The role of exosomes in metastasis and progression of melanoma

Journal Pre-proofs Anti-tumour Treatment The Role of Exosomes in Metastasis and Progression of Melanoma Raghavendra Gowda, Bailey M. Robertson, Soumya Iyer, John Barry, Saketh S. Dinavahi, Gavin P. Robertson PII: DOI: Reference:

S0305-7372(20)30013-X https://doi.org/10.1016/j.ctrv.2020.101975 YCTRV 101975

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Cancer Treatment Reviews Cancer Treatment Reviews

Received Date: Revised Date: Accepted Date:

19 January 2019 16 January 2020 18 January 2020

Please cite this article as: Gowda, R., Robertson, B.M., Iyer, S., Barry, J., Dinavahi, S.S., Robertson, G.P., The Role of Exosomes in Metastasis and Progression of Melanoma, Cancer Treatment Reviews Cancer Treatment Reviews (2020), doi: https://doi.org/10.1016/j.ctrv.2020.101975

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The Role of Exosomes in Metastasis and Progression of Melanoma Raghavendra Gowda1, 5,6,7, Bailey M. Robertson1, Soumya Iyer1,5, John Barry1, Saketh S. Dinavahi1,5, and Gavin P. Robertson* 1,2,3,4,5,6,7.

Departments of 1 Pharmacology, 2 Pathology, 3 Dermatology and 4 Surgery, 5 The Penn State Melanoma and Skin Cancer Center, 6 Penn State Melanoma Therapeutics Program, 7 Foreman Foundation for Melanoma Research, The Pennsylvania State University College of Medicine, Hershey, PA 17033. *Corresponding Author: Gavin P. Robertson, Department of Pharmacology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033. Phone: (717) 531-8098; Fax: (717) 531-0480; E-mail: [email protected] 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). Running title: The Role of Exosomes in Melanoma. Conflict of Interest: None Key words: Exosome, Multivesicular bodies, Melanoma, Metastasis, Angiogenesis, drug delivery.

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.

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Introduction Melanoma is the most deadly form of skin cancer due to its highly metastatic nature [1]. The disease progresses in a stepwise manner as evolving tumor cells become invasive and leave the epithelium of the epidermis to enter adjacent tissues [2]. 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% [3]. Improved surveillance and early detection of metastatic melanoma using specific markers of initiation and progression are required to improve clinical intervention, and patient survival [4]. 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 from the cancer type they originate [5]. Exosomes have been known to carry the messenger proteins required for direct bone marrow– derived cells toward a prometastatic phenotype, and are hence called the “messengers of metastasis” [6]. Exosomes are extracellular vesicles, which play a vital role in the intercellular communication within the tumor microenvironment, metastasis and drug resistance [7, 8]. Exosomes can transport proteins, affect changes in signaling pathways, maturation and differentiation, as well as induce genetic change in cells [7, 8]. Exosomes have also been shown to be relatively stable and resist proteolytic and nuclease activity [9]. The proteins and nucleic acids enclosed within these vesicles are protected from degradation [9]. 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 [10]. They contain proteins, nucleic acids, and metabolites and can mediate intercellular communication to modulate various biological functions as illustrated in Figure 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 [11]. Size based classification consists of small EVs (sEVs) of <100200nm and medium/large EVs of >200nm [11]. 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 [11]. These EVs are currently isolated through a variety of ultracentrifugation 3

and filtering steps dependent on the downstream application of the isolated EVs [11]. In this review, we are focusing on sEVs which will be referred to as exosomes throughout the remainder of this review. 1. 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 [12]. After production, they diffuse into the local tissue through the lymphatic system and eventually enter the circulatory system [13]. The membrane composition of exosomes is enriched with endosome related membrane transport and fusion proteins such as Rab GTPases, flotillin, annexins and integrins [14]. These vesicles carry repertoires of nucleic acids, including mRNAs, miRNAs and other ncRNAs, as well as DNA [10, 15-18]. 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 (Figure 1) [10]. Several studies have provided evidence for ESCRT independent packaging of exosomes [19] [20, 21]. These pathways depend on lipids such as sphingosine 1 phosphate (S1P) and tetraspanin-enriched microdomains [20, 22]. Pre-melanosomal protein -Pmel17 appears to be independent of the ESCRT pathway [23]. The biogenesis and tracking of exosomes is altered in cancer and reports have shown enhanced secretion of exosomes by the cells undergoing unconditional stress [24, 25] [26, 27]. Cancer cells undergo stress due to uncontrolled growth and cell damage when treated with chemotherapeutics [28]. For example, in melanoma, p53 activation regulates the transcription of the TSAP6 gene, which, in turn, increases the exosome production [26, 29]. Similarly, increased expression of heparanse enzymes, Rab 27a and Rab 27b facilitates the secretion of exosomes in melanoma and other cancer types [30]. 2. 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 [31]. The heterogeneity of exosomal contents is due to selective sorting of exosomes from the donor cells [32]. Exosomes played a role 4

