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The role of exosomes in metastasis and progression of melanoma

  • Raghavendra Gowda
    Affiliations
    Departments of Pharmacology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States

    Penn State Melanoma and Skin Cancer Center, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States

    Penn State Melanoma Therapeutics Program, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States

    Foreman Foundation for Melanoma Research, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States
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  • Bailey M. Robertson
    Affiliations
    Departments of Pharmacology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States
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  • Soumya Iyer
    Affiliations
    Departments of Pharmacology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States

    Penn State Melanoma and Skin Cancer Center, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States
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  • John Barry
    Affiliations
    Departments of Pharmacology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States
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  • Saketh S. Dinavahi
    Affiliations
    Departments of Pharmacology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States

    Penn State Melanoma and Skin Cancer Center, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States
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  • Gavin P. Robertson
    Correspondence
    Corresponding author at: Department of Pharmacology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, United States.
    Affiliations
    Departments of Pharmacology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States

    Departments of Pathology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States

    Departments of Dermatology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States

    Departments of Surgery, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States

    Penn State Melanoma and Skin Cancer Center, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States

    Penn State Melanoma Therapeutics Program, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States

    Foreman Foundation for Melanoma Research, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States
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Published:January 22, 2020DOI:https://doi.org/10.1016/j.ctrv.2020.101975

      Highlights

      • Tumor-derived or tumor-associated exosomes (usually 30–100 nm in diameter) released by the tumor cells are important facilitators of intercellular communication involved in the pathogenesis, development, progression, and metastasis of cancer.
      • Exosomes are become attractive new biomarkers for the diagnosis and prognosis of cancer.
      • Clinical trials to determine the feasibility of exosomes for use as a biomarker, biological target, or drug delivery vehicle to treat a wide variety of human disease conditions are being explored.
      • This review discusses the multifaceted role of melanoma-derived exosomes in promoting the process of metastasis by modulating the invasive and angiogenic capacity of malignant cells.

      Abstract

      The mechanisms of melanoma metastasis have been the subject of extensive research for decades. Improved diagnostic and therapeutic strategies are of increasing importance for the treatment of melanoma due to its high burden of mortality in the advanced stages of the disease. Intercellular communication is a critical event for the progression of cancer. Collective evidence suggests that exosomes, small extracellular membrane vesicles released by the cells, are important facilitators of intercellular communication between the cells and the surrounding environment. Although the emerging field of exosomes is rapidly gaining traction in the scientific community, there is limited knowledge regarding the role of exosomes in melanoma. This review discusses the multifaceted role of melanoma-derived exosomes in promoting the process of metastasis by modulating the invasive and angiogenic capacity of malignant cells. The future implications of exosome research and the therapeutic potential of exosomes are also discussed.

      Keywords

      Introduction

      Melanoma is the most deadly form of skin cancer due to its highly metastatic nature [
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      ]. The disease progresses in a stepwise manner as evolving tumor cells become invasive and leave the epithelium of the epidermis to enter adjacent tissues [
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      ]. The depth of invasion correlates with a poor prognosis and disease dissemination. Melanoma patients receiving a diagnosis of metastatic disease have a decrease in 5-year survival rate from 91.3% to 16.0% [
      • Miller Kimberly D.
      • Siegel Rebecca L.
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      ]. Improved surveillance and early detection of metastatic melanoma using specific markers of initiation and progression are required to improve clinical intervention, and patient survival [
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      ]. Exosomes have the potential to fill this niche. Use of exosomes as a prognostic biomarker for cancer diagnosis and targeted therapy stems from the fact that they closely represent the cells of the cancer type from which they originate [
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      ]. Exosomes have been known to carry the messenger proteins required to direct bone marrow–derived cells toward a prometastatic phenotype, and are hence called the “messengers of metastasis” [
      • Somasundaram R.
      • Herlyn M.
      Melanoma exosomes: messengers of metastasis.
      ].
      Exosomes are extracellular vesicles, which play a vital role in the intercellular communication within the tumor microenvironment, metastasis and drug resistance [
      • Webber J.
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      Cancer exosomes trigger fibroblast to myofibroblast differentiation.
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      Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.
      ]. Exosomes can transport proteins, affect changes in signaling pathways, maturation and differentiation, as well as induce genetic change in cells [
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      • Steadman R.
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      Cancer exosomes trigger fibroblast to myofibroblast differentiation.
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      Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.
      ]. Exosomes have also been shown to be relatively stable and resist proteolytic and nuclease activity [
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      • Dong M.
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      ]. The proteins and nucleic acids enclosed within these vesicles are protected from degradation [
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      ]. However, the exact mechanisms mediating the complex roles of exosomes in cancer continue to be elucidated. Several reviews have focused on exosomes but none have discussed the role of these vesicles in melanoma. Since this is an emerging and evolving field of research with regard to melanoma, areas with research potential will be highlighted for additional study in the future.
      Extracellular vesicles (EVs) are formed from cavitations in endosomes and are released when the multivesicular bodies (MVBs) fuse with the plasma membrane [
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      ]. They contain proteins, nucleic acids, and metabolites and can mediate intercellular communication to modulate various biological functions as illustrated in Fig. 1. Within the past ten years, EVs have expanded into several subtypes that can be characterized based on size, biochemical composition, or description of conditions and cell of origin [
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      ]. Size based classification consists of small EVs (sEVs) of < 100–200 nm and medium/large EVs of greater than 200 nm [
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      ]. However, biochemical composition is also used to verify presence of EVs such as confirming the presence of CD63+/CD81+ or by staining EVs with an Annexin A5 stain [
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      ]. These EVs are currently isolated through a variety of ultracentrifugation and filtering steps dependent on the downstream application of the isolated EVs [
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      ]. In this review, we are focusing on sEVs which will be referred to as exosomes throughout the remainder of this review.
      Figure thumbnail gr1
      Fig. 1Biogenesis of Exosomes. Exosomes are formed by the cells when intracellular organelles called multivesicular bodies (MVBs) fuse with the plasma membrane. During this process, various cellular contents like proteins (e.g., receptor, cytoplasmic proteins, tetraspanin), nucleic acids (e.g., DNA, mRNA, miRNA), and lipids (e.g., cholesterol, ceramide) are packed into the exosomes. The fate of the MVBs can be, either fusion with lysosomes resulting in the degradation of cargo or alternatively with the plasma membrane, resulting in the release of the cargo to the extracellular milieu. Various molecules like the ESCRT machinery, tetraspanins and ceramides are dominating the biogenesis of exosomes. With respect to the cargo contents, exosome secreted from the recipient cells are known to modulate different biological processes in the target cells. The mechanism responsible for the uptake of exosomes includes exosome fusion, endocytosis and receptor activation through receptor-ligand interactions and antigen presenting cells.

      Exosome biogenesis and secretion in cancer

      Exosomes are ubiquitous in the body and are generated by all mammalian cell types in both healthy and diseased tissue [
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      Prostasomes: extracellular vesicles from the prostate.
      ]. After production, they diffuse into the local tissue through the lymphatic system and eventually enter the circulatory system [
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      Exosomal-like vesicles are present in human blood plasma.
      ]. The membrane composition of exosomes is enriched with endosome related membrane transport and fusion proteins such as Rab GTPases, flotillin, annexins and integrins [
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      ]. These vesicles carry repertoires of nucleic acids, including mRNAs, miRNAs and other ncRNAs, as well as DNA [
      • Thery C.
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      Exosomes: composition, biogenesis and function.
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      Exosomes from human saliva as a source of microRNA biomarkers.
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      Biogenesis and secretion of exosomes.
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      Molecular characterization of exosome-like vesicles from breast cancer cells.
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      ].
      To date, the molecular mechanisms involved in exosome packaging have not been fully delineated, but appear to be uniquely regulated through various signaling networks depending on cell type. The biogenesis of exosomes seems similar to that of lysosomal bound multi-vesicular bodies (MVBs) since the endosomal sorting complexes required for transporting (ESCRT) proteins, which are crucial for packing MVBs, are also present (Fig. 1) [
      • Thery C.
      • Zitvogel L.
      • Amigorena S.
      Exosomes: composition, biogenesis and function.
      ]. Several studies have provided evidence for ESCRT independent packaging of exosomes [

      Johnson DS, Bleck M, Simon SM. Timing of ESCRT-III protein recruitment and membrane scission during HIV-1 assembly. Elife. 2018;7.

      ,
      • Kajimoto T.
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      Ongoing activation of sphingosine 1-phosphate receptors mediates maturation of exosomal multivesicular endosomes.
      ,
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      Tetraspanins in extracellular vesicle formation and function.
      ]. These pathways depend on lipids such as sphingosine 1 phosphate (S1P) and tetraspanin-enriched microdomains [
      • Kajimoto T.
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      • Zhang L.
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      Ongoing activation of sphingosine 1-phosphate receptors mediates maturation of exosomal multivesicular endosomes.
      ,
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      Dual loss of ER export and endocytic signals with altered melanosome morphology in the silver mutation of Pmel17.
      ]. Pre-melanosomal protein -Pmel17 appears to be independent of the ESCRT pathway [
      • Theos A.C.
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      A lumenal domain-dependent pathway for sorting to intralumenal vesicles of multivesicular endosomes involved in organelle morphogenesis.
      ].
      The biogenesis and tracking of exosomes is altered in cancer and reports have shown enhanced secretion of exosomes by the cells undergoing stress [
      • Pitson S.M.
      • Powell J.A.
      Modification of the tumour microenvironment via exosomal shedding of sphingosine 1-phosphate receptor 2 by breast cancer cells.
      ,
      • Bae S.
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      Exosomes derived from cancerous and non-cancerous cells regulate the anti-tumor response in the tumor microenvironment.
      ,
      • Yu X.
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      The regulation of exosome secretion: a novel function of the p53 protein.
      ,
      • Kucharzewska Paulina
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      ]. Cancer cells undergo stress due to uncontrolled growth and cell damage when treated with chemotherapeutics [
      • Reuter S.
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      ]. For example, in melanoma, p53 activation regulates the transcription of the TSAP6 gene, which, in turn, increases the exosome production [
      • Yu X.
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      ,
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      Exosome secretion, including the DNA damage-induced p53-dependent secretory pathway, is severely compromised in TSAP6/Steap3-null mice.
      ]. Similarly, increased expression of heparanse enzymes, Rab 27a and Rab 27b facilitates the secretion of exosomes in melanoma and other cancer types [

      Ostrowski M, Carmo NB, Krumeich S, Fanget I, Raposo G, Savina A, et al. Rab27a and Rab27b control different steps of the exosome secretion pathway. Nature cell biology. 2010;12:19-30; sup pp 1-13.

      ].

