeutic target for breast cancer (300). Tyagi et al. study examined exosomal miR-4466 from N2 neutrophils in smokers and non-smokers. They observed that the expression of exosomal miR-4466 from neutrophils was increased in smokers versus non-smokers. Therefore, neutrophil-derived exosomal miR-4466 can be used as a promising predictor of metastatic disease in smokers (301) ( Figure 6 ).

T lymphocytes, thymus-dependent lymphocytes, are pluripotent stem cells derived from bone marrow (302). These cells are an integral part of adaptive immunity. T cells have different subsets with varied functions, and can play a role through direct contact between cells or the transfer of secreted molecules (303). According to the phenotype, T cells can be mainly divided into CD4⁺ Th cells, CD8⁺ cytotoxic T cells, follicular helper T cells, and regulatory T cells. Following maturation in the thymus, T cells migrate to peripheral tissues (304).

A growing number of studies have shown that immune cells participate in cell communication by secreting exosomes (305). Among immune cell-derived exosomes, those derived from T cells participate in the anti-tumor effect of cancer immunotherapy by mimicking the effect of parental cells (303). T cells produce exosomes that reflect their characteristics, such as direct killing of target cells, regulating B cells to produce antibodies, and producing cytokines (e.g., IL-7, IL-10, IL-12, IL-17, INF-γ) (306), thus creating the optimal microenvironment for paracrine and autocrine immune cells. T cell-derived exosomes can also play an important role in intercellular signal transduction and activate other immune cells, thereby participating in the corresponding immune regulation process (303).

Programmed cell death 1 (PD-1) is widely expressed in tumor-infiltrating lymphocytes in triple-negative breast cancer, and cell-surface PD-1 transduces negative signals for effector T cell activity during cell-cell contact (307). PD-1 is secreted in the form of exosomes from activated T cells and can remotely interact with cell-surface or exosomal programmed death ligand 1 (PD-L1). This interaction restores tumor surveillance by attenuating PD-L1-induced suppression of tumor-specific cytotoxic T cell activity and exosome PD-1 anti-PD-L1 function. Overall, it enhances the activity of cytotoxic T cells (308).

Paclitaxel (PTX) is a chemotherapeutic drug with limited use due to its systemic toxicity. Chimeric antigen receptor-T (CAR-T) cell-derived exosomes (CAR-T-Exo) contain tumor-targeted CAR and cytotoxic particles (granzyme B [GZMB] and perforin [PRF]), which can be used in the treatment of tumors and are considered potential carriers of paclitaxel (268). Zheng et al. reported that CAR-T-derived exosomes can deliver paclitaxel, reprogram the TME, and reverse immunosuppression to increase the levels of CD8⁺T cells, IFN-γ, and TNF-α, thereby enabling the treatment of non-small cell lung cancer (309) ( Figure 6 ).

Huang et al. performed omics analysis of CD4+T cell-derived exosomes from patients with rheumatoid arthritis (RA) and found that the expression of dihydropyrimidinase associated protein 3 (DPYSL3) was significantly up-regulated and the expression of proteasome activating complex subunit 1 (PSME1) was significantly down-regulated. These differentially expressed genes may be involved in the pathogenesis of RA, thus DPYSL3 and PSME1 are expected to be biomarkers for RA diagnosis (310). Xu et al. found that miR-186–5p in CD8 T cell-derived exosomes caused renal inflammation and tissue damage. miR-186–5p directly activates TLR7/8 signaling axis in renal tubules to cause renal inflammation, which reveals the specific pathogenic mechanism and reason of the pathogenic role in T cell-mediated renal dysfunction and provides new ideas for the treatment of nephropathy (311). T cell-derived exosomes are still in the exploratory stage, and continuous efforts are still needed to achieve clinical transformation. We believe that with the continuous efforts of scholars, T cell-derived exosomes can become a powerful tool for disease treatment.

