Exosomes are extracellular vesicles with an average diameter of 100 nm. Exosomes contain transmembrane proteins (e.g., CD63 and CD81), transporters (e.g., Rab-GTPase), and receptors (e.g., integrins) in addition to carrying nucleic acids, proteins, and lipids (Kalluri and LeBleu 2020). Generally, exosomes are generated and secreted by the fusion of multivesicular bodies (MVBs) with the plasma membrane (Liu et al. 2021). MVBs are generated by the entry of endosomes into cells by endocytosis. Endosomes containing various cargos undergo a maturation process and are incorporated into intraluminal vesicles (ILVs) to form MVBs. MVBs are matured through the exchange of vesicles from various organelles, including the Golgi and mitochondria, and mature MVBs release exosomes through fusion with the plasma membrane. Secreted exosomes play an important role in intercellular interactions and maintaining homeostasis through cargo transport (Desdín-Micó and Mittelbrunn 2017).

Several studies have reported that tumor-derived exosomes (TDEs) interact with blood vessels and immune cells in the tumor microenvironment to suppress immune activity, promote tumor growth, progression, and metastasis, and increase angiogenesis (Zhang et al. 2018; Wang et al. 2019; Pritchard et al. 2020; Zhao et al. 2020; Morrissey et al. 2021; Zheng et al. 2021; Du et al. 2022; Li et al. 2022; Zhang et al. 2022a). In particular, lncARSR, a cargo of renal cell carcinoma-derived exosomes, activates the STAT3 signaling pathway in macrophages and promotes M2 polarization. M2 polarization of macrophages by lncARSR reduces immune activity, including phagocytosis, thereby promoting tumor growth and progression (Zhang et al. 2022a). Another study reported that cervical cancer-derived exosomes promote angiogenesis by delivering the tyrosine kinase TIE2 to macrophages. TIE2 in macrophages promotes tumor angiogenesis through interaction with ANG2 (Du et al. 2022). miR-3591-3p has been reported to be absent in normal brain tissue and neurons but is present at high levels in the cerebral spinal fluid and exosomes of glioma patients (Li et al. 2022). In addition, miRNA-373 is present at high levels in the serum of receptor-negative breast cancer patients (Eichelser et al. 2014). Two studies have suggested that TDEs may be utilized as diagnostic biomarkers for cancer. Additionally, many studies are underway on cancer treatment through drug delivery using exosomes (Kim et al. 2016; Yong et al. 2019; Liang et al. 2020). In this review, we describe the biogenesis of exosomes, the components of their cargo, the functions of TDEs in tumor growth and progression, and recent clinical trials using exosomes. We show that exosomes are potential therapeutic targets in cancer and can serve as biomarkers for the early diagnosis of cancer.