      Characteristics of melanoma exosomes

      The composition of an exosome is not a mere reflection of the donor cell but may vary significantly based on the physiological conditions the cell is experiencing [
      • Saleem S.N.
      • Abdel-Mageed A.B.
      Tumor-derived exosomes in oncogenic reprogramming and cancer progression.
      ]. The heterogeneity of exosomal contents is due to selective sorting of exosomes from the donor cells [
      • Villarroya-Beltri C.
      • Baixauli F.
      • Gutierrez-Vazquez C.
      • Sanchez-Madrid F.
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      ]. Exosomes played a role in cellular debris disposal along with a significant role in long distance cellular interactions [
      • Gajos-Michniewicz A.
      • Duechler M.
      • Czyz M.
      MiRNA in melanoma-derived exosomes.
      ]. Exosomes similar to EVs may also contain RNA, membrane proteins, cytosolic proteins, and raft-like structures in the exosome membrane [
      • Raposo G.
      • Stoorvogel W.
      Extracellular vesicles: exosomes, microvesicles, and friends.
      ]. According to the Exocarta database (http://www.exocarta.org), which catalogs the contents of exosomes, more than 9769 proteins, 4946 mRNAs, 2838 miRNAs and 1116 lipids have already been identified in exosomes from the different cell types of various organisms. It is important to note that the Exocarta database does not take methods of exosome purification and validation into account, which may result in a difference in exosome contents compared to what would be present in reality. Additionally, Exocarta does not differentiate between Exosomes and EVs; therefore, the above mentioned composition may overlap between them. Melanoma exosomes have similarities and differences when compared to neoplastic exosomes from other cancer types [
      • Namee N.M.
      • O'Driscoll L.
      Extracellular vesicles and anti-Cancer drug resistance.
      ,
      • Simpson R.J.
      • Jensen S.S.
      • Lim J.W.
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      ]. Melanoma exosomes in particular are normally secreted by keratinocytes as a means of interacting with melanoctyes by increasing the expression and activity of melanosome proteins [
      • Mannavola F.
      • D'Oronzo S.
      • Cives M.
      • Stucci L.S.
      • Ranieri G.
      • Silvestris F.
      • et al.
      Extracellular Vesicles and Epigenetic Modifications Are Hallmarks of Melanoma Progression.
      ]. Current data regarding membrane characteristics, RNA, and protein profiles of melanoma exosomes is discussed next.

      Membrane composition of melanoma exosomes

      The exosomal membrane contains some of the same lipids as the parent cell membrane and contains the same proteins and RNA [
      • Shen B.
      • Wu N.
      • Yang J.M.
      • Gould S.J.
      Protein targeting to exosomes/microvesicles by plasma membrane anchors.
      ]. Sphingomyelin, lyso-phophatidyethanolamine, lyso-phosphatidylcholine and cholesterol enriched raft-like domains are present on most cancer exosomes including those from melanoma cells [
      • Rappa G.
      • Mercapide J.
      • Anzanello F.
      • Pope R.M.
      • Lorico A.
      Biochemical and biological characterization of exosomes containing prominin-1/CD133.
      ]. Tetraspanin proteins are also enriched in exosomes along with elevated sphingomyelin. CD81, CD63, and CD9 tetraspanins have been found in exosomes isolated from melanoma lines with osteotropic potential [
      • Mannavola F.
      • Tucci M.
      • Felici C.
      • Passarelli A.
      • D'Oronzo S.
      • Silvestris F.
      Tumor-derived exosomes promote the in vitro osteotropism of melanoma cells by activating the SDF-1/CXCR4/CXCR7 axis.
      ]. There are also multiple associated proteins, many of which are cell adhesion molecules [
      • Kowal J.
      • Tkach M.
      • Thery C.
      Biogenesis and secretion of exosomes.
      ,
      • Yue S.
      • Mu W.
      • Erb U.
      • Zoller M.
      The tetraspanins CD151 and Tspan8 are essential exosome components for the crosstalk between cancer initiating cells and their surrounding.
      ]. Although the lipid composition and raft-like structures are expected to affect the physiological response of exosomes such as circulation time, uptake, and targeting [
      • Kooijmans S.A.
      • Vader P.
      • van Dommelen S.M.
      • van Solinge W.W.
      • Schiffelers R.M.
      Exosome mimetics: a novel class of drug delivery systems.
      ], this has yet to be demonstrated. The effect of subtle changes in lipid chemistry, tetraspanin webs, and other membrane structures in exosome roles also remains to be fully explored.

      RNA in melanoma exosomes

      RNA presence was not widely considered in exosomes until 2007 by a study which looked at exosomes from mast cells [
      • Valadi H.
      • Ekstrom K.
      • Bossios A.
      • Sjostrand M.
      • Lee J.J.
      • Lotvall J.O.
      Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.
      ]. Since then, there has been considerable interest in the RNA carried by exosomes. RNA expression tends to be similar to that of the parent cancer cells, which likely shows elevated levels of oncogenes and low levels of tumor suppressor genes, suggesting that exosomes function in the transport of tumor promoting RNA [
      • Valadi H.
      • Ekstrom K.
      • Bossios A.
      • Sjostrand M.
      • Lee J.J.
      • Lotvall J.O.
      Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.
      ]. The majority of work in exosomes derived from cancer cells suggests they may function as tumor promoters.
      Differential RNA expression seems to be a characteristic of the tumor [
      • Pfeffer S.R.
      • Grossmann K.F.
      • Cassidy P.B.
      • Yang C.H.
      • Fan M.
      • Kopelovich L.
      • et al.
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      ,
      • Liao J.
      • Liu R.
      • Yin L.
      • Pu Y.
      Expression profiling of exosomal miRNAs derived from human esophageal cancer cells by Solexa high-throughput sequencing.
      ]. For example, circulating exosomal microRNA levels increased in ovarian and lung cancer while normal controls did not have detectable microRNA levels [
      • Taylor D.D.
      • Gercel-Taylor C.
      MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer.
      ,
      • Rabinowits G.
      • Gercel-Taylor C.
      • Day J.M.
      • Taylor D.D.
      • Kloecker G.H.
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      ]. The enhanced levels of let-7a, miR-182, miR-221, miR-222, miR-31, miR-19b-2, miR-20b and miR-92a-2, miR-21, miR-15b, miR-210, miR-30b, miR-30d, and miR-532-5p were found in exosomes released from A375 melanoma cells in comparison with normal melanocytes [
      • Xiao D.
      • Ohlendorf J.
      • Chen Y.
      • Taylor D.D.
      • Rai S.N.
      • Waigel S.
      • et al.
      Identifying mRNA, microRNA and protein profiles of melanoma exosomes.
      ]. There was also other differential expression of RNAs involved in cell growth, development, migration, metastasis and apoptosis including miR-125, miR-346 and miR-193 that were variably expressed in exosomes isolated from A375M melanoma cells [
      • Xiao D.
      • Ohlendorf J.
      • Chen Y.
      • Taylor D.D.
      • Rai S.N.
      • Waigel S.
      • et al.
      Identifying mRNA, microRNA and protein profiles of melanoma exosomes.
      ]. In addition, the levels of miR125b were reported to be higher in exosomes isolated from the serum of advanced stage melanoma compared to those from healthy controls [
      • Alegre E.
      • Sanmamed M.F.
      • Rodriguez C.
      • Carranza O.
      • Martin-Algarra S.
      • Gonzalez A.
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      ,
      • Tengda L.
      • Shuping L.
      • Mingli G.
      • Jie G.
      • Yun L.
      • Weiwei Z.
      • et al.
      Serum exosomal microRNAs as potent circulating biomarkers for melanoma.
      ]. The research into melanoma exosome RNA expression profiling gives a glimpse into the role of exosomes in genetic transport and transfer, which could be explored in future research. Specifically, to determine whether exosomal RNA is involved in the silencing of anticancer pathways; whether it promotes migration, invasion and other cancer promoting events; the role of RNAs in these responses; and by their involvement and approaches to influence the balance of tumor promoting vs. tumor suppressing roles.

