The occurrence of ESCC is a result of a continuous accumulation of mutations in esophageal cells and oncogenic alteration in the TME (75). The TME involves blood vessels, the extracellular matrix (ECM), cytokines, and stromal cells and is indispensable in tumorigenesis because it provides necessary conditions for tumor growth and manipulates the interaction between cancer cells and their surroundings (76). Exosomes act as an essential role in the formation and reprogramming of the TME, as has been widely documented in many cancer types (77–79). In ESCC, Li et al. found that exosomal FMR1 antisense RNA 1 (FMR1-AS1) could remodel the TME in ESCC (65). The study confirmed that the FMR1-AS1 exosomes were secreted from cancer stem-like cells (CSCs) of ESCC, which transferred stemness phenotypes to recipient non-CSCs in the TME through the mechanism of activating toll-like receptor 7-nuclear factor κ B (TLR7-NFκB) signaling, upregulating the c-Myc level in recipient cells. Another example of exosomal cargos promoting the TME formation involves circ-0048117-rich exosomes derived from hypoxic ESCC cells, promoting M2 macrophage polarization to alter the components in the microenvironment (71). Researchers indicated that hypoxic exosomes modulated the TME in ESCC via the transformation of endothelial cell phenotypes and transcriptomes, which enhance angiogenesis and metastasis. Moreover, the exchange of exosomes in cancer cells and the stroma is bidirectional, and cancer-associated fibroblasts (CAFs) can also secrete exosomal Sonic Hedgehog to promote the generation of TME in ESCC (72). These findings suggest that exosomes can play an essential role in the formation, remolding, and normal function of the TME and that novel therapies targeting the TME may be a new approach to cancer treatment.

The progression of ESCC results from rapid growth and expansion of cancer cells, which may incur tumor survival and defiance to therapy. Exosomes can influence the growth of ESCC by mediating the apoptosis, cycle, and proliferation rate of ESCC cells (80). Molecular profiling has indicated that exosomal miRNA-19b-3p from EC9706 cells targets PTEN, a well-known tumor suppressor gene, to regulate the apoptosis of ESCC (56). A similar study elucidated that exosomal lncRNA ZNFX1 antisense RNA 1 (ZFAS1) derived from EC109 cells regulates ESCC proliferation, apoptosis, and migration via targeting the miRNA-124/STAT3 signaling pathway (66). Cancer cell-derived exosomes can regulate the ratio of G1-phase cells and influence the cycle and migration ability of ESCC cells (81). Some researchers have demonstrated that the proliferation and apoptosis of ESCC cells are modulated by several other exosomal cargos, including miRNA-103a-2-5p, miRNA-652-5p, lncRNA Family with sequence similarity 225 member A (FAM225A), and lncRNA urothelial cancer-associated 1 (UCA1) (57, 58, 67, 68). Generally, these exosomal contents can work by mediating the expression of proliferation- or apoptosis-related proteins and triggering a subsequent signaling pathway. Importantly, these studies also indicated that the identified exosomal RNAs and other exosomal contents can facilitate the proliferation ability of tumor cells alone and may concurrently alter the potential for migration, angiogenesis, and metastasis. Above all, the activation of cell proliferation cannot entirely be attributed to the expansion and development of ESCC; instead, it results from several steps, including migration and metastasis, angiogenesis, immune response, and therapy resistance. The role of exosomes in these steps of tumor development is explored in the following sections.