B lymphocytes are bone marrow-dependent lymphocytes. Mature B cells migrate out of the peripheral blood, and enter the spleen and lymph nodes. Following stimulation by antigens, they proliferate and differentiate into plasma cells, and participate in humoral immunity. B cells differentiate into effector cells with the synthesis of exosomes, which is initiated upon stimulation by activation signals, in particular T cell “help” via CD40 and IL-4 signaling. B cell-derived exosomes induce antigen-specific, MHC-II-restricted T cell responses, suggesting a role for exosomes in antigen presentation in vivo ( 312). B cell-derived exosomes also contain immunoglobulins that deliver surface B cell receptor-bound antigens into the endosomal/exosomal pathway (313). Saunderson et al. demonstrated that primary B cells release high levels of exosomes in response to CD40 and IL-4 signaling. The absolute number of splenic immune cell subsets was determined to investigate the immune cells that respond to Ag of B cell-derived exosomes. After immunization, the number of NK cells, B cells, CD4 T cells, and CD8 T cells in the spleen was significantly increased (314) ( Figure 6 ). Dan Ma et al. characterized B lymphocyte-derived exosomes in fatal Pneumocystis pneumonia (PCP) and found significant alterations in histone H1.3, vimentin, and tyrosine protein phosphatase non-receptor type 6 (PTPN6) levels. The proinflammatory effects of B-cell-derived exosomes from PCP on CD4+T cell responses were revealed. This finding provides a new idea for the study of PCP (315).

NK cells, which constitute a small population of cells, can kill target cells in a non-specific manner in the human body. This killing activity is innate, does not require prior antigen sensitization, and is not restricted by MHC. NK-Exo expressed various NK receptors/markers, including CD56, CD69, cytotoxic receptors (e.g., NKG2D), NKp44, NKp46, NKp30, CD40L, PD-1, and molecules involved in tumor cell recognition and immune synapse formation (lymphocyte function-associated antigen 1 [LFA-1], DNAM1). They also carry cytotoxic proteins (e.g., PRF, GZMA, GZMB, and Fas ligand [FasL]) and cytokines (e.g., IFN-γ and TNF-α) (316). NK-Exo recognize and kill cancer cells through various mechanisms in vitro and in vivo. They exert an anti-tumor effect due to the presence of PRF and FasL, which trigger the intrinsic pathway and promote the release of cytochrome-c. FasL triggers the extrinsic apoptotic pathway by activating caspase 8 (CASP8), CASP3, and poly(ADP-ribose) polymerase (PARP) (317). NK-Exo contain potent cytotoxic proteins that induce apoptosis in targeted cancer cells. Furthermore, EVs derived from cancer cells carrying NK ligands may evade immune surveillance and responses (318). Zhu et al. found that NK-Exo expressed two typical exosomal proteins (CD63 and ALG-2 interacting protein X [ALIX]) and two functional NK proteins (PRF and FasL) (317). Moreover, NK-Exo can secrete TNF-α, thereby affecting signaling pathways that control cell proliferation. NK-Exo exert a cytotoxic effect on melanoma cells in vitro, without significant side effects on normal NK-Exo cells (317).

Luo et al. found that NK-Exo could activate NK cells from the immunosuppressed TME. They also showed that cisplatin-loaded NK-Exo could enhance the sensitivity of drug-resistant ovarian cancer cells to cisplatin, thus playing an anti-proliferation role (319). Han et al. also reported that paclitaxel embedded in NK-Exo effectively inhibited the proliferation and induced apoptosis of breast cancer cells. Hashemi et al. constructed a drug delivery system combining NK-Exo and the anti-cancer drug sorafenib (NK-Exo-SFB), which exerted an inhibitory effect on breast cancer cells (320). Di Pace et al. analyzed miRNAs in NK-Exo, revealing that let-7b-5p was enriched in exosomes. The let-7b-5p belongs to the let-7 family of miRNAs with key tumor suppressor functions. It has an anti-proliferation effect on pancreatic cancer cells (321). Sun et al. also reported that miR-3607–3p of NK-Exo can inhibit the progression of pancreatic cancer (322). Notably, NK-Exo eliminated leukemia cells isolated from patients with acute and chronic leukemia and inhibited the growth of hematopoietic colonies; these findings led to the development of a cell-free therapy for leukemia (323). Wang et al. demonstrated that treatment with NK-Exo significantly inhibited TGF-β1-induced proliferation and activation of hepatic stellate cells, as well as liver fibrosis, thus providing a new means for the treatment of liver fibrosis (290). Neviani et al. reported that NK exosomes carry the tumor suppressor gene miR-186–5p, which impairs the growth of neuroblastoma cells in vitro and in vivo ( 318). They demonstrated that NK-Exo carrying tumor suppressor gene miR-186p exhibited cytotoxicity against neuroblastoma cell lines ( Figure 6 ).