      Proteins in melanoma exosomes

      The proteins responsible for the biogenesis of exosomes include the ones contributing to membrane fusion, cytoskeleton components, MVB-forming proteins, adhesion proteins, and the tetraspanin family of proteins [
      • Thery C.
      • Zitvogel L.
      • Amigorena S.
      Exosomes: composition, biogenesis and function.
      ,
      • Malla R.R.
      • Pandrangi S.
      • Kumari S.
      • Gavara M.M.
      • Badana A.K.
      Exosomal tetraspanins as regulators of cancer progression and metastasis and novel diagnostic markers. Asia Pac.
      ].
      Various proteins have been identified in melanoma exosomes, several of which are involved in melanoma progression and metastasis [
      • Lazar I.
      • Clement E.
      • Ducoux-Petit M.
      • Denat L.
      • Soldan V.
      • Dauvillier S.
      • et al.
      Proteome characterization of melanoma exosomes reveals a specific signature for metastatic cell lines.
      ]. Not surprisingly, the melanoma exosome protein profile differs from that of melanocytes [
      • Xiao D.
      • Ohlendorf J.
      • Chen Y.
      • Taylor D.D.
      • Rai S.N.
      • Waigel S.
      • et al.
      Identifying mRNA, microRNA and protein profiles of melanoma exosomes.
      ]. In addition to a large increase in total exosomal protein released by melanoma cells over melanocytes, an increase in tumor marker proteins occurs, including the presence of an isoform of Hsp-70 unique to melanoma indicative of tumor progression [
      • Dressel R.
      • Johnson J.P.
      • Gunther E.
      Heterogeneous patterns of constitutive and heat shock induced expression of HLA-linked HSP70-1 and HSP70-2 heat shock genes in human melanoma cell lines.
      ]. Various other proteins like TYRP2, VLA-4, Hsp-70, MHC I, Mart-1, Her2/neu, TRP, CD44, MAPK4K, GTP-binding proteins, ADAM10, annexin A2, and GP100 are also enriched in exosomes derived from malignant melanoma and could serve as possible prognostic biomarkers [
      • Rappa G.
      • Mercapide J.
      • Anzanello F.
      • Pope R.M.
      • Lorico A.
      Biochemical and biological characterization of exosomes containing prominin-1/CD133.
      ,
      • Mears R.
      • Craven R.A.
      • Hanrahan S.
      • Totty N.
      • Upton C.
      • Young S.L.
      • et al.
      Proteomic analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry.
      ,
      • Wolfers J.
      • Lozier A.
      • Raposo G.
      • Regnault A.
      • Thery C.
      • Masurier C.
      • et al.
      Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming.
      ]. However, the potential use of exosomes as melanoma biomarkers has not yet been fully validated in prospective clinical trials.
      An exosomal protein that may play an interesting role in cancer and which appears conserved across cancer types is annexin A2 [
      • Guan M.
      • Chen X.
      • Ma Y.
      • Tang L.
      • Guan L.
      • Ren X.
      • et al.
      MDA-9 and GRP78 as potential diagnostic biomarkers for early detection of melanoma metastasis.
      ,
      • Valapala M.
      • Vishwanatha J.K.
      Lipid raft endocytosis and exosomal transport facilitate extracellular trafficking of annexin A2.
      ]. Annexin A2 and other annexins are known to play a role in tumor development and progression [
      • Lokman N.A.
      • Ween M.P.
      • Oehler M.K.
      • Ricciardelli C.
      The role of annexin A2 in tumorigenesis and cancer progression.
      ]. Recently, annexin A2 was shown to form a tetramer with S100 proteins (a biomarker for melanoma on the cell surface) [
      • Cross S.S.
      • Hamdy F.C.
      • Deloulme J.C.
      • Rehman I.
      Expression of S100 proteins in normal human tissues and common cancers using tissue microarrays: S100A6, S100A8, S100A9 and S100A11 are all overexpressed in common cancers.
      ,
      • Hauschild A.
      • Michaelsen J.
      • Brenner W.
      • Rudolph P.
      • Glaser R.
      • Henze E.
      • et al.
      Prognostic significance of serum S100B detection compared with routine blood parameters in advanced metastatic melanoma patients.
      ,
      • Hauschild A.
      • Engel G.
      • Brenner W.
      • Glaser R.
      • Monig H.
      • Henze E.
      • et al.
      S100B protein detection in serum is a significant prognostic factor in metastatic melanoma.
      ]. This tetramer promotes ceramide-1-phosphate dependent vascular endothelial cell invasion [
      • Hankins J.L.
      • Ward K.E.
      • Linton S.S.
      • Barth B.M.
      • Stahelin R.V.
      • Fox T.E.
      • et al.
      Ceramide 1-phosphate mediates endothelial cell invasion via the annexin a2–p11 heterotetrameric protein complex.
      ]. It would be interesting to see if melanoma cells hijacks the exosomal transport of annexins to promote invasion. Together with annexins, multiple other proteins are associated with small GTP-binding proteins, cytoskeletal and cytoskeletal binding proteins and motor proteins that play a significant role in signal transduction in melanoma [
      • Lazar I.
      • Clement E.
      • Ducoux-Petit M.
      • Denat L.
      • Soldan V.
      • Dauvillier S.
      • et al.
      Proteome characterization of melanoma exosomes reveals a specific signature for metastatic cell lines.
      ].
      Series of ribosomal proteins like RPL4, RPL35, RPL19, RPS11, RPL13 and RPL5, are overexpressed in the melanoma exosomes (http://www.exocarta.org/). Ribosomal proteins can have extra-ribosomal functions such as DNA repair, signaling transduction and apoptosis [
      • Bhavsar R.B.
      • Makley L.N.
      • Tsonis P.A.
      The other lives of ribosomal proteins.
      ]. Ribosomal protein mutations suppress the activity of the AKT pathway thereby resulting in proteasomal degradation of p53 [
      • Antunes A.T.
      • Goos Y.J.
      • Pereboom T.C.
      • Hermkens D.
      • Wlodarski M.W.
      • Da Costa L.
      • et al.
      Ribosomal Protein Mutations Result in Constitutive p53 Protein Degradation through Impairment of the AKT Pathway.
      ]. Increased ribosomal protein expression modulates breast cancer stem cell self-renewal and depletion reduced tumor growth and metastasis mediated through the nitric oxide synthase pathway [
      • Dave B.
      • Granados-Principal S.
      • Zhu R.
      • Benz S.
      • Rabizadeh S.
      • Soon-Shiong P.
      • et al.
      Targeting RPL39 and MLF2 reduces tumor initiation and metastasis in breast cancer by inhibiting nitric oxide synthase signaling.
      ]. It is likely that several different proteins regulate this process, and future research should be focused on understanding the functional role of these proteins. Collectively, the involvement of exosomal proteins illustrates the potentially important role in melanoma progression and metastasis. Furthermore, these studies suggest that it is important to control the secreted exosomes and the possibility of using them as a potential drug delivery vehicle [
      • Ha D.
      • Yang N.
      • Nadithe V.
      Exosomes as therapeutic drug carriers and delivery vehicles across biological membranes: current perspectives and future challenges.
      ].
      More recently, Jang et al. discovered mitochondrial protein present in human melanoma vesicles isolated from patient plasma [
      • Jang S.C.
      • Crescitelli R.
      • Cvjetkovic A.
      • Belgrano V.
      • Olofsson Bagge R.
      • Sundfeldt K.
      • et al.
      Mitochondrial protein enriched extracellular vesicles discovered in human melanoma tissues can be detected in patient plasma.
      ]. In this study, patient tissue from both melanoma lymph node and skin metastases was taken and EVs were isolated and preserved in serum free media [
      • Jang S.C.
      • Crescitelli R.
      • Cvjetkovic A.
      • Belgrano V.
      • Olofsson Bagge R.
      • Sundfeldt K.
      • et al.
      Mitochondrial protein enriched extracellular vesicles discovered in human melanoma tissues can be detected in patient plasma.
      ]. Of the proteins isolated from the EVs in this study, 23.6% are distinct from those found in current EV databases [
      • Jang S.C.
      • Crescitelli R.
      • Cvjetkovic A.
      • Belgrano V.
      • Olofsson Bagge R.
      • Sundfeldt K.
      • et al.
      Mitochondrial protein enriched extracellular vesicles discovered in human melanoma tissues can be detected in patient plasma.
      ].

      Exosomes as mediators of metastasis

      Exosomes can play an important role in metastatic progression [
      • Suchorska W.M.
      • Lach M.S.
      The role of exosomes in tumor progression and metastasis (Review).
      ]. The ability of exosomes to carry proteins and nucleic acids enable them to modulate both the primary tumor and remote tissue to promote metastasis [
      • Azmi A.S.
      • Bao B.
      • Sarkar F.H.
      Exosomes in cancer development, metastasis, and drug resistance: a comprehensive review.
      ]. Exosomes are able to transfer metastatic ability to local cells and ‘educate’ distant cells to help create a pre-metastatic niche conducive to tumor formation at the site [
      • Rana S.
      • Malinowska K.
      • Zoller M.
      Exosomal tumor microRNA modulates premetastatic organ cells.
      ]. The ‘seed and soil’ hypothesis in cancer progression infers that the metastatic tumor cell, or ‘seed’, breaks free from the primary tumor and migrates to a secondary location where it finds a microenvironment or ‘soil’ conducive to growth, [
      • Langley R.R.
      • Fidler I.J.
      The seed and soil hypothesis revisited–the role of tumor-stroma interactions in metastasis to different organs.
      ,
      • Fidler I.J.
      The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited.
      ]. This ‘soil’ can be prepared prior to the metastasizing cell reaching this site in order for the secondary tumor to survive and grow in the metastatic niche [
      • Kharaziha P.
      • Ceder S.
      • Li Q.
      • Panaretakis T.
      Tumor cell-derived exosomes: a message in a bottle.
      ]. Exosomes from cancer cells can increase the invasive nature of cells and facilitate an increase in angiogenesis both locally and remotely as a metastatic aid and there is a notable increase in vascular leakiness seen in the lungs and lymph nodes of exosome treated animals [
      • Hood Joshua L
      • Pan Hua
      • Lanza Gregory M
      • Wickline Samuel A
      Paracrine induction of endothelium by tumor exosomes.
      ]. Additionally, exosomes can direct or inhibit cancer fibroblast differentiation eventually leading to angiogenesis and tumor growth [
      • Webber J.P.
      • Spary L.K.
      • Sanders A.J.
      • Chowdhury R.
      • Jiang W.G.
      • Steadman R.
      • et al.
      Differentiation of tumour-promoting stromal myofibroblasts by cancer exosomes.
      ,
      • Zhang X.
      • Yuan X.
      • Shi H.
      • Wu L.
      • Qian H.
      • Xu W.
      Exosomes in cancer: small particle, big player.
      ]. Tumor derived exosomes were found to also conditionally promote EMT of tumor cells by upregulating Let7a, Let7i, and miR-191, as well as migrating to distant sites in order to prepare pre-metastatic niches [
      • Mannavola F.
      • D'Oronzo S.
      • Cives M.
      • Stucci L.S.
      • Ranieri G.
      • Silvestris F.
      • et al.
      Extracellular Vesicles and Epigenetic Modifications Are Hallmarks of Melanoma Progression.
      ].
      In a groundbreaking study by Peinado et al., MET, a protein involved in melanoma metastasis was transferred from exosomes to the target cells, preparing these cells for metastasis [
      • Peinado H.
      • Aleckovic M.
      • Lavotshkin S.
      • Matei I.
      • Costa-Silva B.
      • Moreno-Bueno G.
      • et al.
      Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.
      ]. Lazar et al. reported that proteins belonging to NRAS, SRC, KIT, EGFR, Mucins and MET pathways are highly enriched in melanoma exosomes, reflecting its importance in metastasis [
      • Lazar I.
      • Clement E.
      • Ducoux-Petit M.
      • Denat L.
      • Soldan V.
      • Dauvillier S.
      • et al.
      Proteome characterization of melanoma exosomes reveals a specific signature for metastatic cell lines.
      ]. Furthermore, the proteins responsible for glycolysis and the response to wound healing, are enriched in exosomes derived from melanoma, suggesting a possible role in these processes as well [
      • Lazar I.
      • Clement E.
      • Ducoux-Petit M.
      • Denat L.
      • Soldan V.
      • Dauvillier S.
      • et al.
      Proteome characterization of melanoma exosomes reveals a specific signature for metastatic cell lines.
      ].

