Melanoma is an extremely malignant tumor accounting for approximately 5% of all tumors, which arises from melanocytes (1). Melanoma mainly involves the skin, and can also occur in eyes, meninges and diverse mucosal surfaces (2). Although melanoma is rare, it is responsible for most skin cancer-related death (3), owing to the great metastatic feature of melanoma cells (4). In addition to the tumor-draining sentinel lymph node (SLN) that has been recognized as the most initial metastasis site, melanoma cells also frequently disseminate to the distant regions and organs (5, 6). The tumor microenvironment (TME), a complex environment consists of various cells, including tumor cells, neutrophils, myeloid-derived suppressor cells (MDSC), immune and stromal cells, is vital for the development of melanoma, and exerts a key role in modulating both tumor immunity and the prognosis of melanoma (7–9). Notably, these impacts are mainly mediated by the crosstalk between melanoma tumor cells and other types of TME cells.

In the past decades, the lipid membrane bound nanoparticles extracellular vesicles (EVs), which can be produced by most cells, have been identified as important mediators during the communication between cells (10). EVs have been suggested to be divided into several subtypes according to their size (small and large EVs), origin (exosomes, ectosomes and apoptotic bodies), biochemical components (CD63⁺/CD81⁺ EVs) and physiological condition (hypoxic EVs) (11, 12). Although the classification of EVs is distinct, and it is difficult to distinguish them accurately, recent technological advances enable a more refined differentiation of the subset of EVs by diverse markers (13). EVs contain a great variety of biomolecules released from their donor cells, such as proteins, nucleic acids, and lipids. By transmitting their cargo to adjacent or distant recipient cells or tissues, EVs are able to trigger the malignant phenotype changes of the receptor cell, including the augmented tumor cell migration and invasion, enhanced angiogenesis, and impaired tumor immunity (14, 15).

Recently, increasing evidence have shown the close relationship between EVs and TME ( Figure 1 ), which is responsible for the tumor progression, including melanoma (15–17). Therefore, in this review, we aimed to summarize the role of EVs in melanoma by targeting TME, and discuss the potential of EVs to be applied as an alternative option for the clinical treatment of melanoma.

In the microenvironment of tumor, stromal cells are vital components and play important roles in the occurrence, development and metastasis of tumor (18, 19). Among them, cancer-associated fibroblasts (CAFs) is the main type of the reactive tumor stroma and contributes to the tumorigenesis, such as melanoma (20, 21). Tumor-related EVs have been demonstrated to be involved in the differentiation of normal fibroblasts (NFs) into CAFs, and then induced a tumor-stimulative stroma ( Table 1 ) (29). Gm26809, a novel lncRNA, was upregulated obviously in cytotoxic T-lymphocytes CTLL2 under the stimulation of EVs derived from B16F0 melanoma cells (30). Moreover, Hu et al. (22) found that the EVs released by B16F0 cell can induce the reprogram of fibroblast NIH/3T3 cells to CAFs, as well as the facilitated Cloundman S91 melanoma cell proliferation and migration, which was mediated by delivering Gm26809 to NIH/3T3 cells. These findings suggest that EVs-Gm26809 might be a potential target for promoting the progression of melanoma. Nevertheless, the potential molecular mechanism about how EVs-Gm26809 regulated the transition of NFs to CAFs remains unclear, and whether melanoma-derived EVs could transfer Gm26809 to other stromal cells required further investigation. The miRNAs with 18–24 nucleotide are major members of the non-coding RNAs, which can control the translation of downstream tumor-associated mRNAs, thus participating the tumor progression (31, 32). The miRNAs secreted by melanoma cells have also been implicated in the transformation of NFs towards CAFs by changing CAFs-related genes expression (33, 34). Similar to EVs, melanosomes with a diameter of 0.5 µm are melanin-containing vesicles that are specifically fabricate by melanocytes (23). As reported before, melanosome-miR-211 released by melanoma cells induced NFs to CAF transition upon absorbed by NFs, whereas the depletion of miR-211 prevented the formation of CAFs. Furthermore, miR-211 was found to target tumor suppressor IGF2R, resulting in the increase of collagen by CAFs so as to promote tumor cell motility (35). Although this study provided an opportunity to attenuate melanoma invasion by blocking the transformation of CAFs, whether melanosomes-miRNA can be absorbed by other cells in the melanoma microenvironment deserves more research. In addition, more efforts to explore EVs-miRNA mediated crosstalk between tumor cells and TME, as well as their potential to act as prognostic biomarkers or novel therapeutic strategy for melanoma should be considered. In addition to the transmission of tumor cell derived EVs to CAFs, EVs secreted by CAFs have also been revealed to exert an essential role in several tumor progression (24, 36). Recent data showed that CAF-released EVs enriched CD9 and CD63, and inhibited the proliferation of melanoma cells remarkably. The patients with CAF-derived CD9-positive EVs displayed a better five-year disease-free survival than patients with CD9-negative, indicating that CD9 expression in CAFs-EVs is a favorable prognostic marker for malignant melanoma patients (25). Furthermore, to develop novel targeted drugs depending on this type of EVs may be beneficial for the clinical treatment of melanoma patients. Moreover, tumor-derived EVs have been revealed to transdifferentiate CAFs by endothelial to mesenchymal transition (EndMT) pathway. By using an in vitro microfluidic model, which enables to observe the synergetic effect of TME in situ, Yeon et al. (37) found that the differentiated CAFs from human umbilical vein endothelial cells (HUVECs) were increased obviously when treated with melanoma-derived EVs, whereas melanoma-secreted EVs could promote EndMT. Predictably, this experimental model is expected to serve as a potent tool in the development of anti-tumor agents by exploring a variety of candidates, not just tumor derived EVs, which can depress the transformation of endothelial cells to CAFs.