Liver cancer cell-derived exosomes, i.e., HCC-derived exosomes (HCC-Exo), have been shown to attenuate the cytotoxicity of T and NK cells and promote immunosuppressed M2 macrophages, N2 neutrophils, and regulatory B cells (329). Yu et al. reported that miR-21–5p from HCC-Exo directly targeted the UTR of Ras homolog family member B (RhoB) in human monocyte-derived leukemia (THP-1) cells and promoted TAM polarization. The evidence indicates that tumor-derived miR-21–5p promotes the malignant progression of HCC, thereby mediating intercellular crosstalk between tumor cells and macrophages. Targeting M2-like TAMs and blocking their associated signaling pathways may provide specific and novel therapeutic approaches to HCC treatment (331). Zhang et al. reported that HCC cells can secrete exosome circular ubiquitin-like PHD and ring finger domain 1 RNA (circUHRF1). The circRNA mainly acts as a miRNA sponge by binding to miRNA and subsequently promoting the expression of miRNA targeted genes. The circUHRF1 degrades miR-449c-5p, thereby upregulating T-cell immunoglobulin mucin family member 3 (TIM-3) expression, inhibiting NK cell-derived IFN-γ and TNF-α, and promoting immunosuppression. These findings provide a potential treatment strategy for patients with HCC (253).

Shen et al. tested the immunoregulatory effect of gastric cancer cell-derived exosomes (AGS-Exo) on MSCs. MSCs were stimulated with AGS-Exo, which led to abnormal activation of the nuclear factor-κB (NF-κB) signaling pathway. The effects of AGS-Exo-stimulated MSCs significantly attenuated the function of T cells and macrophages. Therefore, AGS-Exo affects the immunomodulatory function of MSCs through the NF-κB signaling pathway, thus enhancing the ability of MSCs to activate immune cells, maintain an inflammatory environment, and support tumor growth (332). High mobility group box 1 (HMGB1) is a non-histone chromatin-related protein widely distributed in eukaryotic cells. It is involved in DNA damage repair and maintenance of genome stability. HMGB1 can promote tumorigenesis, while it can also mediate immunogenic cell death during chemoradiotherapy and enhance anti-tumor immunity (333). Liu et al. studied the regulatory effect and potential mechanism of HMGB-1 in AGS-Exo on the polarization of M2-like macrophages. They revealed that HMGB-1 interacts with the transcription factor POU2F1 to inhibit the transcriptional activity of p50 and inactivate the NF-κB signaling pathway, thereby inducing the polarization of M2 macrophages (334).

Vγ9Vδ2 T cells, as a subtype of T cells, express T cell receptors composed of γ and δ chains and play an important role in innate and adaptive immune surveillance (335). AGS-Exo are effectively taken up by Vγ9Vδ2 T cells to induce cell apoptosis. This uptake also reduced the production of cytotoxic cytokines IFN-γ and TNF-α. Li et al. demonstrated that exosomal miR-135b-5p was successfully delivered to Vγ9Vδ2 T cells. Exosomal miR-135b-5p impairs the function of Vγ9Vδ2 T cells by targeting specific protein 1 (SP1) (336). SP1 inhibitor plicamycin was also administered to prevent SP1 function. These results suggested that AGS-Exo impaired the function of Vγ9Vδ2 T cells through the miR-135b-5p/SP1 pathway (336). Targeting the exosomal miR-135b-56/SP1 axis may improve the efficiency of immunotherapy for gastric cancer based on Vγ9Vδ2 T lymphocytes ( Figure 7 ).