      Metastatic spread mediated by melanoma exosomes

      Exosomes are capable of transferring metastatic potential to surrounding cells through the transfer of genetic information and/or pro-metastatic proteins [
      • Fujita Y.
      • Yoshioka Y.
      • Ochiya T.
      Extracellular vesicle transfer of cancer pathogenic components.
      ]. Hao et al. showed that melanoma exosomes were able to transfer a tumor metastasis marker, met 72, from metastatic melanoma (B16) to a poorly metastatic (F1) cell line [
      • Hao S.
      • Ye Z.
      • Li F.
      • Meng Q.
      • Qureshi M.
      • Yang J.
      • et al.
      Epigenetic transfer of metastatic activity by uptake of highly metastatic B16 melanoma cell-released exosomes.
      ]. Concurrently, F1 cells became metastatic when treated with the exosomes released from the B16 cell line. Lung metastases were seen in 100% of both animals receiving an intravenous injection of B16 cells and with exosome treated F1 cells, but no metastases were seen in F1 treated mice. The mechanism underlying this process occurred by recruiting the growth factor receptor-bound protein 2 (GRB2) and SRC to the MET receptor [
      • Schaeper U.
      • Gehring N.H.
      • Fuchs K.P.
      • Sachs M.
      • Kempkes B.
      • Birchmeier W.
      Coupling of Gab1 to c-Met, Grb2, and Shp2 mediates biological responses.
      ].
      Melanoma exosomes are also capable of transferring metastatic potential to bone marrow progenitor cells [
      • Peinado H.
      • Aleckovic M.
      • Lavotshkin S.
      • Matei I.
      • Costa-Silva B.
      • Moreno-Bueno G.
      • et al.
      Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.
      ]. This transfer resulted in significantly increased metastasis once the bone marrow progenitor cells had been ‘educated’. Peinado et al. illustrated that the melanoma-derived exosomes enriched with pro-metastatic proteins increased the levels of MET and phospho MET in BDMCs (bone marrow derived cells), a protein involved in the migration and invasion to the bone marrow cells [
      • Peinado H.
      • Aleckovic M.
      • Lavotshkin S.
      • Matei I.
      • Costa-Silva B.
      • Moreno-Bueno G.
      • et al.
      Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.
      ]. One of the other enriched proteins in melanoma exosomes include Rab27a, which also promotes metastasis [
      • Peinado H.
      • Aleckovic M.
      • Lavotshkin S.
      • Matei I.
      • Costa-Silva B.
      • Moreno-Bueno G.
      • et al.
      Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.
      ]. Knockdown of Rab27a decreased tumor invasion and metastasis of melanoma cells by preventing its mobilization to bone marrow derived cells and also decreased the exosome secretion process [
      • Peinado H.
      • Aleckovic M.
      • Lavotshkin S.
      • Matei I.
      • Costa-Silva B.
      • Moreno-Bueno G.
      • et al.
      Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.
      ]. Mannavola et al. also demonstrated the potential of osteotropic melanoma derived exosomes to stimulate metastatic migration to bone in non-opteotropic melanoma lines [
      • Mannavola F.
      • Tucci M.
      • Felici C.
      • Passarelli A.
      • D'Oronzo S.
      • Silvestris F.
      Tumor-derived exosomes promote the in vitro osteotropism of melanoma cells by activating the SDF-1/CXCR4/CXCR7 axis.
      ]. This study is an excellent example of current work in the field, taking into account the size of melanoma exosomes (30–150 nm) while installing protocols to exclude contamination, such that results definitely show the effect of melanoma exosomes on bone metastasis [
      • Mannavola F.
      • Tucci M.
      • Felici C.
      • Passarelli A.
      • D'Oronzo S.
      • Silvestris F.
      Tumor-derived exosomes promote the in vitro osteotropism of melanoma cells by activating the SDF-1/CXCR4/CXCR7 axis.
      ].
      Treatment of mice with melanoma exosomes can increase vascular permeability in the lungs, paving the way for metastasis [
      • Peinado H.
      • Aleckovic M.
      • Lavotshkin S.
      • Matei I.
      • Costa-Silva B.
      • Moreno-Bueno G.
      • et al.
      Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.
      ]. Melanoma derived exosomes activated the expression of MAPK signaling molecules like MAP3K4, MAP2K5, and MAPK13 in primary melanocytes to promote a metastatic phenotype [
      • Xiao D.
      • Barry S.
      • Kmetz D.
      • Egger M.
      • Pan J.
      • Rai S.N.
      • et al.
      Melanoma cell-derived exosomes promote epithelial-mesenchymal transition in primary melanocytes through paracrine/autocrine signaling in the tumor microenvironment.
      ]. The MAPK pathway is known for the induction of EMT in melanoma and other cancer types [
      • Roberts P.J.
      • Der C.J.
      Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer.
      ,
      • Lin K.
      • Baritaki S.
      • Militello L.
      • Malaponte G.
      • Bevelacqua Y.
      • Bonavida B.
      The Role of B-RAF Mutations in Melanoma and the Induction of EMT via Dysregulation of the NF-kappaB/Snail/RKIP/PTEN Circuit.
      ]. Likewise, melanoma-derived exosomes enriched with several other proteins like TYRP2, VLA4, HSP70 and MET play an important role in migration to bone marrow, lungs, lymph nodes and other sites of metastasis to create a favorable tumor niche by altering the ECM, inducing tissue inflammation by increasing the cytokine levels, invasion to the nearby tissues by inducing vascular permeability, and finally also promoting other proangiogenic events [
      • Peinado H.
      • Aleckovic M.
      • Lavotshkin S.
      • Matei I.
      • Costa-Silva B.
      • Moreno-Bueno G.
      • et al.
      Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.
      ]. Schematic representations of the events responsible for metastasis that are regulated by exosomes are illustrated in Fig. 2. Some reports also suggest that cancer exosomes promote tumor metastasis through tumor immune escape mechanisms and altering the functions of macrophages and dendritic cells [
      • Marton A.
      • Vizler C.
      • Kusz E.
      • Temesfoi V.
      • Szathmary Z.
      • Nagy K.
      • et al.
      Melanoma cell-derived exosomes alter macrophage and dendritic cell functions in vitro.
      ].
      Figure thumbnail gr2
      Fig. 2Melanoma-derived exosomes are messengers for metastatic invasion. Melanoma-derived exosomes enriched with oncoproteins are secreted into the extracellular milieu and migrate to bone marrow, lungs and lymph nodes and by increasing vascular permeability, induce inflammation, promote proangiogenic events and form a pre-metastatic niche by remodeling the ECM.
      Although exosomes are capable of communicating with fellow cancer cells, their pathogenic capabilities are not limited to only these cell types. Under hypoxic conditions, exosomes released from cancer cells are known to have enriched the amounts of cytokines like TGF-β, IL6, and TNF-α, which play a major role in the promotion of metastasis [
      • Ramteke A.
      • Ting H.
      • Agarwal C.
      • Mateen S.
      • Somasagara R.
      • Hussain A.
      • et al.
      Exosomes secreted under hypoxia enhance invasiveness and stemness of prostate cancer cells by targeting adherens junction molecules.
      ]. Furthermore, in situations where stress is applied, such as cryostasis, heat, and oxidative stress, melanoma cells produce a greater number of exosomes when compared to unstressed cells [
      • Harmati M.
      • Gyukity-Sebestyen E.
      • Dobra G.
      • Janovak L.
      • Dekany I.
      • Saydam O.
      • et al.
      Small extracellular vesicles convey the stress-induced adaptive responses of melanoma cells.
      ]. In breast cancer, exosomes secreted by hypoxic tumor cells stimulate the formation of the focal adhesion junction and invasion into the extracellular matrix of lung tissues [
      • Wang T.
      • Gilkes D.M.
      • Takano N.
      • Xiang L.
      • Luo W.
      • Bishop C.J.
      • et al.
      Hypoxia-inducible factors and RAB22A mediate formation of microvesicles that stimulate breast cancer invasion and metastasis.
      ]. Protein transfer from cancer exosomes has also been shown in leukemic exosomes. Human vascular endothelial cells (HUVEC) showed increases in ICAM-1, VCAM-1, and IL-8 after exposure to myelogenous leukemia exosomes [
      • Taverna S.
      • Flugy A.
      • Saieva L.
      • Kohn E.C.
      • Santoro A.
      • Meraviglia S.
      • et al.
      Role of exosomes released by chronic myelogenous leukemia cells in angiogenesis.
      ]. Interestingly, cancer cells showed higher levels of attachment to the HUVEC monolayer with increased expression of the adhesion molecules after exosome treatment. There was also a pro-angiogenic response in the HUVEC line suggesting an exosomal effect on angiogenesis [
      • Whitehead B.
      • Wu L.
      • Hvam M.L.
      • Aslan H.
      • Dong M.
      • Dyrskjot L.
      • et al.
      Tumour exosomes display differential mechanical and complement activation properties dependent on malignant state: implications in endothelial leakiness.
      ]. Melanoma derived exosomes were also internalized in MSCs with 91% efficacy, increased PD-1 expression and elevated metastasis in the murine model [
      • Gyukity-Sebestyen E.
      • Harmati M.
      • Dobra G.
      • Nemeth I.B.
      • Mihaly J.
      • Zvara A.
      • et al.
      Melanoma-Derived Exosomes Induce PD-1 Overexpression and Tumor Progression via Mesenchymal Stem Cell Oncogenic Reprogramming.
      ].

      Invasive potential driven by melanoma exosomes

      An effect related to the metastatic increases in response to exosome treatment is an observed increase in invasive potential [
      • Soung Y.H.
      • Nguyen T.
      • Cao H.
      • Lee J.
      • Chung J.
      Emerging roles of exosomes in cancer invasion and metastasis.
      ]. The invasive character is a critical component of the metastatic process, but the preparation of the metastatic niche is also vital. Since the mechanism by which exosomes initiate invasion is unknown, research into this process as well as the ways in which exosomes discriminate between target cells is needed.
      The proteomic analysis of cancer derived exosomes have shown elevated expression of matrix metalloproteinases, ADAM and ADAMTS, which play a role in the degradation of the extracellular matrix of cancer cells and alleviating its invasion potential [
      • Whitehead B.
      • Wu L.
      • Hvam M.L.
      • Aslan H.
      • Dong M.
      • Dyrskjot L.
      • et al.
      Tumour exosomes display differential mechanical and complement activation properties dependent on malignant state: implications in endothelial leakiness.
      ]. Similarly, there exists a positive correlation between the quantity of exosomes, the amount of lytic enzymes and invasive capabilities [
      • Ramteke A.
      • Ting H.
      • Agarwal C.
      • Mateen S.
      • Somasagara R.
      • Hussain A.
      • et al.
      Exosomes secreted under hypoxia enhance invasiveness and stemness of prostate cancer cells by targeting adherens junction molecules.
      ,
      • Lin R.
      • Wang S.
      • Zhao R.C.
      Exosomes from human adipose-derived mesenchymal stem cells promote migration through Wnt signaling pathway in a breast cancer cell model.
      ]. Xiao et al. demonstrated that melanoma exosome treatment of normal melanocytes resulted in a significant increase in invasiveness [
      • Xiao D.
      • Ohlendorf J.
      • Chen Y.
      • Taylor D.D.
      • Rai S.N.
      • Waigel S.
      • et al.
      Identifying mRNA, microRNA and protein profiles of melanoma exosomes.
      ]. Furthermore, this report also investigated RNA and protein profiles in melanoma exosomes compared to parental cells and found that there was an increase in pro-metastatic factors. These results suggest that the cargo exported within exosomes from melanoma cells confers metastatic ability locally. In addition, Rappa et al. confirmed that bone marrow derived stromal cells exhibit increased invasiveness after exposure to melanoma exosomes [
      • Rappa G.
      • Mercapide J.
      • Anzanello F.
      • Pope R.M.
      • Lorico A.
      Biochemical and biological characterization of exosomes containing prominin-1/CD133.
      ]. Hu et al. also demonstrated that melanoma derived exosomes delivering GM26809 are capable of reprogramming murine fibroblasts into cancer associated fibroblasts [
      • Hu T.
      • Hu J.
      Melanoma-derived exosomes induce reprogramming fibroblasts into cancer-associated fibroblasts via Gm26809 delivery.
      ].
      Other studies further support the association of exosomes with tumor cell migration. For example, Lin et al. found that exosomes derived from adipose mesenchymal stem cells promote the migration of MCF-7, a breast cancer cell line, by stimulating the Wnt/β-catenin signaling pathway [
      • Lin R.
      • Wang S.
      • Zhao R.C.
      Exosomes from human adipose-derived mesenchymal stem cells promote migration through Wnt signaling pathway in a breast cancer cell model.
      ]. In addition, studies suggest that the melanoma exosomes possessing Let-7a are also involved in cancer cell migration and invasion through its specific protein targets, such as LIN28B and HMGA [
      • Xiao D.
      • Barry S.
      • Kmetz D.
      • Egger M.
      • Pan J.
      • Rai S.N.
      • et al.
      Melanoma cell-derived exosomes promote epithelial-mesenchymal transition in primary melanocytes through paracrine/autocrine signaling in the tumor microenvironment.
      ].