Angiogenesis, a critical phase during neoplasia, migration, and metastasis, is a multistep formation of neovascularization through which cancer cells obtain sufficient oxygen, nutrition, and energy (82). Reports have illustrated that some exosomes could play a role in inducing angiogenesis in many cancer types (83). Exosomes can deliver numerous pro-angiogenic bioactive substances, including vascular endothelial growth factor (VEGF), miRNAs, or other bioactive mediators. Published data suggest that exosomal cargos accelerate angiogenesis by suppressing the expression of anti-angiogenesis genes and promoting the expression of pro-angiogenic genes (84, 85). For example, compared with normal exosomes, hypoxic exosomes have played a unique role in facilitating the aggressive behavior of human umbilical vein endothelial cells (HUVECs) both in vitro and in vivo, and HUVECs exposed to hypoxic exosomes induce enhanced proliferation, metastatic dissemination, and vessel formation ability in ESCC (86). Consistent with that study, Zhuang showed that ESCC cell-derived exosomal miRNA-21 potentiates the angiogenesis ability of HUVEC by targeting sprouty RTK signaling antagonist 1 (SPRY1) in ESCC (60). Moreover, exosomal miRNA-21-5p has been shown to significantly promote the angiogenesis of targeted cells via the activation of programmed cell death 4 and downgrading of the signaling pathway or the PTEN/Akt signal pathway in ESCC (59). Similar findings have suggested that exosomal lncRNA FAM225A accelerates ESCC angiogenesis by binding to miRNA-206 and promoting NETO2 and FOXP1 expression (67). Given the pivotal role of angiogenesis in ESCC development and progression, exosome-related research may provide a new avenue to counteract these mechanisms of progression in ESCC, and these discoveries will become even more promising if they are linked to antitumor vascular drugs.

Metastasis is a critical step in tumor growth, and it remains a paramount threshold for cancer treatment and the chief cause of cancer mortality (87). Metastasis is a complicated and intricate process involving several steps such as epithelial-mesenchymal transition (EMT) of cancer cells, migration and infiltration into surrounding tissues, intravascular transport, and recognition and establishment in distant tissues (88, 89). Intercellular communication by delivering exosomes from primary tumor cells to the local microenvironment or distant organs is crucial for the phenotypic change and biological aggressive behavior of cancer cells, forming a pre-metastatic niche, and attachment and implantation to distant organs (90). Esophageal cancer cell-derived exosomes can modulate gene expression of recipient cancer cells, leading to an increased risk of invasion and metastasis. For example, the Sonic Hedgehog (SHH) signaling pathway, which has played important roles during development and in cancer (91, 92), can drive tumorigenesis and progression of ESCC. One study showed that exosomal Sonic Hedgehog derived from cancer-associated fibroblasts (CAFs) could increase the activation of N-cadherin and Vimentin in EC109 cell lines and consequently promoted the growth and migration abilities of ESCC (72). Similarly, exosomes derived by infiltrating T cells from irradiated esophageal carcinoma can incur the EMT in ESCC and facilitate metastasis (93). Conversely, human umbilical cord mesenchymal stem cells can suppress enabled homolog (ENAH) expression and decrease the invasion and migration ability of ESCC via the exosomal delivery of miRNA-375 (62). Exosomes play a pivotal role in forming a premetastatic niche in distant organs in ESCC, like in gastric cancer and breast cancer (94). This role may be attributed partly to the predisposition of early lymphatic metastasis in ESCC; metastatic dissemination in the liver or lung is relatively rare (95). In conclusion, exosomal cargos can exert pro-tumorigenic effects in most steps of ESCC metastasis, thus promoting the metastatic potential of ESCC. The studies discussed here may offer new insights that help the researcher understand the function of exosomes in metastatic ESCC and uncover exosome-based therapies that may curb cancer metastasis.

The elimination of tumor cells relies heavily on the immune system in vivo and exogenous therapies, such as drugs or irradiation (96). The immune system is an intricate network that can guard the body by monitoring, recognizing, and eliminating foreign invaders, such as bacteria, parasites, and endogenous antigens like cancer cells (97). These days, when we talk about the relationship between immune response and tumors, programmed cell death protein 1 (PD-1) is one of the monumental works that are closely associated with it indeed (98). A recent study suggested that O-linked β-N-acetylglucosamine (O-GlcNAc) transferase from stem cells of ESCC can upregulate PD-1 in CD8+ T cells and promote cancer immunosuppression (73). Furthermore, exosomes isolated from serum, plasma, urine, or other body fluids of patients with ESCC, as well as from ESCC cell lines, can reduce B-cell proliferation and induce an increase in interleukin-10 positive regulatory B cells and a high level of PD-1 regulatory B cells (99). Besides, another research demonstrated that exosomal High Mobility Group Box 1 (HMGB1) obtained from ESCC could successfully trigger clonal expansion of PD1 positive tumor-associated macrophages (TAMs), which thereby created conditions for the development of ESCC (74). These findings above contribute to understanding exosomal functions in the immune response and illustrate exosomes’ therapeutic application that promotes antitumor immune responses.

