Cancer is a leading cause of death worldwide. At present, tissue biopsy is considered the gold standard method for the clinical diagnosis of cancer. However, tissue collection may be complex, due to the close proximity of some tumors to major arteries in the body. In addition, obtaining tumor tissues from a specific location may not accurately represent the overall health of the patient (1). By contrast, liquid biopsies, which include biopsies of circulating tumor cells (CTCs), free DNA (cfDNA) and exosomes, provide further information regarding the status of the tumor, and do not require invasive procedures (2). This information may be obtained through frequent non-invasive sampling, which aids in the diagnosis of cancer and monitoring the pathophysiologic condition of the patient (3, 4).

Exosomes are biologically active extracellular vesicles that range from 50–200 nanometers in size, and are produced by live cells (5). Exosomes are located in the majority of bodily fluids, including urine, blood, saliva, ascites, breast milk, amniotic fluid and cerebrospinal fluid (6). Exosomes are responsible for transporting a variety of substances (7, 8), including proteins, nucleic acids, lipids, enzymes and metabolites, and these are regulated by the physiology of the original cell (9). Several mechanisms, including direct fusion with cell membranes, endocytosis routes and ligand-receptor interactions, are involved in the process of intercellular communication (10–12). Notably, exosomes may play a role in intercellular communication.

As tumor cells are primarily responsible for the production of tumor-derived exosomes that contain chemicals which reflect the properties of the parental tumor cells (13), they may exhibit potential as diagnostic markers for tumors (14). Importantly, liquid biopsy of exosomes may be more useful than CTC or circulating tumor DNA (ctDNA), as there is a large volume of exosomes in bodily fluids (up to 10¹¹ exosomes per ml in blood), making them easier to detect. In addition, ~10% of exosomes detected in the bodily fluids of individuals who have advanced malignancies originate from tumor cells (15). At present, research is focused on exosomal RNAs, including microRNAs, circular RNAs (circRNA) and long non-coding RNAs (16–18). Exosomal proteins are either encased inside an inner lumen or embedded on the surface of the exosome. This allows for the categorization of exosome subtypes based on surface biomarkers, without causing any disruption to the structure of the exosomes. Compared with alternate exosomal cargoes, exosomal proteins exhibit numerous advantages, including i) a long half-life and stability inside the exosomes in which they are found (19), and ii) a relatively straightforward isolation procedure, due to identification of exosomal surface proteins in smaller sample sizes (20–22). Through the use of proteomics, protein coverage and sensitivity have been significantly enhanced, which has led to an increase in the breadth of exosomal proteomics data surrounding cancer causation, function and disease association (23). Further investigations are required to determine the specific process of carcinogenesis and the advancement of cancer. The present study aimed to provide a summary of the diagnostic and prognostic functions that exosomal proteins play in a variety of malignancies, and demonstrate the potential uses of exosomal proteins in clinical cancer treatment.

Extracellular vesicles (EVs) are a collective name that refers to nanoscale vesicles that are actively released by cells (24). Exosomes are formed through the fusion of multi-vesicular vesicles with the cell membrane, with a diameter of 50–200 nm, and microvesicles are formed through the direct outgrowth of the cell membrane, with a diameter of 200-2,000 nm. Moreover, apoptotic vesicles are formed through the shrinkage and fragmentation of apoptotic cells, with a diameter of 500-2,000 nm (25) ( Figure 1A ).

In 1983, exosomes were initially isolated from sheep reticulocytes. Through the investigation of the transferrin receptor during the maturation of reticulocytes, Johnston et al. discovered that the mechanism of the transferrin receptor is lost when exosomes are created during the maturation of erythrocytes (26). There are a variety of cell types that are capable of actively producing exosomes, including human umbilical vein endothelial cells, reticulocytes and immune cells, such as lymphocytes, macrophages, dendritic cells, natural killer cells, stem cells, endothelial cells and neuronal cells (27–30). Exosomes may be produced under both healthy and pathological conditions, and exosomal development includes initiation, endocytosis, the creation of multivesicular bodies and release. The creation of exosomes begins with the endocytosis of vesicles, leading to the development of early sorting endosomes via the invagination of the cell membrane. Subsequently, these endosomes evolve into late sorting endosomes (31), and these bud inward to form multivesicular bodies (MVBs). MVBs ultimately fuse with the plasma membrane and discharge their contents into the extracellular environment, and these are referred to as exosomes ( Figure 1A ). Moreover, released exosomes may be directed to other cells using a variety of cell surface proteins, including tetraspanning proteins (32–34).