      Angiogenesis mediated by melanoma exosomes

      As cells produce exosomes, it is reasonable to expect some degree of paracrine signaling in the tumor microenvironment to promote growth and metastasis [
      • Huan J.
      • Hornick N.I.
      • Goloviznina N.A.
      • Kamimae-Lanning A.N.
      • David L.L.
      • Wilmarth P.A.
      • et al.
      Coordinate regulation of residual bone marrow function by paracrine trafficking of AML exosomes.
      ]. A study by Hood et al. showed that exosomes promote angiogenesis by inducing the formation of endothelial spheroids [
      • Hood Joshua L
      • Pan Hua
      • Lanza Gregory M
      • Wickline Samuel A
      Paracrine induction of endothelium by tumor exosomes.
      ]. Increased expression of Wnt5a in melanoma cells induces a rapid release of exosomes loaded with pro-angiogenic factors IL-8, VEGF, MMP2 and immunomodulatory cytokines IL-6 [
      • Ekstrom E.J.
      • Bergenfelz C.
      • von Bulow V.
      • Serifler F.
      • Carlemalm E.
      • Jonsson G.
      • et al.
      WNT5A induces release of exosomes containing pro-angiogenic and immunosuppressive factors from malignant melanoma cells.
      ]. Furthermore, these studies emphasized that an increase in angiogenic factors like VEGF, FGF, TGF, PDGF, and IL-8 play a major role in regulating the networking of quiescence, migration, and proliferation of endothelial cells, which is required for stimulating angiogenesis [
      • Katoh M.
      Therapeutics targeting angiogenesis: genetics and epigenetics, extracellular miRNAs and signaling networks (Review).
      ]. Similarly, melanoma cell derived exosomes containing miRNA-9 were readily internalized by endothelial cells, which later promoted metastasis and angiogenesis by the activation of the JAK-STAT pathway [
      • Gajos-Michniewicz A.
      • Duechler M.
      • Czyz M.
      MiRNA in melanoma-derived exosomes.
      ].
      Exosome induced angiogenesis is not an event tied solely to melanoma but has also been reported in myelogenous leukemia and other cancers [
      • Taverna S.
      • Flugy A.
      • Saieva L.
      • Kohn E.C.
      • Santoro A.
      • Meraviglia S.
      • et al.
      Role of exosomes released by chronic myelogenous leukemia cells in angiogenesis.
      ,
      • Ribeiro M.F.
      • Zhu H.
      • Millard R.W.
      • Fan G.C.
      Exosomes Function in Pro- and Anti-Angiogenesis.
      ]. Under hypoxia conditions, exosomes released from cancer cells can mediate the angiogenic process [
      • Katoh M.
      Therapeutics targeting angiogenesis: genetics and epigenetics, extracellular miRNAs and signaling networks (Review).
      ]. One of the reports suggests that the extracellular vesicles derived from hypoxic tumor glioblastoma cells were enriched with angiogenic stimulatory molecules, such as IL-8 and PDGF [
      • Katoh M.
      Therapeutics targeting angiogenesis: genetics and epigenetics, extracellular miRNAs and signaling networks (Review).
      ]. Exosomes derived from metastatic breast cancer cells also contained multiple miRNAs, like miRNA-210, which play a vital role in angiogenesis [
      • Camacho L.
      • Guerrero P.
      • Marchetti D.
      MicroRNA and protein profiling of brain metastasis competent cell-derived exosomes.
      ]. This illustrates that, while there are many unique characteristics of melanoma exosomes, there are some conserved mechanisms used by multiple cancer types to promote tumor growth and metastasis.

      Promotion of lymph node metastasis mediated by melanoma exosomes

      CTCs and melanoma exosomes have also been shown to preferentially accumulate in the sentinel lymph nodes [
      • Hood J.L.
      • San R.S.
      • Wickline S.A.
      Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis.
      ,
      • Mumford B.S.
      • Robertson G.P.
      Circulating melanoma cells in the diagnosis and monitoring of melanoma: an appraisal of clinical potential.
      ,
      • De Souza L.M.
      • Robertson B.M.
      • Robertson G.P.
      Future of circulating tumor cells in the melanoma clinical and research laboratory settings.
      ]. Once localized in the lymph nodes, accumulated exosomes increased the migration of melanoma cells to the lymph node. It was noted that melanoma cells migrated to sites with high exosome content [
      • Hood J.L.
      • San R.S.
      • Wickline S.A.
      Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis.
      ]. Gene expression in the melanoma cells was investigated to determine the possible role of exosomes in melanoma metastasis to the lymph nodes [
      • Vences-Catalan F.
      • Levy S.
      Immune Targeting of Tetraspanins Involved in Cell Invasion and Metastasis.
      ,
      • Hood J.L.
      • San R.S.
      • Wickline S.A.
      Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis.
      ]. This analysis also showed that three groups of genes were upregulated when treated with exosomes. Those genes represent; (1) cell recruitment proteins (promoting migration to the lymph node); (2) extracellular matrix proteins (potentially involved in the capture and anchoring of metastasizing melanoma cells); and (3) vascular growth factors (promoting angiogenesis for the continued growth of metastasized tumors). Melanoma research is currently leading this area of scientific discovery, but more research is required to fully dissect this process. Still to be determined is the mechanism through which exosomes draw melanoma cells to the lymph nodes and possible lipid, protein, or other molecules eliciting migration. It would be important to determine whether a similar response is responsible for promoting CTC attachment and growth at metastatic sites.

      Effects on cell differentiation mediated by melanoma exosomes

      Exosomes have also been shown to affect cell differentiation [
      • Behbahani G.D.
      • Khani S.
      • Hosseini H.M.
      • Abbaszadeh-Goudarzi K.
      • Nazeri S.
      The role of exosomes contents on genetic and epigenetic alterations of recipient cancer cells.
      ]. Cancer derived exosomes have also been shown to facilitate fibroblast differentiation into carcinoma associated fibroblasts (also known as myofibroblasts) [
      • Webber J.
      • Steadman R.
      • Mason M.D.
      • Tabi Z.
      • Clayton A.
      Cancer exosomes trigger fibroblast to myofibroblast differentiation.
      ,
      • Karagiannis G.S.
      • Poutahidis T.
      • Erdman S.E.
      • Kirsch R.
      • Riddell R.H.
      • Diamandis E.P.
      Cancer-associated fibroblasts drive the progression of metastasis through both paracrine and mechanical pressure on cancer tissue.
      ]. Myofibroblasts are rich in an altered tumor stroma that facilitates tumor growth, angiogenesis, and metastasis [
      • Olumi A.F.
      • Grossfeld G.D.
      • Hayward S.W.
      • Carroll P.R.
      • Tlsty T.D.
      • Cunha G.R.
      Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium.
      ]. Melanoma exosomes decrease differentiation of bone marrow-derived dendritic cells [
      • Yu S.
      • Liu C.
      • Su K.
      • Wang J.
      • Liu Y.
      • Zhang L.
      • et al.
      Tumor exosomes inhibit differentiation of bone marrow dendritic cells.
      ]. Umbilical cord stem cells were shown to differentiate into carcinoma-associated fibroblasts when exposed to gastric cancer exosomes [
      • Gu J.
      • Qian H.
      • Shen L.
      • Zhang X.
      • Zhu W.
      • Huang L.
      • et al.
      Gastric cancer exosomes trigger differentiation of umbilical cord derived mesenchymal stem cells to carcinoma-associated fibroblasts through TGF-beta/Smad pathway.
      ]. Though information on the effect of exosomes on cell differentiation is limited, it appears that exosomes are capable of both inhibiting, and promoting differentiation. More research is needed to identify the mechanism by which exosomes promote/inhibit differentiation.