PEX is involved in drug resistance, immune evasion, metabolic reprogramming, and distant metastasis of pancreatic cancer, and plays a key role in the occurrence and development of this disease. Their extensive differential expression and functional content render PEX a promising screening tool and therapeutic target (337).

Exosomes secreted by cancer-associated fibroblasts in the TME promote tumor proliferation and chemotherapy resistance by inhibiting a tumor suppressor (PTEN) during the treatment of PDAC with gemcitabine. Therefore, exosome inhibitor GW4869 should be used to block the inhibition of PTEN in vivo and improve the anti-tumor effect of chemotherapy drugs (338). Exosomes secreted by PDAC can be used as diagnostic markers. The function of PDAC exosomes is mainly reflected in mediating immune escape, enhancing resistance to gemcitabine therapy, and promoting the progression of PDAC through the release of proteins and miRNAs. Exosomes produced by PDAC cells highly express cytoskeleton-associated protein 4 (CKAP4), a novel dickkopf WNT signaling pathway inhibitor 1 (DKK1) receptor. CKAP4 is highly expressed in patients with pancreatic cancer. Inhibitors of this molecule can prevent binding to DKK1, thereby inhibiting the proliferation and migration of PDAC cells. Hence, CKAP4 is a potential new target for the treatment of PDAC (339) ( Figure 7 ).

Exosomes play a key role in brain tumors. Exosomes produced by glioblastoma enable cell-to-cell communication and promote glioblastoma progression. This occurs by inducing M2 macrophages around the glioblastoma to achieve immune escape. The expression of miRNAs was detected in the cerebrospinal fluid and plasma of patients with glioblastoma. Regardless of the role and activity of these miRNAs, they can be used as diagnostic markers. Specifically, miR-221 and miR-21 have been evaluated in the cerebrospinal fluid of patients with glioblastoma. Similarly, miR-320 and miR-574–3p in plasma can also be used as diagnostic biomarkers (340).

Yang et al. reported that nasopharyngeal carcinoma-derived exosomes (NPC-Exo) highly express PD-L1, which can bind to PD-1 on CD8⁺T cells. This leads to inhibition of CD8⁺T cell activity, promotion of nasopharyngeal carcinoma tumor growth in mice, and evasion of T cell immunity by nasopharyngeal carcinoma cells (341). Yu et al. found that NPC-Exo can help RNF126 (an E3 ubiquitylation ligase that acts as an oncogene) to enter TAMs and bind to and ubiquitate PTEN (a tumor suppressor termed tension homolog) (78). PTEN regulates tumor radiotherapy and chemotherapy resistance and pathogenesis by regulating the PI3K/AKT signaling pathway. PTEN degradation activates the PI3K/AKT pathway and inhibits autophagy. These effects enhance macrophage migration and M2 polarization, thereby promoting tumor growth (78). NPC-Exo associated PD-1 and PTEN provide a basis for early biomarker screening and targeted therapy of nasopharyngeal carcinoma ( Figure 7 ).

MTEX contain the same antigens as the parental cells. Sharma et al. reported that exosomes isolated from the plasma of patients with melanoma contained melanoma-associated antigens compared with those isolated from normal cells. This evidence illustrates that plasma-derived exosomes from patients with melanoma may be a useful biomarker of melanoma in tumor liquid biopsy (259). Whiteside demonstrated that incubation of MTEX with immune-receptor cells resulted in inhibition of the anti-tumor function of these cells. MTEX are involved in immunosuppression in melanoma; therefore, they may play a role in promoting melanoma progression (342). Marton et al. purified and characterized B16F1 melanoma cell-derived exosomes. They found that MTEX affected the proliferation of CD4⁺T cells induced by bone marrow-derived DCs. MTEX also activated macrophages as measured by NF-κB activation. This finding suggests that exosomes play a role in tumor progression and metastasis formation by supporting tumor immune escape mechanisms (343). Gerloff et al. found that exposure to MTEX induced a tumor-promoting TAM phenotype. Sequencing showed that miR-125b-5p was enriched in MTEX. The miR-125b-5p could be delivered to macrophages through MTEX, targeted lysosomal acid lipase A (LIPA) in macrophages, and induced a tumor-promoting TAM phenotype (344).