in cellular debris disposal along with a significant role in long distance cellular interactions [33]. Exosomes similar to EVs may also contain RNA, membrane proteins, cytosolic proteins, and raftlike structures in the exosome membrane [34]. 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 as 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 [35, 36]. 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 [37]. Current data regarding membrane characteristics, RNA, and protein profiles of melanoma exosomes is discussed next. 2.1 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 [38]. Sphingomyelin, lyso-phophatidyethanolamine, lysophosphatidylcholine and cholesterol enriched raft-like domains are present on most cancer exosomes including those from melanoma cells [39]. 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 [40]. There are also multiple associated proteins, many of which are cell adhesion molecules [16, 41]. Although the lipid composition and raft-like structures are expected to affect the physiological response of exosomes such as circulation time, uptake, and targeting [42], 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. 2.2 RNA in melanoma exosomes RNA presence was not widely considered in exosomes until 2007 by a study which looked at exosomes from mast cells [43]. 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 5

that exosomes function in the transport of tumor promoting RNA [43]. 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 [44, 45]. For example, circulating exosomal microRNA levels increased in ovarian and lung cancer while normal controls did not have detectable microRNA levels, indicating an increase in exosomal miRNA [46, 47]. The enhanced levels of let-7a, miR-182, miR-221, miR-222, miR-31, miR-19b-2, miR-20b and miR-92a2, 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 [48]. There was also other differential expressions 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 [48]. 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 [49, 50]. 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; their involvement and approaches to influence the balance of tumor promoting vs. tumor suppressing roles. 2.3 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 [10, 51]. Various proteins have been identified in melanoma exosomes, several of which are involved in melanoma progression and metastasis [52]. Not surprisingly, the melanoma exosome protein profile differs from that of melanocytes [48]. 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 [53]. 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 6

[39, 54, 55]. 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 [56, 57]. Annexin A2 and other annexins are known to play a role in tumor development and progression [58]. Recently, annexin A2 was shown to form a tetramer with S100 proteins (a biomarker for melanoma on the cell surface) [59-61]. This tetramer promotes ceramide-1-phosphate dependent vascular endothelial cell invasion [62]. It would be interesting to see if melanoma 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 [52]. 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 [63]. Ribosomal protein mutations suppress the activity of the AKT pathway thereby resulting in proteasomal degradation of p53 [64]. 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 [65]. It is likely that several different proteins regulate this, and future research should be focused on understanding the functional aspects of these proteins. Collectively, the functional role of exosomal proteins illustrates the potentially important role in melanoma progression and metastasis. Furthermore, these studies suggest that it is important to regulate the secreted exosomes and the possibility of using them as a potential drug delivery vehicle [66]. More recently, Jang et al. discovered mitochondrial protein present in human melanoma vesicles isolated from patient plasma [67]. In this study, patient tissue from both melanoma lymph node and skin metastases was taken and EVs were isolated by preserving in serum free media [67]. Of the proteins isolated from the EVs in this study, 23.6% are distinct from those found in current EV databases [67]. 3. Exosomes as mediators of metastasis 7

Exosomes can play an important role in metastatic progression [68]. The ability of exosomes to carry proteins and nucleic acids enable them to modulate both the primary tumor and remote tissue to promote metastasis [69]. 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 [70]. 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, [71, 72]. 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 [73]. 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 [74]. Additionally, exosomes can direct or inhibit cancer fibroblast differentiation eventually leading to angiogenesis and tumor growth [75, 76]. 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 [37]. 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 [8]. 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 [52]. 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 [52]. 3.1 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 [77]. 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 [78]. 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 [79].

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Melanoma exosomes are also capable of transferring metastatic potential to bone marrow progenitor cells [8]. 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 [8]. One of the other enriched proteins in melanoma exosomes include Rab27a, which also promotes metastasis [8]. 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 [8]. Mannavola et al. also demonstrated the potential of osteotropic melanoma derived exosomes to stimulate metastatic migration to bone in nonopteotropic melanoma lines [40]. 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 [40]. Treatment of mice with melanoma exosomes can increase vascular permeability in the lungs, paving the way for metastasis [8]. Melanoma derived exosomes activated the expression of MAPK signaling molecules like MAP3K4, MAP2K5, and MAPK13 in primary melanocytes to promote a metastatic phenotype [80]. The MAPK pathway is known for the induction of EMT in melanoma and other cancer types [81, 82]. 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 [8]. Schematic representations of the events responsible for metastasis that are regulated by exosomes are illustrated in Figure 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 [83]. 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 TNFα, which play a major role in the promotion of metastasis [84]. Furthermore, in situations where stress is applied, such as cryostasis, heat, and oxidative stress, melanoma cells produce a greater 9