      Exosome mediated regulation of the immune system in melanoma

      Accumulating evidence suggests that exosomes regulate the immune system [
      • Tucci M.
      • Mannavola F.
      • Passarelli A.
      • Stucci L.S.
      • Cives M.
      • Silvestris F.
      Exosomes in melanoma: a role in tumor progression, metastasis and impaired immune system activity.
      ,
      • Viaud S.
      • Thery C.
      • Ploix S.
      • Tursz T.
      • Lapierre V.
      • Lantz O.
      • et al.
      Dendritic cell-derived exosomes for cancer immunotherapy: what's next?.
      ]. Dendritic cell derived exosomes (DEXs) can induce T-cell stimulation when compared with immature DCs [
      • Naslund T.I.
      • Gehrmann U.
      • Qazi K.R.
      • Karlsson M.C.
      • Gabrielsson S.
      Dendritic cell-derived exosomes need to activate both T and B cells to induce antitumor immunity.
      ]. Similarly, DEXs promote CD8+ T cell activation, which also appears to correspond with DEXs interacting with dendritic cells [
      • Naslund T.I.
      • Gehrmann U.
      • Qazi K.R.
      • Karlsson M.C.
      • Gabrielsson S.
      Dendritic cell-derived exosomes need to activate both T and B cells to induce antitumor immunity.
      ]. In addition, DEXs modulate immune function through MHC class I and/or II proteins expressed on the exosomes or through the transfer of cargo to the cells (vesicles with packages of DNA, RNA, microRNA, and proteins) [
      • Pitt J.M.
      • Charrier M.
      • Viaud S.
      • Andre F.
      • Besse B.
      • Chaput N.
      • et al.
      Dendritic cell-derived exosomes as immunotherapies in the fight against cancer.
      ]. Further, melanoma exosomes can induce pro-tumor macrophage polarization and activation [
      • Bardi G.T.
      • Smith M.A.
      • Hood J.L.
      Melanoma exosomes promote mixed M1 and M2 macrophage polarization.
      ] along with altering the transcriptome of CD8+ T-cells by modulating mitochondrial respiration [
      • Bland C.L.
      • Byrne-Hoffman C.N.
      • Fernandez A.
      • Rellick S.L.
      • Deng W.
      • Klinke 2nd, D.J.
      Exosomes derived from B16F0 melanoma cells alter the transcriptome of cytotoxic T cells that impacts mitochondrial respiration.
      ]. Melanoma exosomes also directly activate CD4+ T-cells through the expression and release of miR690 and Rab27a [
      • Zhou J.
      • Yang Y.
      • Wang W.
      • Zhang Y.
      • Chen Z.
      • Hao C.
      • et al.
      Melanoma-released exosomes directly activate the mitochondrial apoptotic pathway of CD4(+) T cells through their microRNA cargo.
      ].
      Several groups have shown that when bone marrow-derived dendritic cells (BMDCs) were treated with acid-eluted peptides isolated from tumors, the dendritic cells triggered immune system activation [
      • Liau L.M.
      • Black K.L.
      • Prins R.M.
      • Sykes S.N.
      • DiPatre P.L.
      • Cloughesy T.F.
      • et al.
      Treatment of intracranial gliomas with bone marrow-derived dendritic cells pulsed with tumor antigens.
      ]. These initial studies were performed using non-melanoma skin cancers; however, these discoveries resulted in vaccine focused clinical trials in metastatic melanoma. Exosomes isolated from the dendritic cell population elicited the same vaccine-like response from the host immune system, raising the possibility that exosomes may have led to this response in the cell based vaccination studies [
      • Zeelenberg I.S.
      • Ostrowski M.
      • Krumeich S.
      • Bobrie A.
      • Jancic C.
      • Boissonnas A.
      • et al.
      Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses.
      ]. Exosomes were used as the delivery agent to increase responsiveness in these studies. This study also shows that antigens delivered by exosomes can be immunogenic [
      • Zeelenberg I.S.
      • Ostrowski M.
      • Krumeich S.
      • Bobrie A.
      • Jancic C.
      • Boissonnas A.
      • et al.
      Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses.
      ]. Lamparski et al. studied a mouse tumor exosome uptake model, where the dendritic cells induced CD8+ T-cell-dependent antitumor effects on syngeneic and allogeneic established mouse tumors. These results suggest that exosomes represent a novel source of tumor rejection antigens for T-cell cross priming, relevant for immunological-based interventions [
      • Lamparski H.G.
      • Metha-Damani A.
      • Yao J.Y.
      • Patel S.
      • Hsu D.H.
      • Ruegg C.
      • et al.
      Production and characterization of clinical grade exosomes derived from dendritic cells.
      ]. Importantly, these studies paved the way for the isolation of clinical grade exosomes from the dendritic cells [
      • Lamparski H.G.
      • Metha-Damani A.
      • Yao J.Y.
      • Patel S.
      • Hsu D.H.
      • Ruegg C.
      • et al.
      Production and characterization of clinical grade exosomes derived from dendritic cells.
      ].
      The clinical trials of the exosome based vaccination in MAGE3+ metastatic melanoma patients suggested that the production of exosomes was feasible and that they could be safely administered [
      • Escudier B.
      • Dorval T.
      • Chaput N.
      • Andre F.
      • Caby M.P.
      • Novault S.
      • et al.
      Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial.
      ]. Results of the study found an objective minor response in one patient, two patients had stable disease, and one patient who continued to be stable for 24 months during the administration of dendritic cell-derived exosomes injections [
      • Escudier B.
      • Dorval T.
      • Chaput N.
      • Andre F.
      • Caby M.P.
      • Novault S.
      • et al.
      Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial.
      ]. These discoveries suggested the safety of dendritic cell-derived exosomes when administered to patients and laid the foundation for future clinical interventions, while also highlighting the feasibility of commercial-scale and clinical grade of production of these particles. Expansion of this area of research should occur quickly in the future potentially leading to better treatments for cancer.

      Exosomes as prognostic indicators of melanoma

      The protein and nucleic acid content within exosomes are defined by the melanoma cells producing them [
      • Kahlert C.
      • Kalluri R.
      Exosomes in tumor microenvironment influence cancer progression and metastasis.
      ]. For this reason, scientists have studied the dysregulation of exosome production in melanoma and how this leads to cancer growth and dissemination [
      • Kucharzewska Paulina
      • Belting Mattias
      Emerging roles of extracellular vesicles in the adaptive response of tumour cells to microenvironmental stress.
      ,
      • Wendler Franz
      • Bota-Rabassedas Neus
      • Franch-Marro Xavier
      Cancer becomes wasteful: emerging roles of exosomes in cell-fate determination.
      ]. Studies are also exploring the prognostic value of exosomes in cancers [
      • Sandfeld-Paulsen B.
      • Aggerholm-Pedersen N.
      • Baek R.
      • Jakobsen K.R.
      • Meldgaard P.
      • Folkersen B.H.
      • et al.
      Exosomal proteins as prognostic biomarkers in non-small cell lung cancer.
      ,
      • Saadatpour L.
      • Fadaee E.
      • Fadaei S.
      • Nassiri Mansour R.
      • Mohammadi M.
      • Mousavi S.M.
      • et al.
      Glioblastoma: exosome and microRNA as novel diagnosis biomarkers.
      ,
      • Wang Z.
      • Chen J.Q.
      • Liu J.L.
      • Tian L.
      Exosomes in tumor microenvironment: novel transporters and biomarkers.
      ]. Since exosomes are present in the circulation, the next section explores the utility of exosomes for prognostic and diagnostic purposes [
      • Ge R.
      • Tan E.
      • Sharghi-Namini S.
      • Asada H.H.
      Exosomes in Cancer Microenvironment and Beyond: have we Overlooked these Extracellular Messengers?.
      ].
      The use of exosomes as prognostic indicators is not linked solely to the cargo they transport, but there is also interest in using the exosomes themselves as a biomarker [
      • Alegre E.
      • Zubiri L.
      • Perez-Gracia J.L.
      • Gonzalez-Cao M.
      • Soria L.
      • Martin-Algarra S.
      • et al.
      Circulating melanoma exosomes as diagnostic and prognosis biomarkers.
      ]. While the literature on the value of exosomes as prognostic indicators in melanoma is limited, the use of exosomes and the proteins they carry have been studied as predictive biomarkers.
      Exosomal levels of MET, TYRP2, VLA-4, Hsp-90, and Hsp-70 were reported to be upregulated in melanoma exosomes and investigated as potential melanoma biomarkers [
      • Peinado H.
      • Aleckovic M.
      • Lavotshkin S.
      • Matei I.
      • Costa-Silva B.
      • Moreno-Bueno G.
      • et al.
      Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.
      ]. Furthermore, several studies reported increased levels of tetraspanins in the exosomes of melanoma patients suggesting their potential use as biomarkers [
      • Logozzi M.
      • De Milito A.
      • Lugini L.
      • Borghi M.
      • Calabro L.
      • Spada M.
      • et al.
      High levels of exosomes expressing CD63 and caveolin-1 in plasma of melanoma patients.
      ,
      • Yoshioka Yusuke
      • Konishi Yuki
      • Kosaka Nobuyoshi
      • Katsuda Takeshi
      • Kato Takashi
      • Ochiya Takahiro
      Comparative marker analysis of extracellular vesicles in different human cancer types.
      ]. Specifically, the exosomes from melanoma patients have higher levels of CD63 compared to healthy controls [
      • Logozzi M.
      • De Milito A.
      • Lugini L.
      • Borghi M.
      • Calabro L.
      • Spada M.
      • et al.
      High levels of exosomes expressing CD63 and caveolin-1 in plasma of melanoma patients.
      ]. Yoshioka et al. found that the levels of CD63 are higher in exosomes derived from melanoma compared to those derived from noncancerous cells, suggesting a potential use of exosomal CD63 as a potential protein marker for cancer [
      • Yoshioka Yusuke
      • Konishi Yuki
      • Kosaka Nobuyoshi
      • Katsuda Takeshi
      • Kato Takashi
      • Ochiya Takahiro
      Comparative marker analysis of extracellular vesicles in different human cancer types.
      ].
      Extracellular vesicles purified from lymphatic drainage of melanoma tumors are also enriched in proteins present in melanoma progression [
      • Garcia-Silva S.
      • Benito-Martin A.
      • Sanchez-Redondo S.
      • Hernandez-Barranco A.
      • Ximenez-Embun P.
      • Nogues L.
      • et al.
      Use of extracellular vesicles from lymphatic drainage as surrogate markers of melanoma progression and BRAF (V600E) mutation.
      ]. For example, V600EBRAF mutation can be detected in these extracellular vesicles and its presence correlated with patients at risk of relapse [
      • Garcia-Silva S.
      • Benito-Martin A.
      • Sanchez-Redondo S.
      • Hernandez-Barranco A.
      • Ximenez-Embun P.
      • Nogues L.
      • et al.
      Use of extracellular vesicles from lymphatic drainage as surrogate markers of melanoma progression and BRAF (V600E) mutation.
      ]. Exosomal levels of 11 genes that are enriched in acidic conditions also significantly correlated with poor prognosis and ability to metastasize [
      • Boussadia Z.
      • Lamberti J.
      • Mattei F.
      • Pizzi E.
      • Puglisi R.
      • Zanetti C.
      • et al.
      Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules.
      ]. Furthermore, acidic environment induces an abundant release and intra-tumoral uptake of exosomes, which may prove a signature of melanoma progression [
      • Boussadia Z.
      • Lamberti J.
      • Mattei F.
      • Pizzi E.
      • Puglisi R.
      • Zanetti C.
      • et al.
      Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules.
      ].
      Exosomes can also be considered as biomarkers for treatment response. A recent study in melanoma and NSCLC patients treated with a combination of PD1/PD-L1 antibodies demonstrated a significant correlation of exosomal PD-L1 mRNA expression levels to treatment response [
      • Del Re M.
      • Marconcini R.
      • Pasquini G.
      • Rofi E.
      • Vivaldi C.
      • Bloise F.
      • et al.
      PD-L1 mRNA expression in plasma-derived exosomes is associated with response to anti-PD-1 antibodies in melanoma and NSCLC.
      ]. Patients with higher levels of PD-L1 mRNA in plasma-derived exosomes had a better response with the PD1/PD-L1 combination. Additionally, when the PD-L1 expression in exosomes increased with treatment, patients underwent disease progression demonstrating the biomarker potential of exosomes [
      • Del Re M.
      • Marconcini R.
      • Pasquini G.
      • Rofi E.
      • Vivaldi C.
      • Bloise F.
      • et al.
      PD-L1 mRNA expression in plasma-derived exosomes is associated with response to anti-PD-1 antibodies in melanoma and NSCLC.
      ].
      Collectively, these studies suggest that the proteins or RNA in exosomes can be used as biomarkers. A clinical trial to investigate the use of exosomes for predictive and prognostic purposes in advanced gastric cancers is currently underway [

      Pazo-Cid R. Circulating Exosomes As Potential Prognostic And Predictive Biomarkers In Advanced Gastric Cancer Patients (“EXO-PPP Study”). ClnicalTrialsgov. 2014;NCT01779583.