Xu et al. reported that exosomes derived from prostate cancer cells can transport IL-8 and bind to peroxisome proliferator activated receptor α (PPARα) in CD8⁺T cells (345). Through this process, they activate the uncoupling protein 1 (UCP1), decomcause fatty acids for thermogenesis, interfere with energy metabolism, hinder the function of CD8⁺T cells, and promote immune escape (345). Ding et al. studied exosomes derived from bladder cancer cell line T24; the exosomes blocked the function of NK cells by inhibiting the expression of NKG2D, NKP30, CD226, PRF, and GZMB receptors on NK cells, The miR-221–5p and miR-186–5p in exosomes derived from T24 cells interfere with the stable expression of DNAX-activation protein 10 (DAP10), CD96, and PRF mRNA in NK cells (346). Therefore, they may be targets for the treatment of bladder cancer ( Figure 7 ).

Exosomal miR-20a-5p is released by breast cancer cells and transferred to CD8⁺ T cells, where it inhibits their function by targeting the nuclear protein coactivator of histone transcription (NPAT) (347). NPAT is a cell cycle gene highly expressed in immature CD8 T cells, The miR-20a-5p binds to the 3’-UTR of NPAT, inducing the development of resistance to anti-PD-1 therapy. These findings suggest that exosomal miR-20a-5p derived from triple-negative breast cancer plays an important role in promoting immune escape and immunotherapy resistance by inducing CD8⁺T cell dysfunction (347). Breast cancer-derived exosomes containing tumor cell-derived PD-L1 interact with PD-1-producing T cells to significantly reduce responses to immune checkpoint blockade agents. The drug macitane acts as a powerful helper of CD8⁺T cell anti-tumor response by inhibiting tumor cell-derived EV-PD-L1. Lee et al. reported that macitane inhibits the secretion of tumor-derived EV-PD-L1 in breast cancer cells by targeting endothelin receptor A (ETA) and can reduce the binding of PD-1 to EV-PD-L1, thereby synergizing the effect of anti-PD-L1 antibodies. Enhanced CD8⁺T cell-mediated tumor killing. These findings strongly support that macitane, which has been approved for clinical use, can be used to improve and/or overcome the inadequate response to PD-1/PD-L1 blockade therapy (348) ( Figure 7 ).

CD4⁺T cells play a great role in tumor immunity, and can bind to DC-Exo generated by tumor cells to induce tumor immunity. Li et al. reported that LEX did not exert the expected effect when binding to CD4⁺T cells due to insufficient costimulatory ability, CD4⁺T-LEX-CD8086 was constructed, which could stimulate antigen-specific CD8⁺ cytotoxic T cell responses to leukemia cells. These data indicated that CD4⁺T cell vaccines using leukemia cell-derived exosomes modified by costimulatory molecules may be effective in immunotherapy for leukemia (200). Huang et al. reported that TGF-β1-silenced leukemia cell-derived exosomes (LEX-TGF-β1si) targeted DCs. The treatment effectively promoted the proliferation of CD4⁺T cells and the secretion of Th1 cytokines in vitro, and induced tumor-specific cytotoxic T cell responses to achieve an anti-leukemia effect. This evidence suggests that LEX-TGF-β1si targeting DCs are a promising immunotherapy option for leukemia (349) ( Figure 7 ).