Endosomal sorting complexes (ESCRT) are required for translocation-dependent processes. Significantly, ESCRT and non-ESCRT mechanisms (35) play a key role in the production of exosomes. ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III are the four functional subcomplexes involved in the ESCRT mechanism, and this is comprised of ~30 proteins. Particularly, the aforementioned subcomplexes are required for exosomal biogenesis. Lipids and associated proteins, such as the transmembrane tetraprotein CD63, play key roles in the ESCRT pathway, and these do not require any other proteins. Results of previous studies demonstrated that specific structures, such as lipid rafts and proteins with a four-transmembrane structural domain, may play a crucial role in the creation of certain exosomes (36, 37). Notably, exosomes transfer information to receptor cells via three primary routes ( Figure 2 ); namely, i) Receptor-ligand interactions; ii) direct fusion with the cell membrane; and iii) endocytosis via phagocytosis.

At present, research is focused on exosomal proteins due to their unique biological functions and the key roles that they play in regulating the tumor microenvironment (TME). Some exosomal proteins are embedded on the surface of the membrane, and some are completely encased in the membrane ( Figure 1B ). Importantly, certain proteins, such as CD63, TSG101 and Alix, have been recognized as biomarkers for exosomes, while others, such as Calnexin, may function as negative markers for exosome identification (38). Specific proteins on exosomes, such as epidermal growth factor receptor (EGFR), Ephrin type-A receptor 2 (EphA2) and Epithelial cell adhesion molecule (EpCAM), are increasingly used to distinguish between tumor-derived exosomes and non-tumor-derived exosomes (39). Metastatic ovarian cancer cells release numerous exosomes carrying E-cadherin, which is an inducer of angiogenesis (40). Expression levels of exosomal lipopolysaccharide-binding protein and E-cadherin were used to identify non-small cell lung cancer (NSCLC) and ovarian cancer cells with metastatic phenotypes (41). Results of a previous study revealed that in patients with head and neck squamous cell carcinoma, exosomal Programmed cell death ligand 1 (PD-L1) expression levels were associated with disease progression, UICC stage and lymph node invasion (42). Moreover, the detection of PD-L1 positive exosomes in blood samples from patients with pancreatic ductal adenocarcinoma was associated with poorer survival (43). As a key immune checkpoint molecule, the expression levels of PD-L1 directly reflect the ability of tumor cells to evade immune surveillance. It has been found that revealed that PD-L1 inhibits T cell function and promotes immune escape through binding to PD-1 on the surface of T cells (44). In addition, epigenetic studies revealed that the demethylation status of the PD-L1 promoter is positively correlated with its expression levels (45). Collectively, these studies suggest that exosomal surface proteins may exhibit potential in the diagnosis and prognosis prediction of cancer; thus, exhibiting potential in monitoring treatment response. In addition, exosomal surface proteins may contribute to further understanding the mechanisms underlying exosome biogenesis (46–51), targeting (52, 53) and interaction (54–57). The main types of exosomal surface proteins and their functions are displayed in Table 1 .