      ]. Another trial is monitoring the level of exosomes in the circulation to determine if it could be utilized clinically in colorectal cancer as high levels of exosomes correlated with shorter survival and poor prognosis [
      • Silva J.
      • Garcia V.
      • Rodriguez M.
      • Compte M.
      • Cisneros E.
      • Veguillas P.
      • et al.
      Analysis of exosome release and its prognostic value in human colorectal cancer.
      ]. If a clinically validated exosome-based biomarker system was to emerge for any particular cancer, it could also be used for melanoma as the principles for exosome quantification are similar across cancer types.

      Exosomes for melanoma treatment

      Exosomes could be used to modulate the immune system in melanoma patients for treatment. Exosomes might also be used as therapeutic delivery systems for treating melanoma [
      • Kosaka N.
      • Takeshita F.
      • Yoshioka Y.
      • Hagiwara K.
      • Katsuda T.
      • Ono M.
      • et al.
      Exosomal tumor-suppressive microRNAs as novel cancer therapy: “exocure” is another choice for cancer treatment.
      ,
      • Collet G.
      • Grillon C.
      • Nadim M.
      • Kieda C.
      Trojan horse at cellular level for tumor gene therapies.
      ]. An exosomal delivery system would share many of the advantages of the synthetic vesicles used for drug delivery called liposomes, as both are constructed from lipids and have low toxicity [
      • Brys A.K.
      • Gowda R.
      • Loriaux D.B.
      • Robertson G.P.
      • Mosca P.J.
      Nanotechnology-based strategies for combating toxicity and resistance in melanoma therapy.
      ,
      • Gowda R.
      • Madhunapantula S.V.
      • Sharma A.
      • Kuzu O.F.
      • Robertson G.P.
      Nanolipolee-007, a novel nanoparticle-based drug containing leelamine for the treatment of melanoma.
      ,
      • Tran M.A.
      • Watts R.J.
      • Robertson G.P.
      Use of liposomes as drug delivery vehicles for treatment of melanoma.
      ]. In contrast, an exosome has membrane proteins, nucleic acids, and other cellular components, including cytoskeletal proteins that alter the properties of the exosome [
      • Kooijmans S.A.
      • Vader P.
      • van Dommelen S.M.
      • van Solinge W.W.
      • Schiffelers R.M.
      Exosome mimetics: a novel class of drug delivery systems.
      ,
      • Ha D.
      • Yang N.
      • Nadithe V.
      Exosomes as therapeutic drug carriers and delivery vehicles across biological membranes: current perspectives and future challenges.
      ]. These differences could be important for decreasing uptake of these particles by the reticuloendothelial system and might increase the uptake into cancer cells if targeted to these cells [
      • Ohno S.
      • Takanashi M.
      • Sudo K.
      • Ueda S.
      • Ishikawa A.
      • Matsuyama N.
      • et al.
      Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells.
      ]. Another advantage of exosomes over synthetic vesicles is that they can preferentially sort biogenic and synthetic substances depending on the extracellular niche of the donor cells [
      • Ohno S.
      • Takanashi M.
      • Sudo K.
      • Ueda S.
      • Ishikawa A.
      • Matsuyama N.
      • et al.
      Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells.
      ]. By modulating the membrane composition, they can selectively target certain cells. Exosomes can be loaded with therapeutic cargos using electroporation [
      • Tian Y.
      • Li S.
      • Song J.
      • Ji T.
      • Zhu M.
      • Anderson G.J.
      • et al.
      A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy.
      ], chemical based transfection using Lipofectamine 2000 [
      • Shtam T.A.
      • Kovalev R.A.
      • Varfolomeeva E.Y.
      • Makarov E.M.
      • Kil Y.V.
      • Filatov M.V.
      Exosomes are natural carriers of exogenous siRNA to human cells in vitro.
      ], incubation of exosomes with a particular cargo [
      • Jang S.C.
      • Kim O.Y.
      • Yoon C.M.
      • Choi D.S.
      • Roh T.Y.
      • Park J.
      • et al.
      Bioinspired exosome-mimetic nanovesicles for targeted delivery of chemotherapeutics to malignant tumors.
      ], transfection of exosome producing cells and cell activation [
      • Zhang Y.
      • Liu D.
      • Chen X.
      • Li J.
      • Li L.
      • Bian Z.
      • et al.
      Secreted monocytic miR-150 enhances targeted endothelial cell migration.
      ]. Also, methods have been developed to load both lipophilic and hydrophilic substances into exosomes [
      • Vlassov A.V.
      • Magdaleno S.
      • Setterquist R.
      • Conrad R.
      Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials.
      ]. The methods for using exosomes as targeted delivery vehicles are illustrated in Fig. 3. This emerging area will likely see extensive development in the near future.
      Figure thumbnail gr3
      Fig. 3Methods using exosomes for targeted drug delivery. Melanoma derived exosomes can be either used for treatment by loading drug into the parental exosomes or for diagnostic purposes by screening cargo carried by exosomes of particular cell types.

      Exosomal delivery of RNA

      Exosomes have been studied as delivery vehicles for RNA [
      • van den Boorn J.G.
      • Dassler J.
      • Coch C.
      • Schlee M.
      • Hartmann G.
      Exosomes as nucleic acid nanocarriers.
      ]. Therapeutic efficacy of exosomes loaded with siRNA can lead to selective gene silencing of the MAPK-1 gene [
      • Wahlgren J.
      • De L.K.T.
      • Brisslert M.
      • Vaziri Sani F.
      • Telemo E.
      • Sunnerhagen P.
      • et al.
      Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes.
      ]. Similar methodology was used to induce knockdown of RAD51-, RAD52- and BACE protein levels in cancer cells to decrease cell viability [
      • Shtam T.A.
      • Kovalev R.A.
      • Varfolomeeva E.Y.
      • Makarov E.M.
      • Kil Y.V.
      • Filatov M.V.
      Exosomes are natural carriers of exogenous siRNA to human cells in vitro.
      ]. Exosome-delivered tumor suppressor miRNAs, miR-143 and let- 7a, let-7b inhibited the growth of skin, prostate and breast cancer cells, respectively [
      • Pfeffer S.R.
      • Grossmann K.F.
      • Cassidy P.B.
      • Yang C.H.
      • Fan M.
      • Kopelovich L.
      • et al.
      Detection of Exosomal miRNAs in the Plasma of Melanoma Patients.
      ,
      • Hannafon B.N.
      • Carpenter K.J.
      • Berry W.L.
      • Janknecht R.
      • Dooley W.C.
      • Ding W.Q.
      Exosome-mediated microRNA signaling from breast cancer cells is altered by the anti-angiogenesis agent docosahexaenoic acid (DHA).
      ,
      • Hessvik N.P.
      • Sandvig K.
      • Llorente A.
      Exosomal miRNAs as Biomarkers for Prostate Cancer.
      ].
      For neurodegenerative diseases, siRNA loaded into exosomes has mediated knockdown of target genes in neural cell types for treatment of these conditions [
      • Alvarez-Erviti L.
      • Seow Y.
      • Yin H.
      • Betts C.
      • Lakhal S.
      • Wood M.J.
      Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes.
      ]. Cells were engineered to express Lamp2b, an exosomal membrane protein, fused with homing peptides to facilitate active targeting of the exosomes by the immune system. The study analyzed nucleic acid delivery to the brain and evaluated therapeutic efficacy for treating Alzheimer’s disease. These results demonstrate the therapeutic potential of exosomes as an siRNA delivery vehicle [
      • Alvarez-Erviti L.
      • Seow Y.
      • Yin H.
      • Betts C.
      • Lakhal S.
      • Wood M.J.
      Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes.
      ].
      Exosomes carrying shRNAs against the hepatitis C virus (HCV) replication machinery resulted in a decrease in HCV infection of liver cells further supporting the therapeutic potential of this approach [
      • Pan Q.
      • Ramakrishnaiah V.
      • Henry S.
      • Fouraschen S.
      • de Ruiter P.E.
      • Kwekkeboom J.
      • et al.
      Hepatic cell-to-cell transmission of small silencing RNA can extend the therapeutic reach of RNA interference (RNAi).
      ]. Exosomes carrying natural miRNAs in various disease models have also been studied. The exosomal delivery of miR-214 to hepatic stellate cells decreased the expression of CCN2, a gene known to be important in regulating liver fibrosis [
      • Chen L.
      • Charrier A.
      • Zhou Y.
      • Chen R.
      • Yu B.
      • Agarwal K.
      • et al.
      Epigenetic regulation of connective tissue growth factor by MicroRNA-214 delivery in exosomes from mouse or human hepatic stellate cells.
      ]. Therefore, exosomes can be loaded with both synthetic and natural RNA and could potentially be used to gain therapeutic advantage.