Due to the dual role of TEX in promoting tumor growth and as a therapeutic carrier, to overcome the tumor-promoting risk of TEX during treatment (324), immunotherapy combining immune system activation with immune escape inhibitors has been shown to be a new effective tumor suppression strategy, Fan et al. modified two antibodies (anti-PD-L1 and anti-CD40) on the surface of exosomes by co-culture. First, anti-PD-1 blocks immune checkpoint molecules by binding to the PD-L1 receptor on tumor cells. Second, anti-CD40 will direct exosomes to target the CD40 receptor on the membrane of dendritic cells (DC). After DC receives a positive costimulatory signal, exosomes will be taken up by DC and release cGAMP through lysosome-mediated exosome permeability to produce type I interferon (IFN-I) and pro-inflammatory cytokines. The two activation of dendritic cells (DCs) and the blocking of PD-L1 in tumor cells have improved the efficacy of combined cancer immunotherapy for tumor suppression (350). In addition to the use of dual agents, chimeric peptide exosomes are also a novel strategy for therapy, Cheng et al. engineered with chimeric peptides for dual plasma membrane and nuclear targeting photosensitizer delivery and synergistic photodynamic therapy (PDT), engineered chimeric peptide exosomes (ChiP-Exo) can achieve membrane targeting and, to some extent, lead to cell death, as the presence of nuclear localization signal (NLS) peptides can also enhance nuclear delivery. Nuclear ChiP-Exo activates reactive oxygen species (ROS) in situ to disrupt the nucleus, resulting in stable and synergistic PDT. This exosome-based dual-stage light-guided subcellular dual-target PDT strategy has shown greatly enhanced therapeutic effects in inhibiting tumor growth, providing new ideas for the development of individualized biomedicine for precise tumor treatment (351). Similarly, Trivedi et al. used dual-targeted hyaluronic acid nanoparticles to manipulate exosome contents by gene transfection into tumor cells. Studies have found that changes in miRNA levels in exosomes can mediate the repolarization of macrophages to a more pro-inflammatory/anti-tumor M1 phenotype, indicating that gene transfer of exosomes can support an anti-tumor environment, thereby reducing tumorigenesis (352).

Liquid biopsy is of great significance in the early diagnosis, treatment staging and prognosis of cancer (50). In recent years, tumor-derived exosomes (TEX) have become a popular biomarker and potential candidate for non-invasive liquid biopsy and diagnosis of a variety of cancers due to their tumor-specific antigen (TSA) (353). Blood is a commonly used specimen for testing, and future studies may focus on fluids other than blood (e.g., ascites, urine and cerebrospinal fluid, etc.) (354). Early biomarkers of ovarian cancer are limited, and it is difficult to detect ovarian cancer at an early stage due to the deep anatomical position of the ovary (355). Therefore, the study of exosomes provides a new method for the diagnosis of ovarian cancer (356). Cheng et al. reported the proteomic and lipidomic analysis of exosomes derived from ovarian cancer cells (SKOV-3) and ovarian surface epithelial cells (HOSEPiC) and found that Cholesterol Ester (ChE), Zymosterol (ZyE), V collagen alpha 2 chain (COL5A2) and lipoprotein lipase (LPL) than from HOSEPiC usually secrete body content is richer, therefore, outside the body protein and lipid secretion has certain application value in the early diagnosis of ovarian cancer (355). In addition, the popularization of liquid biopsy also provides a new idea for the diagnosis of thyroid cancer (PTC) (357). The diagnosis of PTC is generally performed by fine needle aspiration biopsy, but this method is limited in use, with low accuracy and tissue trauma (358). The circular RNA (circRNA) in exosomes has shown great value in cancer diagnosis (359). Dai et al. detected hsa_circ_0082002 and hsa_circ_0003863 in serum exosomes from healthy people, benign thyroid tumors and PTC without Hashimoto’s thyroiditis. It was found that the levels of exosomal hsa_circ_0082002 and hsa_circ_0003863 were positively correlated with lymph node metastasis and vascular invasion of PTC, Therefore, exosomal circRNA has the potential to be used as a tumor marker for the diagnosis of PTC (360). In summary, cancer cell-derived exosomes (glioma, nasopharyngeal carcinoma, liver, gastric, bladder, prostate, breast, and leukemia) provide new diagnostic methods and targets for the treatment of cancer, thereby potentially improving medical care.