Many studies have reported that a variety of stimuli, including hypoxia, UV, and serum, promote exosome production in cancer and immune cells (Fig. 1A) (Wysoczynski and Ratajczak 2009; Gurunathan et al. 2021; Ng et al. 2022). Exosomes are generated by endosomal maturation and contain various cargos, including DNA, RNA, proteins, and lipids (Fig. 1B and C). Endosomal maturation is initiated by the endocytosis of several molecules, which results in the formation of early endosomes (Krylova and Feng 2023). Early endosomes undergo internal budding via the endosomal sorting complex required for transport (ESCRT) pathway, which results in the formation of MVBs (Fig. 2). Briefly, the ESCRT pathway progresses through a series of processes from the ESCRT-0 to ESCRT-III complex to generate the ILV inside the MVB (Schöneberg et al. 2017). The ESCRT-0 complex, composed of the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs, such as Vps27 in yeast) and signal-transducing adaptor molecule (STAM, such as Hse-1 in yeast), binds to the ubiquitinated cargo and recruits the ESCRT-I complex (Bache et al. 2003a; 2003b). The ESCRT-I complex is a long-coiled stem tetramer composed of tumor susceptibility gene 101 (TSG101, also known as Vps23 in yeast), vacuolar protein sorting-associated proteins (Vps28 and Vps37), and multivesicular body factor 12 (Mvb12) (Kostelansky et al. 2006; Krylova and Feng 2023). TSG101 and Vps28 are known to bind to ESCRT-0 and ESCRT-II, respectively (Lu et al. 2003; Kostelansky et al. 2006). ESCRT-II is a Y-shaped complex composed of EAP20, EAP30, and EAP45 (also known as Vps25, Vps22 and Vps36 in yeast, respectively) (Langelier et al. 2006). ESCRT-II is known to promote the inner budding of endosomes together with ESCRT-I and induces ESCRT-III complex assembly (Babst et al. 2002; Teo et al. 2004; Langelier et al. 2006). The ESCRT-III complex is composed of the charged MVB protein (CHMP, which is known to be composed of Vps20, Snf7, Vps24 and Vps2 in yeast) family, and it is known to be involved in the formation of the ILVs of MVBs (Henne et al. 2011; Ju et al. 2021). In addition, Snf7 (known as CHMP4 in humans) recruits BCK1-like resistance to osmotic shock protein-1 (Bro-1), an adaptor protein of ESCRT-III. Bro-1 mediates the recruitment of the deubiquitinating enzyme Doa-4, resulting in the deubiquitination of cargos (Luhtala and Odorizzi 2004). After inner membrane fission in endosomes, the ESCRT-III complex is degraded by the ATPase Vps4 (Babst et al. 1998; Scott et al. 2005; Ju et al. 2021). The generation of ILVs in MVBs is dependent on not only the ESCRT-dependent pathway but also the ESCRT-independent pathway, which includes nSMase2-ceramide and caveolin-1 (Trajkovic et al. 2008; Hayer et al. 2010). In addition, mitochondrial vesicles can fuse with MVBs (Liang et al. 2023). The generated MVBs follow one of two fates: i) intracellular degradation by lysosomal fusion (Luzio et al. 2010) or ii) secretion of ILVs by MVB–PM fusion (Krylova and Feng 2023). Under normal conditions, mitochondrial vesicles in MVBs are degraded by the fusion of MVBs with lysosomes. When lysosomal function is impaired, mitochondrial vesicles in MVBs are secreted in the form of extracellular vesicles (Liang et al. 2023). The processes of MVB–PM fusion and ILV secretion require MVBs to carry and dock in the PM (Krylova and Feng 2023). RAB GTPase is known to play an important role in vesicle transport in the cell (Borchers et al. 2021). Munc13-4 and Slp4 are effectors of RAB27A (Fukuda 2013). RAB27A/Slp4 is involved in the docking of MVBs to the PM, and Munc13-4 mediates MVB–PM fusion through interactions with SNARE complexes (Snap25, VAMP, and syntaxin) (Fukuda 2003; 2013; Kasai et al. 2005; He et al. 2016).

Figure 1. The exosome biogenesis pathway. (A) Various stimuli and conditions in tumor microenvironment promote exosome production in cancer cells and immune cells. Tumor-derived exosomes (TDEs) or immune cell-derived exosomes (IDEs) promote or inhibit tumor growth and progression. (B) The exosome biogenesis pathway. Exosomes are produced via the endosomal maturation pathway. Endosomal maturation involves multiple processes, including endocytosis, early endosome sorting, and the formation of multivesicular bodies (MVBs, late endosomes). Early endosomes exchange molecular cargo with the Golgi through vesicles. Early endosomes undergo maturation, resulting in MVBs. The intraluminal vesicles (ILVs) within MVBs are generated by budding into the early endosomal membrane, which is mediated by the ESCRT pathway. MVBs secrete ILVs (exosomes) through fusion with the plasma membrane. In another pathway, MVBs are degraded through fusion with lysosomes. (C) The cargo components of exosomes. Exosome cargos contain various DNA, RNA, proteins and lipids.

Figure 2. The endosomal sorting complexes required for transport (ESCRT) pathway. The ESCRT pathway mediates the generation of ILVs within MVBs. (A) First, the ESCRT-0 complex (composed of Hrs and STAM) binds to ubiquitinated proteins in early endosomes. (B) ESCRT-0 recruits ESCRT-I (composed of TSG101, Vps28, Vps37 and Mvb12) and ESCRT-II (composed of EAP25, EAP30 and EAP45) to induce the inner budding of endosomes. The square box illustrates internal budding by the ESCRT complex viewed from above. (C) After inner budding, ESCRT-III (composed of many various CHMP families) binds to and assembles the ESCRT-II complex and deubiquitinates the protein by recruiting deubiquitination enzymes such as Doa4 (not shown in the figure). (D and E) The ESCRT-III complex is degraded by the Vps4 complex, resulting in the formation of ILVs.

Exosomes contain a variety of cargo, including DNA, RNA, proteins, and lipids. These cargos mediate cell–cell communication, cell homeostasis maintenance, tissue repair and regeneration and antigen presentation. Here, we describe in detail the types of cargo and their roles.

DNA

The exosome cargo contains sequence-independently fragmented genomic DNA and mitochondrial DNA (mtDNA). Although the exact mechanism by which DNA is packaged inside exosomes has not yet been revealed, it has been reported that exosomes maintain cellular homeostasis by removing cytoplasmic DNA (Takahashi et al. 2017). Nanoflow cytometry revealed that DNA (200 bp to 5,000 bp) exists as both single- and double-stranded DNA without histone proteins in exosomes (Liu et al. 2022). Another reported role of exosomal DNA is that T cell-derived exosomal DNA can prime dendritic cells. Primed dendritic cells protect against pathogen infection by activating the cGAS-STING signaling pathway (Torralba et al. 2018).