Currently, chemotherapy is regarded as the most effective therapy for ESCC after surgery, and tumor recurrence can be attributed mainly to chemotherapy resistance (100, 101). Tumors can achieve drug resistance in many ways, including via information exchange by exosomes (102). A recent study indicated that exosomes carrying lncRNA prostate androgen-regulated transcript 1 (PART1) derived from Gefitinib-resistant cells confer cisplatin resistance in ESCC (69). Furthermore, several studies have elucidated that many other exosome-shuttled cargos, such as lncRNA POU class 3 homeobox 3 (POU3F3), miRNA-21, and miRNA-193, are involved in Cisplatin resistance in ESCC (61, 63, 70). Apart from roles in chemoresistance, exosomes reportedly regulate radiation therapy and induce radiation-induced bystander effect (RIBE) (103). Exosomal miRNA-339-5p can mediate the radiosensitivity of ESCC by downregulating cell division cycle 25A (Cdc25A) and can predict outcomes in preoperative radiotherapy (64). These discoveries capture the role of exosomes in therapy resistance and shed light on how engineered exosomes may deliver therapeutic agents for ESCC treatment in the future.

ESCC is considered silent cancer because it lacks characteristic manifestations in the early stage. Patients are frequently diagnosed in middle or late stages, delaying the optimal time for treatment and causing a high mortality rate (104). Therefore, it is paramount to find early diagnostic methods to identify patients with ESCC to benefit from early interventions (105). Currently, the gold standard for the diagnosis of ESCC is tissue biopsy under endoscopy, an invasive inspection with correspondingly high costs (106). A non-invasive diagnostic method in early-stage ESCC is urgently needed. Researchers have made great efforts to screen for ESCC biomarkers; possible candidates include circulating tumor cells (CTCs), serum miRNAs, small extracellular vesicles (sEVs), as well as circulating tumor DNA (ctDNA) (107, 108). Exosomal cargos serve as promising tumor biomarkers because they reflect the donor cell and their presence in various biological fluids (109–111). New research into the potential application of exosomes as tumor biomarkers has emerged and yielded valuable information for additional in-depth exploration (112, 113). Growing evidence has confirmed that exosomal RNAs outperform peripheral blood-free RNAs in cancer diagnosis because of several advantages: First, exosomes exist in all biological fluids and are easily accessible compared with plasma. Second, exosomal cargos can be well protected from degradation by enzymes or elimination by the biological barrier. Third, the components of exosomes have high homology with donor cells, which may encourage a higher specificity of exosome-based detection. Last, the concentration of exosomal cargos is higher than the expression of plasma RNAs (114–116). Here, we focus on state-of-the-art exosomal cargos in ESCC.

The distinct expression of exosome-shuttling contents between cancer cells and normal cells supports the application of exosomes as biomarkers for ESCC. Among these exosomal compounds, exosome-carrying miRNAs are most investigated. For example, Zeng corroborated that exosome-shuttled miRNA-19b-3p separated from patients with ESCC is significantly upregulated compared with healthy controls, suggesting that serum exosome-encapsulated miRNA-19b-3p highlights the potential utility of exosomal RNAs in the early detection of ESCC (56). A study from 51 patients with ESCC and 41 with benign diseases showed that plasma exosomal miRNA-21 levels were significantly elevated in ESCC versus benign diseases so that they were suitable to be biomarkers for early diagnosis of ESCC (117). Except for the effect of exosomes on the differential diagnosis of ESCC, lymph node metastasis and TNM grade have been associated with the expression of some exosomal cargos that may serve as independent prognostic factors of ESCC. Lu et al. suggested that tumor cells-derived exosomes markedly upregulated the expression of hsa-circ-0048117 under the condition of hypoxia—a change that may be positively correlated with advanced T and N stages serves as a biomarker for progression (71). Similar studies have revealed that downregulated exosomal miRNA-339-5p and miRNA-652-5p in the serum are related to advanced TNM stages and a higher lymph node metastasis rate (58, 64). Moreover, several studies have indicated that higher serum exosomal miRNA-182, miRNA-766-3p, lncRNA POU3F3, and has-circ-0026611 levels in patients with ESCC are positively related to poor prognosis (70, 118–120). Except for exosomal RNAs, a small amount of exosome-carrying protein has been reported previously. The over-expression of Stathmin-1, regarded as microtubule depolymerization protein, is related to the process of tumor spread, adverse clinical outcomes, and chemoresistance in many types of cancer, especially squamous cell carcinoma, by controlling cell division, proliferation, and migration (121–124). In ESCC, Yan et al. corroborated that the average expression of stathmin-1 elevated in oncogenic exosomes, and the serum stathmin-1 level in patients with ESCC was obviously higher than that of healthy individuals (125). In addition, elevated concentration of stathmin-1 was related to lymphatic metastasis and late staged cancer.