Apart from CAFs, mesenchymal stem cells (MSCs) is another essential stromal cells in the TME. EVs secreted by mesenchymal stem cells (MSCs) have shown huge potential for treating tumors because they can precisely locate TME (26). MSC-derived EVs was demonstrated to induce the apoptosis of melanoma cells via transmitting miR-138-5p, and then targeted SOX4. These results indicated the anti-tumor role of miR-138-5p/SOX4 axis during the malignancy of melanoma cells, and validated the potential therapeutic value of MSC-EVs based therapy for melanoma patients (27). In addition, Yang et al. (38) found that NEAT1 loaded in bone marrow mesenchymal stem cell-derived EVs could accelerate the progression of melanoma through inducing macrophages to M2 polarization, providing a novel target for the treatment of melanoma. In melanoma, epithelial-mesenchymal transition (EMT) is usually occurred when epithelial cells transit into mesenchymal cells, thereby promoting tumor metastasis and therapy resistance (28). Chen et al. (39) demonstrated that MSC-EVs carried miR-22-3p could reduce the expression of EMT related gene LGALS1 in melanoma cells so as to inhibit the EMT process of tumor epithelial cells. However, this study is lack of animal studies, and the therapeutic potential of MSC-EVs with miR-22-3p needed further investigation, especially the clinical trials.

Angiogenesis is a biological process responsible for the formation of new blood vessels, thereby delivering oxygen and nutrients to tissues and organs in human body. Currently, angiogenesis has been considered to be a fundamental procedure in inducing benign tumors to malignant phenotype, such as invasion and metastasis (40). In TME studies, EVs released by tumor cells have been revealed to form the premetastatic niche by accelerating angiogenesis, indicating the vital role of EVs in the communication between tumor and angiogenesis ( Table 2 ) (46, 47). It’s well known that the uPA/uPAR system components (urokinase-type plasminogen activator, uPA; uPA receptor, uPAR) are regarded as critical biomarkers for malignancy, including melanoma (41, 48). By utilizing subcutaneously implanted matrigel plugs containing EVs derived from wild type, uPAR- and uPAR+ melanoma cells, the group treated with uPAR+ EVs displayed more vascularized and micro-vessels, suggesting uPAR expressing melanoma EVs are crucial activators of angiogenesis (49). Therefore, depleting uPAR expression in tumor derived EVs is a promising method for treating melanoma, and the uPAR might be a useful biomarker when obtained EVs from melanoma patients by applying liquid biopsy. Hypoxia is a primary characteristic of solid tumors that is involved in tumor angiogenesis (50, 51). Notably, hypoxia also increases the secretion of EVs from tumor cells and alters the carriers of EVs (42). Tang et al. (52) found that hypoxic melanoma-derived small extracellular vesicles (sEVs) could enhance the angiogenic ability of CAFs by delivering the HSP90/p-IKKα/β complex to activate the IKK/IκB/NF-κB/CXCL1 signaling pathway in CAFs. Despite these finding offered a deeper understanding of the occurrence of angiogenesis in melanoma progression and provided promising targets, more clinical studies are necessary for estimating the therapeutic values of the HSP90/IKK enriched sEVs. The typical M2-like phenotype of TAMs can be induced by cytokines derived from Th2 cell, and IL-13 is the primary cytokine among them. Accordingly, targeting IL-13 receptor and ligand in the TME can block M2 polarization and then inhibit tumor growth (53). As reported, tumor cell-derived EVs are able to recruit and polarize TAMs to activate angiogenic signaling pathways via extensive molecule, including bFGF, TNF-α, and VEGF (43, 54). Given that, Negrea et al. (55) developed a novel nanosystem consists of Il-13-LCL-SIM (Il-13-conjugated long-circulating liposomes with SIM) to target TAMs and PEG-EV-DOX (PEG stabilized EVs with DOX) to target melanoma cells. In this study, it was shown that Il-13-LCL-SIM mainly impaired the pro-angiogenic functions of TAMs and reduced the expression of key proangiogenic proteins (such as VEGF), which further sensitized TME to the killing effects of PEG-EV-DOX. Therefore, combing EVs based therapy with other targeted strategies, including targeted angiogenesis, is promised to be an advantageous method to improve the efficacy of EVs-drugs in melanoma, which may also be applied in other malignant tumors.