RNA

Exosomes contain mRNAs and noncoding RNAs (e.g., miRNAs, lncRNAs and circRNAs). Exosomal mRNA was first discovered through microarray analysis in MC/9 and HMC-1 cell lines. Obese mouse cell-derived exosomal mRNAs were transferred to human cells and translated into proteins (Valadi et al. 2007). RNAs are sorted into exosomes by directly binding RNA binding proteins (RBPs), including hnRNP. Certain miRNAs, including miR-575, miR-125a-3p, and miR-198, are more abundant in exosomes than in cells. The GGAG motif is present at a high level (approximately 75%) in exosomal miRNA sequences and is loaded into exosomes through interaction with sumoylated hnRNP2AB1 (Villarroya-Beltri et al. 2013). In addition, hnRNPA2B1 loads not only miRNAs but also noncoding RNAs, including circNEIL3 (Pan et al. 2022) and the lncRNA LNMAT2 (Chen et al. 2020), into exosomes. hnRNP-Q, called SYNCRIP, specifically binds to the GGCU sequence of miRNA and loads it into exosomes (Santangelo et al. 2016). The role of hnRNPs in recognizing specific RNA sequences suggests that RNA loading into exosomes is not random.

In cancer, exosomes contain tumor-specific mRNAs. The serum exosomal hnRNPH1 mRNA level in hepatocellular carcinoma patients is significantly greater than that in healthy controls, suggesting that hnRNPH1 is a potential biomarker for hepatocellular carcinoma diagnosis (Xu et al. 2018). Huang’s research team developed exoRBase (http://www.exoRBase.org), a web-accessible database, by analyzing circulating blood exosome RNA data. ExoRBase contains a large amount of informative RNA data (58,330 circRNAs, 15,501 lncRNAs, and 18,333 mRNAs) (Li et al. 2018). Therefore, exosomal RNA can be used as a biomarker for diagnosing various diseases.

Proteins

Exosomal proteins are generally loaded into exosomes via the ESCRT pathway. In intestinal epithelial cells, IL-1β is produced by GSDMD-induced NLRP3 inflammasome complex activation and is loaded into exosomes and secreted. The IQ domain of IQGAP1, a Rab GTPase activation-like protein, binds to the C-terminus of GSDMD, and IQGAP1/GSDMD recruits TSG101 to mediate LPS + ATP-induced exosomal IL-1β release (Liao et al. 2023). V-catenin is a transcriptional coactivator activated by Wnt that promotes tumor growth and metastasis (Zhang and Wang 2020). Immunoprecipitation assays have shown that β-catenin and Vps4A bind directly to the liver cancer cell lines Huh7 and SMMC7721. Vps4A promotes exosome loading through direct binding to β-catenin. Inhibition of Vps4A reduces exosome loading and the PM localization of β-catenin (Han et al. 2019).

In addition, proteins are also loaded into exosomes through an ESCRT-independent pathway. LAMP2A loads proteins containing the KFERQ motif into exosomes. HIF1A containing the KFERQ motif is loaded into exosomes through an ESCRT-independent pathway, such as the ceramide pathway (Ferreira et al. 2022).

Lipids

Lipid cargos, including fatty acids and cholesterol, regulate various biological processes by mediating intercellular communication and changing cellular functions (Record et al. 2014). In NASH patients, plasma exosomes contain high levels of ceramide. Treatment with palmitate, an ER stress inducer, increases exosome release through IRE1a activation in hepatocytes. Palmitate-induced hepatocyte-derived exosomes increase the migration of macrophages (Kakazu et al. 2016).

Tumor-derived exosomal lipids induce immune dysfunction in DCs. MC-38 cell-derived exosomes are captured by BMDCs, resulting in high lipid accumulation. Treatment with the exosome inhibitor GW4869 reduces lipid accumulation in DCs and partially rescues immune function. RNA sequence analysis has demonstrated that TDEs inhibit DC priming by activating PPAR-α signaling in DCs. PPAR-α deletion increases CD8+ T-cell infiltration. Therefore, these findings suggest that TDEs induce immune dysfunction and immune evasion in DCs through PPAR-α activation (Yin et al. 2020).

TDE cargos contain various molecules that promote the growth and progression of cancer cells. TDEs interact with immune cells, endothelial cells, and cancer cells in tumor microenvironments. In addition, various immune cell-derived exosomes (IDEs) promote or inhibit tumor growth by interacting with cancer cells, endothelial cells, and other immune cells in the tumor microenvironment. Here, we describe the role of TDEs in the tumor microenvironment (Fig. 3).