Thus, several exosomal cargos have been identified as biomarkers for ESCC in applying possible diagnosis and potential prognosis, as described in Table 2 . It is noteworthy that few literature reports address the diagnostic value of these molecules; the diagnostic efficacy of most candidates, apart from few miRNAs like miRNA-21, deserve additional validation. Much work remains before exosomal cargos can be applied as ideal biomarkers of ESCC. In addition, readers should note that some of the articles we referenced were published before the MISEV2018 guidelines were issued, which means the research methods they applied might not meet the standard of the guidelines. For example, few works failed to further validate sEV-specific markers, which is not recognized by the standards of EV isolation protocols in MISEV2018. Therefore, we hope readers accommodate the term “small extracellular vesicle” to replace “exosome”,

In addition to having a diagnostic role in cancer, exosomes have potential use in disease therapy. The characteristic property in transferring selected payloads to recipient cells has translated into potential applications for treating many diseases, including cancer and cardiovascular diseases (134–136). However, recent articles published on the potential application of exosomes in the treatment of ESCC are sparse. Thus, this section mainly summarizes exosome-based therapy in other cancers and introduces the exosomes that may be potential targets for ESCC therapy in the future. Researchers harness engineered exosomes to deliver chemotherapeutic agents, achieving better performance than traditional vectors like liposomes (49). Theoretically, exosomes have the following advantages: First, the membrane structure of an exosome can protect pharmacological agents from degradation. Second, exosomes naturally exist in all biological fluids, and thus they can be well tolerated when introduced into the body. They can efficiently penetrate biological barriers and deliver targeted cargo with minimal immune clearance. Third, some exosomes have receptor-targeting features resulting from the heterogeneity of exosomal surface proteins, enabling targeted delivery of therapeutic agents for cancer. Last, because they are shed by all cells as part of their normal physiology, exosomes may induce less toxicity and minimize other adverse reactions even with repeated injection (137–140). Therefore, exosomes may have a bright future as nanocarriers for cancer treatment.

Currently, researchers are committed to designing exosomes to encapsulate therapeutic agents and conducting studies that yield valuable information about the application of exosomes for the administration of diseases. For example, gene-engineered exosome-thermosensitive liposomes can block the CD47 immune checkpoint and improve the macrophage-mediated elimination of cancer cells (30). Pan et al. indicated that urinary exosome-based engineered nanovectors could help deliver targeted homologous treatment in prostate cancer and may exemplify a novel, efficient, and facile therapy strategy (29). So far, evidence about the function of engineered exosomes in cancer therapy is primarily from cancers like gastric cancer or prostate cancer, not from ESCC; relevant data in ESCC are, at best, sparse. However, despite the many unanswered questions about their clinical application, exosomes show great potential to facilitate ESCC therapy. In addition, accumulating research has suggested that engineered exosomes in vitro can play a paramount role in different experimental settings, but more exploration is needed before these findings translate into clinical practice (141). Also, research must guarantee the homogeneity of exosomes by standardizing protocols for exosome isolation, preparation, and route of administration (8). Currently, inefficient isolation methods of exosomes cannot provide sufficient exosomes to meet cancer therapy requirements (142). Identifying ways to prevent exosomes from being taken up by other cells and to drive the engineered vehicles to targeted cells or organs remains a considerable challenge (143–145). Overall, engineered exosomes provide a promising therapeutic option for cancer treatment, although the utility of this strategy in clinical practice requires additional exploration.