Wnt signaling proteins are highly conserved proteins that are tightly involved in the developmental processes, and also multiple diseases, including cancers (56, 57). As an atypical Wnt signaling, WNT5A can promote the spread to distant tissues or organs and the formation of metastatic foci of melanoma, whereas the high expression of WNT5A commonly predicted a poor prognosis in melanoma patients (44, 58). In the endogenous WNT5A low expressed melanoma cells, the stimulation with rWNT5A led to a great release of EVs carrying the immunoregulatory cytokine IL-6, and more important the pro-angiogenic agents, including IL-8, VEGF and MMP2. In particular, these EVs might accelerate the angiogenesis processes and create an immunosuppressive TME of melanoma, thus inducing more rapid tumor growth and distinct metastasis (59). Consequently, targeting WNT5A thereby inhibiting the secretion of related EVs might be a promising strategy for the treatment of melanoma patients. However, more studies are needed to assess the potential side effect caused by the silence of WNT5A. Previous studies have proved that JAK2/STAT3 signaling pathway can regulate proangiogenic modulators expression, such as VEGFa, FGF2, and MMP9 (60, 61). Additionally, SOCS1 (suppressor of cytokine signaling 1) is a powerful inhibitor of JAK2/STAT3 signaling, however, SOCS1 is decreased in a great number of tumors and closely related to tumor angiogenesis (45, 62). Zhou et al. (63) demonstrated that melanoma cell-derived EVs could deliver miR-155 to fibroblasts NIH/3T3 and then elevated the expression of proangiogenic factors, including VEGFa, FGF2, and MMP9, by directly targeting SOCS1, thus triggering the proangiogenic switch of CAFs. Furthermore, in vitro and in vivo assays showed that treatment with EVs with overexpressed miR-155 promoted angiogenesis, whereas the knockdown of miR-155 in melanoma cell-released EVs mitigated angiogenesis obviously. This discovery may provide a new therapeutic target for anti-angiogenic therapy in the treatment of melanoma. Nonetheless, the inhibition of miR-155 in melanoma cell-derived EVs cannot diminish the proangiogenic regulators to the original expression level, combined with other therapies may be a feasible option to obtain better clinical benefits.

The TME is infiltrated by multiple immune cells, including lymphocytes (T cells, B cells, NK cells, and T regulatory cells), dendritic cells (DCs), tumor associated macrophages (TAMs), myeloid-derived suppressor cells (MDSC), as well as granulocytes (neutrophils, basophils, eosinophils, and mast cells). However, it has been recognized that tumor cells can regulate signaling pathways involved these immune cells and switch them to an immunorepressive manner, thus inducing enhanced tumor growth (64, 65). EVs comprise several tumor antigens which may result in the immunosuppression, yet increasing studies have indicated that tumor derived EVs are essential mediators between tumor cells and immune response ( Figure 2 ) by releasing immune-associated factors to the TME ( Table 3 ) (76, 77).

In addition, T regulatory cells, as immunosuppressive cells, inhibit the antitumor immune response and secrete various immunosuppressive cytokines, promoting the tumor immune escape (78). Melanoma cell derived EVs induce immune suppression by promoting T regulatory cell expansion as well as the demise of antitumor CD8+ effector T cells, thereby enabling tumor cell escape (79). Another study showed, the transfer of miRNA-214 from Melanoma derived EVs to T-cells down-regulates PTEN while favoring the expansion and migration of T regulatory cells in TME (80).