Figure 3. Communication between tumors and various immune cells through exosomes. TDEs containing miR-31-5p, miR-1247-3p, lncRNA UFC1 and S100A4 induce tumor growth and metastasis. TDEs containing miR-25-3p, miR-130b-3p, miR-425-5p and lncARSR promote M2 polarization of macrophages. Many IDEs containing miR-16-5p, miR-765, PD-1, TNF, FasL and TRAIL, derived from M1 macrophage, NK cells, dendritic cells, and CD8+ T cells, inhibit tumor growth and progression. In constrast, M2 macrophage-derived exosomes containing miR-21-5p, miR-155-5p, miR-221, miR-942 and Apo E promote tumor progression. In particular, miR-155-5p and Apo E induce immune escape and proliferation in cancer cells. The pink boxes mean TDEs and the green boxes mean IDEs.

TDEs in tumors

TDEs play an important role in the tumor metastasis process. Melanoma-derived exosomes promote tumor growth and increase metastasis to the lungs. High levels of MET protein are present in the exosomes of the melanoma cell line, B16-F10, and are transferred to bone marrow progenitor cells. These observations suggest that melanoma-derived exosomal MET protein is delivered to bone marrow progenitor cells and supports tumor growth and metastasis (Peinado et al. 2012). Exosomes derived from the HCC cell line MHCC97 activate the TGF-β/Smad signaling pathway in HepG2 cells and induce epithelial-to-mesenchymal transition (EMT). Exosomes derived from MHCC97 cells treated with LY2109761, a pharmacological inhibitor of TGF-β, increase E-cadherin expression and decrease vimentin expression in HepG2 cells. In animal experiments, MHCC97-derived exosomes promoted metastasis to the lungs. Therefore, it is thought that HCC-derived exosomes induce EMT and promote metastasis to the lung through activation of the TGF-β/Smad signaling pathway (Qu et al. 2019). Under hypoxic conditions, miR-31-5p in lung adenocarcinoma-derived exosomes directly binds to and inhibits SATB2. The inhibition of SATB2 induces MEK/ERK signaling activation and increases the migration and invasion of lung adenocarcinoma cells. In a xenograft model, exosomal miR-31-5p promoted metastasis to the lung. In addition, miR-31-5p is present at significantly higher levels in the plasma exosomes of lung adenocarcinoma patients, suggesting that it is a potential biomarker for lung adenocarcinoma diagnosis (Yu et al. 2021). Liver cancer-derived exosomal miR-1247-3p converts fibroblasts into cancer-related fibroblasts. miR-1247-3p directly binds to and suppresses B4GALT3 mRNA in fibroblasts, inducing b1 integrin/NF-κB pathway activation to increase IL-6 and IL-8 secretion. As a result, IL-6 and IL-8 enhance the stemness, EMT, and chemoresistance of liver cancer cells and promote metastasis to the lungs. In addition, high levels of miR-1247-3p are present in the serum exosomes of hepatocellular carcinoma (HCC) patients. Interestingly, serum exosomal miR-1247-3p is present at higher levels in patients with lung metastatic HCC than in patients with nonmetastatic HCC. Therefore, exosomal miR-1247-3p may be a diagnostic biomarker for liver cancer (Fang et al. 2018). The lncRNA UFC1 is expressed at high levels in non-small cell lung cancer (NSCLC) cell line A549-derived exosomes and serum exosomes from NSCLC patients. High levels of the lncRNA UFC1 promote proliferation and invasion of the NSCLC cell line, H1299, and knockdown of the lncRNA UFC1 induces cell cycle arrest and apoptosis. RNA immunoprecipitation, qPCR, and western blotting have revealed that PTEN expression is suppressed through direct binding to the lncRNAs UFC1 and EZH2. Therefore, lncRNA UFC1 induces epigenetic silencing of PTEN expression and the progression of NSCLC through EZH2 inhibition (Zang et al. 2020).