Research has focused on the role of exosomes in tumors due to the key role they play in intercellular communication. Exosomes that originate from tumor cells or stromal cells are associated with all phases of cancer development. These stages include tumor growth and cell proliferation, the prevention of cell death, angiogenesis, immune evasion, invasion and metastasis. It has been shown that exosomes may play a key role in the intricate biological interactions that take place between tumor cells and the TME (58). Interestingly, there are numerous different physiologically active chemicals that are carried by exosomes. These compounds are critical signals for reprogramming the TME for the induction of carcinogenesis (59). For example, delta-like 4 protein (DLL4) plays a key role in the promotion of cancer-associated TME alterations (60) and the expansion of vascular branching. The presence of DLL4 in individuals with colorectal cancer (CRC) may be associated with aggressiveness of the tumor and negative clinical outcomes (61). Moreover, tumor cell-derived exosomes may function as catalysts for epithelial-to-mesenchymal transition (EMT) and tumor metastasis. This is accomplished by stimulating quiescent cancer cells to actively metastasize via the use of a wide range of signaling molecules, such as Notch1 and HIF1α (62, 63). It has been shown that tumor-derived exosomes may assist tumor cells in evading monitoring of the immune system, developing resistance to chemotherapy and further promoting the growth of the tumor. Results of previous studies revealed that tumor-derived exosomes decrease the cytotoxicity of natural killer cells and cytotoxic T-lymphocytes. Particularly, these cells are essential components of the immune response, which plays a key role in preventing the growth of tumors (64, 65). Previous research has shown that tumor-derived exosomes may promote the polarization of immature macrophages into M2 macrophages and exhibit anti-inflammatory activity, both of which are beneficial to the continued spread of the tumor. Interestingly, angiogenesis is a fundamental physiological process during carcinogenesis that involves numerous steps. A wide range of angiogenic factors, including vascular endothelial growth factor, interleukin, transforming growth factor-β and fibroblast growth factor, are located inside exosomes (66). These factors play a crucial role in promoting the proliferation and migration of endothelial cells, and are essential in the generation of tumor angiogenesis (67, 68).

Given their disease-specific alterations, exosomal proteins have emerged as potential biomarkers for cancer. Exosomes that are produced from tumors carry a large amount of data regarding the biology of cancer cells (69). Levels of exosomal proteins in patients with cancer are considerably higher than those observed in healthy individuals. As a result, the identification of proteins in exosomes is sensitive and advantageous for the early diagnosis of cancer. Thus, the identification of exosomal proteins may exhibit potential in the diagnosis of cancer and in predicting patient prognosis (70). The tumor-derived exosomal protein analysis process is displayed in Figure 3 , and Table 2 summarizes the exosomal proteins that may exhibit potential as diagnostic markers in various tumors.

Exosomal proteins not only exhibit potential as a molecular marker for the diagnosis of cancer, but also demonstrate therapeutic potential due to their unique biological characteristics. At present, research is focused on use of engineered exosomes for the targeted distribution of anti-cancer therapy. Results of a previous study revealed that modified exosomes that include adriamycin may be more effective in targeting tumors and inhibiting their growth (117). An additional investigation was conducted in which the parental cells of exosomes were modified to produce Lamp2b linked to αv integrin-specific iRGD peptide. Importantly, this peptide demonstrated sufficient tumor-targeting characteristics in prostate, breast and cervical cancer models (118). In addition, The study noted that a subtype of MVBs, known as inhibitory protein structural domain 1-mediated microvesicles, aids in the transportation of NOTCH receptors to target cells and the stimulation of downstream gene expression (119). Votteler et al. discovered an innovative method for hybridizing exosomes using envelope protein nanocages, also known as EPNs. Through the process of membrane attachment and self-assembly, extracellular polymeric nanoparticles (EPNs) transfer their payload into the cytoplasm of recipient cells (120). This is accomplished through binding a variety of designed proteins.

In addition, the immunological activity of exosomes may allow them to be used as drug transporters in cancer immunotherapy or as vaccines (121). As exosomes are responsible for transporting a large number of tumor antigens, they may play a role in antigen presentation, exhibiting potential as cancer vaccines (122). Moreover, Hsp70 plays a key role in activating dendritic cells and monocytes, which in turn stimulates immunological responses that are mediated by tumor-derived exosomes (123). Hsp70 also plays a key role in promoting the release of granzyme B from natural killer cells, ultimately leading to the induction of apoptosis in tumor cells (124). Interestingly, HSP70 may function as an exosome surface antigen, eliciting anti-tumor antibody responses. Thus, exosomal proteins exhibit potential in the treatment of a wide range of malignancies. Clinical trials of exosomes in cancer therapy have focused on the following areas, with specific applications including as drug delivery vehicles, immunomodulators, diagnostic markers, and combination therapy tools. Most of the trials are currently in the early stages (phase I/II), but the multifunctional properties of exosomes make them have great potential in personalized cancer therapy ( Table 3 ). With advances in bioengineering technology, more precise clinical applications may be realized in the future.