Extracellular matrix (ECM) is a major non-cellular component in TME (118). It has been well demonstrated that ECM is a macromolecular network comprising collagens, elastin, fibronectin, proteoglycans/glycosaminoglycans, and laminins, which acts as a physical scaffold and is exerts essential roles in tumor cell differentiation, migration, and homeostasis (119, 120). The remodeling of ECM arises under physiological and pathological conditions and is regulated by multiple enzymes, such as metalloproteinases. Some EVs have been observed within the matrix and involved in the remodeling of ECM, that will impel the modification of the TME, and the formation of pre-metastatic niches (121, 122). Palmulli et al. (123) found that melanoma-derived sEVs are able to physically interact with collagen I, the primary fibrous component of the ECM, and then enhanced the activity of metalloproteases at the surface of EVs and the remodeling of the ECM, which is involved in the advancement of malignant tumors. Accordingly, blocking the binding between tumor derived EVs with collagen I may be a feasible therapeutic strategy for melanoma patients ( Figure 3 ). However, whether EVs are capable of binding to other collagens or other components of ECM to remodel TME is still a challenge. Matrix metalloproteinases (MMPs) are zinc-dependent proteins, which take part in the tissue remodeling and involve tumor progression (124, 125). The membrane type 1 matrix metalloproteinase (MT1-MMP) is a critical member of metalloproteinases that facilitate tumor progression by remodeling the ECM (126, 127). From the culture medium of melanoma (G361) cells, EVs carrying the full-length and the proteolytically processed forms of MT1-MMP were isolated and identified (128). Interestingly, the EVs could activate pro-MMP-2 and degrade type 1 collagen, indicating EVs-MT1-MMP possessed functional activities. This provided a novel mechanism by which melanoma cells could remodel the ECM in TME, thus targeting MT1-MMP in EVs, or depressing the release of tumor cell derived EVs might be a promising strategy for melanoma treatment.

Owing to the essential roles of TME in the occurrence and development of tumors, including melanoma, targeting of the TME has recently been regarded as a great promising therapeutic strategy to improve the anti-cancer efficacy (129, 130). Along with the clinical approval of several drugs and targeted therapies that were designed toward the angiogenesis, immune checkpoints, T and NK cells, additional molecules directly target the cellular or non-cellular components of TME are also under development. In this review, we have highlighted the potential of EVs mediated the crosstalk within the TME to be the therapeutic agents for melanoma patients. Moreover, emerging studies have also revealed that plasma-derived EVs can act as biomarkers for the advancement of melanoma, such as the circulating EVs-PD-1/PD-L1, thus providing a novel direction for the lipid biopsy. Although EVs-related therapies and biomarkers have shown huge application prospects, some shortages still needed to be overcome before its clinical use. For example, the heterogeneity in size, composition and sources of EVs lead to the difficulty to isolate a specific subset with desirable factors, which will reduce the accuracy of targeting therapy and bring some unexpected side effects. Therefore, the isolation and characterization methods should be further optimized to obtain more ‘pure’ EVs. Besides, the natural EVs commonly face problems such as the low production and complex composition. Furthermore, the efficiency of EVs may also be declined due to the low infiltration, insufficient drug delivery, captured by immune cells, and the inadequate immune response (131–133). Recently, a number of engineering methods have been used to obtain generous artificial and modified EVs to improve EVs efficacy and practicability, which have shown encouraging results.

In addition to carry endogenous cargoes, because of the inherent protection of the cargo and capable of enhancing solubility, stability, and specificity, EVs have been proposed as a drug delivery system to load single or multiple drugs to exert anti-tumor effect. In the clinical treatment of melanoma, ICBs are widely used. Currently, several monoclonal antibodies, such as pembrolizumab and nivolumab (anti-PD-1), and ipilimumab (anti-CTLA-4) have been studied and become the appropriate therapy to improve metastatic melanoma survival (134–138). However, there are still around 50% of patients fail to achieve clinical benefits (139). Utilizing EVs to delivery these antibodies to the TME, thus directly targeting tumor cells or T cells may be an alternative method to improve the efficacy of ICBs in melanoma. Moreover, modifying EVs to enhance their melanoma TME targeting will be favorable for increasing the accumulation and sustain of ICIs in the TME. However, few engineered EVs loaded with ICIs or other anti-tumor modulators specially targeting melanoma has been developed, whereas more efforts are needed.

Taken together, although the therapeutic application of EVs in melanoma clinical treatment is still restricted by the deficiency of feasible and standardized methods to obtain moderate EVs from melanoma patients biofluids and tumor cells, the limited loading efficiency of multiple agents, and the difficulty to manufacture sufficient melanoma specific EVs, EVs are still the great promising therapeutic manner for melanoma. With the progress of technology and new findings, EVs will finally shed light on the melanoma diagnosis and treatment in the era of precision medicine.

YL: Conceptualization, Methodology, Software Data curation, Writing- Original draft preparation. FL: Visualization, Investigation, Supervision. Writing- Reviewing and Editing. All authors have read and agreed to the published version of the manuscript.