TDEs in the tumor microenvironment

The NSCLC-derived exosomal S100A4 suppresses T-cell immune activity and promotes tumor progression. S100A4 is known as a calcium-binding protein that promotes cancer growth and metastasis (Fei et al. 2017) and is expressed at high levels in the exosomes of the NSCLC cell line, A549. Moreover, NSCLC-derived exosomal S100A4 suppresses T-cell immune activity and promotes NSCLC progression through increased STAT3 and PD-L1 expression (Wu et al. 2022). Renal cancer-derived exosomes (RDEs) induce tumor immune tolerance. RDEs contain high levels of HSP70 and the tumor-specific antigen G250. RDE is captured by myeloid-derived suppressor cells (MDSCs) and induces HSP70-mediated MDSC activation. RDE-induced MDSCs suppress the cytotoxic effect of CD8+ T cells in renal cancer. Therefore, these findings suggest that HSP70 in RDE induces tumor immune tolerance by inducing the activation of MDSCs and inhibiting the activity of CD8+ T cells (Gao et al. 2020). Cervical cancer (Cca)-derived exosomal miR-1468-5p induces tumor immune escape through lymphatic immunosuppressive reprogramming. High levels of miR-1468-5p have been detected in Cca-derived exosomes. Cca-derived exosomal miR-1468-5p is delivered to human skin lymphatic endothelial cells and suppresses the immune response of CD8+ T cells through JAK2/STAT3 pathway activation via HMBOX1 inhibition. Cca-derived exosomal miR-1468- 5p-mediated CD8+ T-cell immunosuppression increases tumor growth in mice. In addition, higher levels of miR-1468-5p have been detected in the serum exosomes of Cca patients than in those of normal individuals. This finding suggests that miR-1468-5p may be a biomarker for diagnosing cervical cancer (Zhou et al. 2021a). In addition, exosomal miRNAs are potential biomarkers for cancer. Exosomal miRNAs (miR-25-3p, miR-130b-3p, and miR-425-5p) derived from the colon cancer cell line HCT116 promote M2 polarization of macrophages through PTEN inhibition. M2 macrophages promote angiogenesis and liver metastasis through secretion of the cytokines VEGF, IL-10, and IL-4. Higher levels of miR-25-3p, miR-130b-3p, and miR-425-5p have been observed in the serum exosomes of colorectal cancer (CRC) patients and metastatic CRC patients than in those of normal individuals (Wang et al. 2020).

Exosomes derived from hypoxic colorectal cancer (CRC) cells are reportedly delivered to endothelial cells and promote angiogenesis. Under hypoxic conditions, exosomes derived from the human CRC cell lines HT29 and HCT116 promote proliferation, migration and tumor angiogenesis by activating β-catenin in endothelial cells (Huang and Feng 2017). Exosomes derived from head and neck squamous cell carcinoma (HNSCC) cells induce functional reprogramming of endothelial cells and increase angiogenesis. Under hypoxic conditions, exosomes derived from PCI-13 cells, an HNSCC cell line, are loaded with proangiogenic proteins (uPA, coagulation factor III, and MMP-9), which increase the mRNA levels of angiogenesis-related genes (VEGF and IGFBP-3) when delivered to human umbilical vein endothelial cells (HUVECs). HNSCC-derived exosomes promote HUVEC proliferation, migration, and tube formation, leading to functional reprogramming (Ludwig et al. 2018). miR-92a-3p is abundant in retinoblastoma (RB) cell-derived exosomes, which are delivered to endothelial cells and promote angiogenesis through KLF2 inhibition. Knockdown of miR-92a-3p decreases the mRNA levels of inflammatory cytokines, including IL-1 and IL-6, in endothelial cells and decreases endothelial cell migration and tube formation. Similarly, overexpressing miR-92a-3p increases inflammatory cytokine levels, endothelial cell migration, and tube formation in endothelial cells (Chen et al. 2021).

The lncRNA BCYRN1 is highly expressed in urinary exosomes from bladder cancer (BCa) patients and in BCa-derived exosomes, where it induces lymph node metastasis. RNA pull-down assays revealed that the lncRNA BCYRN1 directly binds to hnRNPA1. In addition, the lncRNA BCYRN1/hnRNPA1 induced VEGF-C secretion through canonical Wnt pathway activation. As a result, the lncRNA BCYRN1 forms a feed-forward loop with the hnRNPA1/WNT5A/VEGF3 axis, suggesting that the VEGFR3 pathway is important for lymph node metastasis (Zheng et al. 2021).