      Exosomal delivery of small molecule drugs

      Exosomes as therapeutic delivery vehicles are not only limited to RNA but could also be used for small molecule drug delivery [
      • Tian Y.
      • Li S.
      • Song J.
      • Ji T.
      • Zhu M.
      • Anderson G.J.
      • et al.
      A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy.
      ,
      • Yang T.
      • Martin P.
      • Fogarty B.
      • Brown A.
      • Schurman K.
      • Phipps R.
      • et al.
      Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio.
      ]. This is an area of research that could be of particular value to the medical community and used in a manner similar to liposomal delivery [
      • Gowda R.
      • Jones N.R.
      • Banerjee S.
      • Robertson G.P.
      Use of Nanotechnology to Develop Multi-Drug Inhibitors For Cancer Therapy.
      ,
      • Gowda R.
      • Kardos G.
      • Sharma A.
      • Singh S.
      • Robertson G.P.
      Nanoparticle-Based Celecoxib and Plumbagin for the Synergistic Treatment of Melanoma.
      ,
      • Chen Y.C.
      • Gowda R.
      • Newswanger R.K.
      • Leibich P.
      • Fell B.
      • Rosenberg G.
      • et al.
      Targeting cholesterol transport in circulating melanoma cells to inhibit metastasis.
      ,
      • Gowda R.
      • Dinavahi S.S.
      • Iyer S.
      • Banerjee S.
      • Neves R.I.
      • Pameijer C.R.
      • et al.
      Nanoliposomal delivery of cytosolic phospholipase A2 inhibitor arachidonyl trimethyl ketone for melanoma treatment.
      ]. Exosomes loaded with the anti-inflammatory drug curcumin showed increased bioavailability and the formulation-protected mice from LPS (lipopolysaccharide) induced shock [
      • Sun D.
      • Zhuang X.
      • Xiang X.
      • Liu Y.
      • Zhang S.
      • Liu C.
      • et al.
      A novel nanoparticle drug delivery system: the anti-inflammatory activity of curcumin is enhanced when encapsulated in exosomes.
      ]. These particles have also been used for the delivery of doxorubicin and paclitaxel [
      • Tian Y.
      • Li S.
      • Song J.
      • Ji T.
      • Zhu M.
      • Anderson G.J.
      • et al.
      A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy.
      ,
      • Yang T.
      • Martin P.
      • Fogarty B.
      • Brown A.
      • Schurman K.
      • Phipps R.
      • et al.
      Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio.
      ]. Tian et al. used exosomes to deliver doxorubicin into a mouse tumor tissue model showing the accumulation of doxorubicin containing exosomes at the targeted organ and led to suppressed tumor growth [
      • Tian Y.
      • Li S.
      • Song J.
      • Ji T.
      • Zhu M.
      • Anderson G.J.
      • et al.
      A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy.
      ]. Similarly, Yang et al. showed that encapsulating the anticancer drugs paclitaxel and doxorubicin into exosomes derived from endothelial cells could be used for brain delivery across the blood–brain barrier [
      • Yang T.
      • Martin P.
      • Fogarty B.
      • Brown A.
      • Schurman K.
      • Phipps R.
      • et al.
      Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio.
      ].

      Exosomal delivery of proteins

      Exosomes naturally function in the transport and transfer of proteins and are capable of inducing physiological responses [
      • Munson P.
      • Shukla A.
      Exosomes: Potential in Cancer Diagnosis and Therapy.
      ]. Currently, there is not much research utilizing exosomes for protein transport for treatment of any disease. Only one study showed successful delivery of exosomes loaded with the antioxidant protein catalase across the blood brain barrier for the treatment of Parkinson’s disease [
      • Haney M.J.
      • Klyachko N.L.
      • Zhao Y.
      • Gupta R.
      • Plotnikova E.G.
      • He Z.
      • et al.
      Exosomes as drug delivery vehicles for Parkinson's disease therapy.
      ]. However, many proteins are upregulated in melanoma cells and their exosomes, suggesting that increased protein levels can lead to cancer progression and metastasis. Therefore, the down-regulation of tumor suppressor proteins in exosomes could also be a useful area of future research.

      Clinical trials using exosomes for cancer therapy

      The advantage of using exosomes as a non-invasive biomarker or as a method of drug delivery is being evaluated clinically [
      • Mahmoodzadeh Hosseini H.
      • Halabian R.
      • Amin M.
      • Imani Fooladi A.A.
      Texosome-based drug delivery system for cancer therapy: from past to present.
      ]. Clinical trials to determine the feasibility of exosomes for use as a biomarker, biological target, or drug delivery vehicle to treat a wide variety of human disease conditions are being explored [
      • Mahmoodzadeh Hosseini H.
      • Halabian R.
      • Amin M.
      • Imani Fooladi A.A.
      Texosome-based drug delivery system for cancer therapy: from past to present.
      ]. Clinical trials using exosomes for cancer therapeutics are listed in Table 1. Some studies have used exosomes to stimulate the immune response of melanoma stage IIIb/IV and non-small cell lung cancer III/IV patients by loading MAGE3 into exosomes derived from dendritic cells [
      • Escudier B.
      • Dorval T.
      • Chaput N.
      • Andre F.
      • Caby M.P.
      • Novault S.
      • et al.
      Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial.
      ,
      • Morse M.A.
      • Garst J.
      • Osada T.
      • Khan S.
      • Hobeika A.
      • Clay T.M.
      • et al.
      A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer.
      ].
      Table1Clinical trials using exosomes for cancer treatment.
      TrialLoaded agentIndication/EffectsDisease typeReferences
      Exosomes with antisense moleculeIGF-1R/AS ODNActivation of immune systemGlioma of BrainNCT01550523
      Plasma derived exosomes____Prognostic biomarkerCutaneous ulcersNCT02565264
      Exosomes comparing drug sensitive vs resistant patients____Prognostic markersMelanomaNCT02310451
      Exosomes from blood and tissue____Prognostic markersPancreatic cancerNCT02393703
      Grape derived exosomesGrape extractChemotherapy and radiotherapyHead and Neck cancerNCT01668849
      Patients’ Whitcomb, (loaded with MAGE3)MAGE3Effect on immune response on the patientMelanoma stage III/IV and Non-small cell lung cancer III/IV
      • Escudier B.
      • Dorval T.
      • Chaput N.
      • Andre F.
      • Caby M.P.
      • Novault S.
      • et al.
      Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial.
      ,
      • Morse M.A.
      • Garst J.
      • Osada T.
      • Khan S.
      • Hobeika A.
      • Clay T.M.
      • et al.
      A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer.
      Chemotherapeutic agent metronomic cyclophosphamide with Patient’s exosomesmetronomic cyclophosphamideImmunomodulatory responsesUnrespectable Non-small lung cancerNCT01159288
      Mesenchymal Stromal Cells-derived Exosomes loaded with KRAS G12D siRNAsiRNACancer treatmentPancreatic cancerNCT03608631
      Curcumin loaded exosomesCurcuminCancer treatmentColorectal cancerNCT01294072
      Patient’s exosomes combined with GM-CSFGM-CSFSpecific CD8+ responseColorectal cancer
      • Dai S.
      • Wei D.
      • Wu Z.
      • Zhou X.
      • Wei X.
      • Huang H.
      • et al.
      Phase I clinical trial of autologous ascites-derived exosomes combined with GM-CSF for colorectal cancer.
      Exosomes can function as therapeutic cell-free vaccines for the treatment of various cancers [
      • Escudier B.
      • Dorval T.
      • Chaput N.
      • Andre F.
      • Caby M.P.
      • Novault S.
      • et al.
      Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial.
      ]. Clinical trials have been completed using dendritic cell-based exosomes (Dex) injected intradermally [
      • Escudier B.
      • Dorval T.
      • Chaput N.
      • Andre F.
      • Caby M.P.
      • Novault S.
      • et al.
      Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial.
      ]. In a phase 1 trial of autologous Dex in metastatic melanoma, four out of 15 patients (27%) derived clinical benefit (disease stabilization or objective response) with one other patient showing a mixed response [
      • Escudier B.
      • Dorval T.
      • Chaput N.
      • Andre F.
      • Caby M.P.
      • Novault S.
      • et al.
      Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial.
      ]. Another early phase 1 trial enrolled patients with advanced non-small cell lung cancer (NSCLC); two experienced disease stabilization, two of the remaining six patients showed progression-free survival for at least 12 months [
      • Morse M.A.
      • Garst J.
      • Osada T.
      • Khan S.
      • Hobeika A.
      • Clay T.M.
      • et al.
      A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer.
      ]. Both phase 1 trials demonstrated the feasibility of using monocyte-derived DCs pulsed with tumor-associated antigens under GMP conditions and reported excellent safety profiles (all grade ≤ 2 adverse events) [
      • Morse M.A.
      • Garst J.
      • Osada T.
      • Khan S.
      • Hobeika A.
      • Clay T.M.
      • et al.
      A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer.
      ]. Most recently, a phase 2 trial in NSCLC patients with stage IIIb and IV, who had failed to respond to first-line chemotherapy demonstrated a median time to progression and overall survival of 2.2 and 15 months, respectively [
      • Besse B.
      • Charrier M.
      • Lapierre V.
      • Dansin E.
      • Lantz O.
      • Planchard D.
      • et al.
      Dendritic cell-derived exosomes as maintenance immunotherapy after first line chemotherapy in NSCLC.
      ]. Seven out of 22 (32%) patients experienced stable disease for at least 4 months [
      • Besse B.
      • Charrier M.
      • Lapierre V.
      • Dansin E.
      • Lantz O.
      • Planchard D.
      • et al.
      Dendritic cell-derived exosomes as maintenance immunotherapy after first line chemotherapy in NSCLC.
      ]. Only one patient in the study experienced grade 3 hepatotoxicities. Improved clinical outcomes among Dex-immunized subjects have been observed to correlate the induction of natural killer cell cytolytic functions, possibly as a result of transduction by functional substrates on the surface of Dex [
      • Besse B.
      • Charrier M.
      • Lapierre V.
      • Dansin E.
      • Lantz O.
      • Planchard D.
      • et al.
      Dendritic cell-derived exosomes as maintenance immunotherapy after first line chemotherapy in NSCLC.
      ].

      Needed areas for future melanoma exosome research

      Exosomes have been called the “messengers of metastasis” [
      • Somasundaram R.
      • Herlyn M.
      Melanoma exosomes: messengers of metastasis.
      ]. Exosomes play a critical role in the metastatic process from invasion to preparation of the metastatic niche [
      • Soung Y.H.
      • Nguyen T.
      • Cao H.
      • Lee J.
      • Chung J.
      Emerging roles of exosomes in cancer invasion and metastasis.
      ]. Therefore, exosomes are being studied for their role in cancer to promote disease progression and metastasis. Most current research involves dissecting the role of exosomes in cancer development. These studies must now be translated into the development of treatments for melanoma, perhaps using exosomes themselves as a drug delivery platform.

      Grant support

      The Foreman Foundation for Melanoma (Gavin Robertson), The Geltrude Foundation (Gavin Robertson), The Penn State Melanoma and Skin Cancer Center (Raghavendra Gowda), Gilbert Memorial Fund (Raghavendra Gowda), The James Paul Sutton Medical Research Fund (Raghavendra Gowda), The Penn State Chocolate Tour Cancer Research Fund (Raghavendra Gowda & Gavin Robertson).

      Declaration of Competing Interest

      None

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