As a novel form of liquid biopsy markers, exosomal proteins may demonstrate potential in the diagnosis of cancer. In recent years, tumor-derived exosomal proteins have demonstrated unique diagnostic value, and key advantages of these proteins include the following factors: i) Disease-specific enrichment, where the protein composition of exosomes highly reflects the physiological and pathological status of their parent cells, particularly tumor cell-derived exosomes (TEXs). These exosomes carry a large number of tumor-related proteins, such as EGFR, HER2, PD-L1, MET and GPC1, which may be used as cancer-specific markers; ii) a high level of stability, making them suitable for long-term storage and the detection of clinical samples. The lipid bilayer membrane structure of exosomes protects the internal proteins from proteases in the blood. Notably, this is more stable than free proteins or ctDNA due to the strong anti-degradation ability; iii) the potential clinical application of minimally invasive/non-invasive testing. Especially, small volumes of bodily fluid, such as 1 ml of blood or urine, is required for routine tests, mitigating limitations associated with tissue biopsy and sampling; and iv) multi-dimensional diagnostic information integration. Multiple protein marker combinations may be analyzed at the same time to improve diagnostic accuracy.

Although exosomal proteins exhibit potential in the diagnosis of cancer, limitations may remain, leading to limited use in clinical practice. Limitations may include the following factors: i) Complexities in the standardization of isolation and purification techniques. At present, exosome isolation requires numerous techniques, such as ultracentrifugation, size-exclusion chromatography, polymer precipitation and immunoaffinity capture. However, these methods exhibit notable differences in recovery rate, purity and integrity of exosomes. Contaminants, such as lipoproteins and protein aggregates in blood samples, often co-precipitate with exosomes, affecting downstream analysis. Although the International Society for Extracellular Vesicles (ISEV) has proposed the MISEV guideline, a globally recognized standardized process is yet to be developed, resulting in poor comparability of data; ii) analytical challenges associated with tumor heterogeneity. Importantly, exosomes in bodily fluids may be derived from tumor cells, immune cells and platelets. Tumor-derived exosomes may account for >1% of total exosomes, and therefore require enrichment with highly specific markers. In addition, the composition of exosomal proteins changes continuously with tumor progression and treatment response, and a dynamic monitoring system is required; and iii) complexities in large-scale application and the translation to clinical practice. The majority of studies include small-scale cohorts and lack validation through multi-center, prospective clinical trials. High-precision separation and detection technologies, such as nanoflow or single exosome analysis are costly, and there may be barriers associated with their use in clinical practice.

Exosomal proteins exhibit high levels of potential in the diagnosis of cancer; however, interdisciplinary collaboration, optimization of technical processes and the establishment of a globally uniform standardization system are required for their use in clinical practice. Future research should focus on developing high-purity and high-throughput separation technologies, such as microfluidic chips and aptamer capture. In addition, further investigations focused on the establishment of a multi-omics integrated analysis process are required, to integrate proteins, RNA and metabolites. Future research should also focus on the use of AI-assisted marker screening to improve the efficiency of data analysis. The aforementioned investigations may lead to evolution of the field of liquid biopsy, revolutionizing the early diagnosis and treatment of cancer.

As emerging tumor markers and therapeutic carriers, exosomal proteins have demonstrated revolutionary potential in the diagnosis and treatment of cancer. In the diagnosis of cancer, the unique molecular features and high stability of exosomes may provide a novel basis for early screening, precise typing and the dynamic monitoring of tumors. As a natural delivery system, exosomal proteins may exhibit potential in the treatment of cancer, leading to the development of targeted therapies and immunomodulation. However, future investigations are required to overcome key challenges, including isolation standardization, up-scaling of production and clinical validation. With the integration and development of interdisciplinary technologies, such as nanotechnology, multi-omics analysis and artificial intelligence, exosomal proteins may lead to considerable developments in the diagnosis and treatment of cancer, aiding the development of precise and personalized medicines. Notably, future investigations should focus on establishing a standardized technical system through multi-center clinical trials, and exploring the specific molecular mechanisms associated with exosomes.