IDEs: macrophage-derived exosomes

M1 macrophage-derived exosomes inhibit tumor progression. M1 macrophage-derived exosomes reduce PD-L1 expression in gastric cancer by delivering miR-16-5p to gastric cancer cells. Suppressed PD-L1 expression in gastric cancer enhances T-cell-mediated immune responses, resulting in the inhibition of tumor formation (Li et al. 2020). M2 macrophage-derived exosomes promote tumor growth and metastasis in the tumor microenvironment. High levels of miR-21-5p and miR-155-5p have been detected in M2 macrophage-derived exosomes. Exosomal miR-21-5p and miR-155-5p are delivered to colon cancer cells and promote cancer cell migration and invasion via BRG-1 inhibition (Lan et al. 2019). In another study, M2 macrophage-derived exosomal miR-21-5p was shown to be delivered to renal cancer cells and promote metastasis. miR-21-5p promotes the phosphorylation of AKT via PTEN inhibition in renal cancer cells (Zhang et al. 2022b). M2 macrophage-derived exosomal miR-155-5p is delivered to colon cancer cells and promotes cell proliferation and inhibits apoptosis. miR-155-5p increases the production of IL-6, an anti-inflammatory cytokine, and promotes immune escape by ZC3H12B inhibition in colon cancer cells (Ma et al. 2021). In addition, M2 macrophage-derived exosomes promote the proliferation and G1/S transition of epithelial ovarian cancer cells. miR-221-3p is expressed at high levels in M2 macrophage-derived exosomes and inhibits CDKN1B. High levels of miR-221-3p have been detected in M2 macrophage-derived exosomes. Exosomal miR-221-3p is delivered to colon cancer cells and promotes tumor growth and progression by inhibiting CDKN1B. Inhibition of CDKN1B by miR-221-3p is associated with poor prognosis in colon cancer patients (Li and Tang 2020). In addition, M2 macrophage-derived exosomes promote tumor growth and metastasis by increasing angiogenesis. M2 macrophage-derived exosomal miR-155-5p and miR-221-5p are delivered to vascular endothelial cells, increase angiogenesis and promote the growth of pancreatic adenocarcinoma. miR-155-5p and miR-221-5p promote angiogenesis by suppressing the expression of E2F2, a negative regulator of angiogenesis in endothelial cells (Yang et al. 2021). High levels of miR-942 have been detected in M2 macrophages of lung adenocarcinoma patients. miR-942 is loaded into exosomes and delivered to LUAD cells and vascular endothelial cells, where it promotes cancer metastasis and angiogenesis (Wei et al. 2022). Apo E is present at high levels in M2 macrophage-derived exosomes and reduces the expression of proteins that mediate innate immune responses, such as IFN-β, in cancer cells. Mechanistically, Apo E induces an ATPase inhibition-mediated decrease in MHC-I levels through direct binding to BiP in cancer cells. Therefore, these findings suggest that Apo E induces a decrease in CD8+ T-cell-mediated tumor immunity and inhibits immune checkpoint blockade through BiP-mediated inhibition of MHC-I expression in cancer cells (Zheng et al. 2023).

IDEs: dendritic cell-derived exosomes

Mature dendritic cell (DC)-derived exosomes induce T-cell activation. MHC-II and ICAM-1 are present at high levels in mature DC-derived exosomes and are delivered to naïve CD4+ T cells and induce priming (Segura et al. 2005). This finding suggests that T-cell priming by DC-derived exosomes may inhibit tumor growth and progression.

In addition, DC-derived exosomes induce NK-cell activation. Compared with immature DC-derived exosomes, mature DC-derived exosomes contain higher levels of TNF, FasL, and TRAIL. Exosomal TNF induces NK-cell activation by interacting with the TNF receptor of NK cells. Exosomal TNF, FasL, and TRAIL induce apoptosis when delivered to tumor cells (Munich et al. 2012).

IDEs: NK cell-derived exosomes

NK cell-derived exosomal Fas ligands are delivered to melanoma cells to induce apoptosis. The expression of cleaved caspase-3 and cleaved PARP, which are related to apoptosis, is increased by the transfer of NK cell-derived exosomes to B16F10 cells. In addition, NK cell-derived exosome-mediated cytotoxicity is not observed in normal cells, including Phoenix-A, a human kidney epithelial cell line (Zhu et al. 2017). NK cell-derived exosomes exhibit cytotoxic effects on several cancer cells, including A549 and HeLa cells. IL-15+IL-12 cotreatment enhances the cytotoxic activity of NK cell-derived exosomes. In addition, treatment with IL-15+IL-12 causes the accumulation of miRNAs (particular miR-146b and miR-23a) and changes the protein profile of NK cell-derived exosomes. In particular, high levels of CD226 have been detected in exosomes derived from IL-15+IL12-stimulated NK cells. Blocking CD226 with an antibody suppressed NK cell-derived exosome-mediated cytolytic activity (Enomoto et al. 2022). NK cell-derived exosomes also regulate immune responses. Exosomal miR-10b-5p and miR-92a-3p induce Th1 polarization and T-cell activation through transcriptional inhibition of Gata3. NK cell-derived exosomes mediate the polarization of monocytes and monocyte-derived DCs, increasing the secretion of IFN-γ and improving their presentation function (Dosil et al. 2022).

IDEs: T-cell-derived exosomes

In the TME, T-cell-derived exosomes play an important role in activating immune cells and regulating immune responses against tumors. Exosomes derived from activated CD4+ T cells contain CD4+ T-cell markers, including CD4, TCR, and LFA-1. T-cell-derived exosomes are delivered to DCs through interactions between TCRs/pMHC-II and LFA-1/CD54. In addition, CD4+ T-cell-derived exosomes suppress CD4+ T-cell proliferation and the immune response of CD8+ T cells to melanoma (Zhang et al. 2011).

However, according to another study, CD4+ T-cell-derived exosomes induce CD8+ T-cell activation and increase cell proliferation. CD4+ T-cell-derived exosomal miR-25-3p, miR-155-5p, miR-215-p, and miR-375 improve CD8+ T-cell-mediated antitumor effects. In a melanoma mouse model, CD4+ T-cell-derived exosomes inhibited tumor growth through CD8+ T-cell activation (Shin et al. 2022). Considering these conflicting reports, further studies are needed to clarify the role of CD4+ T-cell-derived exosomes in the TME.

PD-1 is present in exosomes derived from activated CD8+ T cells and is delivered to cancer cells and promotes cell death. Exosomal PD-1 binds to PD-L1 in the triple-negative breast cancer cell lines MDA-MB-231 and BT549, alleviating PD-L1-mediated T-cell immune dysfunction and inhibiting tumor growth (Qiu et al. 2021). CD8+ T-cell-derived exosomes suppress the development of estrogen-induced endometrial cancer. CD8+ T-cell-derived exosomal miR-765 is delivered to endometrial cancer cells and indirectly suppresses PLP2 by controlling several miRNA clusters, including miR-3584-5p and miR-7-5p. Since PLP2 promotes the proliferation and metastasis of cancer cells through Notch signaling activation, CD8+ T-cell-derived exosomal miR-765 may be a potential treatment strategy for endometrial cancer (Zhou et al. 2021b).

Since exosomes transport various cargos, research on the development of anticancer drugs using exosomes is actively underway. Compared with nanomaterials, such as liposomes, exosomes have high bioavailability and low cytotoxicity (Dai et al. 2020). Furthermore, exosomes can be used as biomarkers for the diagnosis of cancer (Wang et al. 2022). Here, we describe various cutting-edge anticancer strategies using exosomes.

Drug delivery

Anticancer drugs, including doxorubicin and paclitaxel, cause serious side effects because they cause the death of not only cancer cells but also normal cells. Anticancer drug-loaded exosomes can be delivered to cancer cells specifically, resulting in fewer side effects and greater treatment efficacy. Compared with free doxorubicin, doxorubicin delivered via exosomes reduces the level of doxorubicin accumulated in the heart by approximately 40%. Compared with free doxorubicin (Toffoli et al. 2015), exosomes loaded with doxorubicin promote the apoptosis of Y79 cells, a retinoblastoma cell line (Farhat et al. 2022). Sonicated paclitaxel-loaded macrophage-derived exosomes show more than 50-fold greater cytotoxicity to multidrug-resistant (MDR) cancer cells. Exosomes are absorbed 30 times more into PC12 cells than are nanoparticles, suggesting that exosomes can efficiently deliver drugs to cancer cells. In addition, paclitaxel-loaded exosomes inhibited growth and metastasis in an LLC mouse model (Kim et al. 2016). There are several strategies for drug loading into exosomes, including co-incubation with exosomes and drugs, ultrasound, and electroporation. (Zhang et al. 2023). Co-incubation with exosomes and drugs is used when loading hydrophobic drugs into exosomes. Because exosomes are encapsulated phospholipids, hydrophobic drugs can be loaded into exosomes through diffusion (Luan et al. 2017). Unlike co-incubation, ultrasound and electroporation are strategies for loading drugs by artificially creating a passage in the exosome membrane. Hydrophilic drugs can be loaded into exosomes through the created passage (Tian et al. 2023).

These findings indicate that exosomes have better cellular uptake and lower toxicity, making them a highly efficient drug delivery strategy compared to other drug delivery vehicles, especially in cancer therapy. Several clinical trials on anticancer strategies using exosomes are underway (Table 1). KrasG12D is a common mutation in metastatic pancreatic cancer (Bannoura et al. 2022). Clinical studies delivering exosomes loaded with KrasG12D- siRNA for the treatment of KrasG12D mutant metastatic pancreatic cancer are currently in phase I (NCT03608631). CDK-004, an exosome encapsulated with lipid-tagged oligonucleotides, is designed to target M2 macrophages and repolarize them into M1 macrophages via STAT6 antisense oligonucleotide. In the Phase I study, the therapeutic effects of CDK-004 delivery are evaluated in patients with advanced hepatocellular carcinoma and patients with liver metastases from either gastric or colorectal cancer (NCT05375604). The delivery of Dabrafenib and Trametinib using exosomes for melanoma treatment is currently in a phase II clinical trial (NCT01972347). The therapeutic effects of metronomic cyclophosphamide and tumor antigen-loaded dendritic cell-derived exosomes are being evaluated in non-small cell lung cancer (NCT01159288). In phase III clinical trials, the delivery of Trastuzumab or Atezolizumab using exosomes for breast cancer or triple negative breast cancer treatment is ongoing (NCT01772472 or NCT02425891, respectively).

Table 1 . Clinical trials for drug delivery and the development of cancer diagnostic biomarkers using exosomes.

Drug delivery
Drug	Cancer	Phase	NCT code
KRASG12D-siRNA	Metastatic pancreatic cancer	Phase I	NCT03608631
exoASO-STAT6 (CDK-004)	Hepatocellular carcinoma	Phase I	NCT05375604
Dabrafenib & trametinib	Melanoma	Phase II	NCT01972347
Metronomic cyclophosphamide (mCTX)	Lung cancer	Phase II	NCT01159288
Trastuzumab	Breast cancer	Phase III	NCT01772472
Atezolizumab	Triple negative breast cancer	Phase III	NCT02425891
Identification of biomarkers for diagnosis
Cancer	Host samples for exosomes		NCT code
Lung cancer	Cancer tissue, peripheral blood		NCT03542253
Pancreatic cancer	Portal venous blood		NCT03821909
Prostate cancer	Urine		NCT03911999
Gastric cancer	Plasma		NCT01779583
Rectal cancer	Serum		NCT03874559
Thyroid cancer	Urine		NCT02862470
Non-small cell lung cancer	Serum		NCT02921854
Non-small cell lung cancer	Serum		NCT05218759

Biomarkers for diagnosis

Exosomes can be used as biomarkers to diagnose cancer. Analysis of patient liquid biopsies, including plasma, is easier to perform and less invasive than analysis of tissue biopsies. High levels of miR-423-5p are present in the serum exosomes of gastric cancer patients and are significantly associated with lymph node metastasis. Kaplan-Meier analysis has shown that the level of miR-423-5p is inversely related to the survival rate of patients, suggesting that the level of miR-423-5p is associated with poor patient prognosis (Yang et al. 2018). High levels of miR-191, miR-21 and miR-451a have been detected in serum exosomes from patients with early-stage pancreatic cancer. In particular, the level of miR-21 in serum exosomes is closely related to patient survival (Goto et al. 2018). In addition, survivin can be used as a potential diagnostic biomarker because survivin is present at high levels in the serum exosomes of breast cancer patients (Khan et al. 2014). Currently, exosomes derived from liquid biopsies, including serum, are being studied as diagnostic biomarkers for various cancers (Table 1). A clinical trial on cancer biomarkers revealed that exosomal micro-RNA is highly prevalent in early lung cancer tissues and the peripheral blood of lung cancer patients (NCT03542253). Exosome mRNA analysis research is underway using RNA-seq on portal vein blood from pancreatic cancer patients (NCT03821909). A clinical study involving prostate cancer patients aims to identify candidate miRNAs that can serve as diagnostic biomarkers in urine-derived exosomes (NCT03911999). Plasma-derived exosome analysis in gastric cancer patients aims to characterize the exosomal molecular profiles from plasma (NCT01779583). For rectal cancer patients who have received chemoradiotherapy, the clinical study aims to characterize biomarker levels in serum-derived exosomes, with the ultimate goal of identifying the roles of these exosomes using rectal cancer organoid and mouse models (NCT03874559). Urine-derived exosome analysis targeting thyroid cancer patients aims to identify prognostic indicators, including proteins, and to find new therapeutic targets (NCT02862470). A clinical study on biomarker identification in serum-derived exosomes from non-small cell lung cancer patients (NCT02921854) demonstrated negative (CD244, CXCL10, and IL-10) and positive (CR2 and IFNGR2) prognostic biomarkers in these exosomes (Vaes et al. 2021). Further analysis of serum-derived exosomal miRNAs to identify diagnostic biomarkers is ongoing in another clinical study of non-small cell lung cancer (NCT05218759).

Preclinical and clinical trials for drug delivery using exosomes are underway for cancer therapy. Drug delivery using exosomes is more efficient and has fewer side effects than free drug delivery in cancer therapy. TDEs promote tumor growth and metastasis by inducing tumor immune evasion and increasing tumor angiogenesis. Because cancer cells release a greater number of exosomes than normal cells and have different cargo components, TDEs have the potential to be used as biomarkers for cancer diagnosis. Many studies on TDEs have revealed several miRNAs and proteins that can serve as biomarkers for cancer diagnosis. Overall, exosomes may serve as biomarkers for cancer diagnosis and potential therapeutic drug delivery agents.

The authors declare that they have no conflict of interest.

The Figures 1, 2, and 3 are illustrated with BioRender.com.