number of exosomes when compared to unstressed cells [85]. 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 [86]. 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 [87]. 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 proangiogenic response in the HUVEC line discussed in the section below detailing the exosomal effect on angiogenesis [9]. Melanoma derived exosomes were also internalized in MSCs with 91% efficacy, increased PD-1 expression and elevated metastasis in the murine model [88]. 3.2 Invasive potential increased by melanoma exosomes An effect related to the metastatic increases in response to exosome treatment is an observed increase in invasive potential [89]. 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 [9]. Similarly, there exists a positive correlation between the quantity of exosomes, the amount of lytic enzymes and invasive capabilities [84, 90]. Xiao et al. demonstrated that melanoma exosome treatment of normal melanocytes resulted in a significant increase in invasiveness [48]. 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 [39]. Hu et al. also demonstrated that melanoma derived exosomes delivering GM26809 are capable of reprogramming murine fibroblasts into cancer associated fibroblasts [91]. 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 10

migration of MCF-7, a breast cancer cell line, by stimulating the Wnt/β-catenin signaling pathway [90]. In addition, other 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 [80]. 3.3 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 [92]. A study by Hood et al. showed that exosomes promote angiogenesis by inducing the formation of endothelial spheroids [74]. 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 [93]. 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 [94]. Similarly, melanoma cells 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 [33]. Exosome induced angiogenesis is not an event tied solely to melanoma but has also been reported in myelogenous leukemia and other cancers [87, 95]. Under hypoxia conditions, exosomes released from cancer cells can mediate the angiogenic process [94]. 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 [94]. Exosomes derived from metastatic breast cancer cells also contained multiple miRNAs, like miRNA-210, which play a vital role in angiogenesis [96]. 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. 3.4 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 [97-99]. 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 [97]. Gene expression in the melanoma cells was investigated to determine the possible role of exosomes in melanoma metastasis to the lymph nodes [100] [97]. 11

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. 3.5 Effects on cell differentiation mediated by melanoma exosomes Exosomes have also been shown to affect cell differentiation [101]. Cancer derived exosomes have also been shown to facilitate fibroblast differentiation into carcinoma associated fibroblasts (also known as myofibroblasts) [7, 102]. Myofibroblasts are rich in an altered tumor stroma that facilitates tumor growth, angiogenesis, and metastasis [103]. Melanoma exosomes decrease differentiation of bone marrow-derived dendritic cells [104]. Umbilical cord stem cells were shown to differentiate into carcinoma-associated fibroblasts when exposed to gastric cancer exosomes [105]. 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. 4. Exosome mediated regulation of the immune system in melanoma patients. Accumulating evidence suggests that exosomes regulate the immune system [106, 107]. Dendritic cell derived exosomes (DEXs) can induce T-cell stimulation when compared with immature DCs [108]. Similarly, DEXs promote CD8+ T cell activation, which also appears to correspond with DEXs interacting with dendritic cells [108]. 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) [109]. Further, melanoma exosomes can induce pro-tumor macrophage polarization and activation [110] along with altering the transcriptome of CD8+ T-cells by modulating mitochondrial respiration [111]. Melanoma exosomes also directly activate CD4+ T-cells through the expression and release of miR690 and Rab27a [112]. 12

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 [113]. 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 [114]. 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 [114]. 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 immuno-interventions [115]. Importantly, these studies paved the way for the isolation of clinical grade exosomes from the dendritic cells [115]. 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 [116]. 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 [116]. 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. 5. Exosomes as prognostic indicators of melanoma The protein and nucleic acid content within exosomes are defined by the melanoma cells producing them [117]. For this reason, scientists have studied the dysregulation of exosome production in melanoma and how this leads to cancer growth and dissemination [27, 118]. Studies are also exploring the prognostic value of exosomes in cancers [119-121]. Since exosomes are present in the circulation, the next section explores the utility of exosomes for prognostic and diagnostic purposes [122].

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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 [123]. 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 [8]. Furthermore, several studies reported increased levels of tetraspanins in the exosomes of melanoma patients suggesting their potential use as biomarkers [124, 125]. Specifically, the exosomes from melanoma patients have higher levels of plasma CD63+ compared to healthy controls [124]. 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 [125]. Extracellular vesicles purified from lymphatic drainage of melanoma tumors are also enriched in proteins present in melanoma progression [126]. For example, V600EBRAF mutation can be detected in these extracellular vesicles and its presence correlated with patients at risk of relapse [126]. Exosomal levels of 11 genes that are enriched in acidic conditions also significantly correlated with poor prognosis and ability to metastasize [127]. Furthermore, acidic environment induces an abundant release and intra-tumoral uptake of exosomes, which may prove a signature of melanoma progression [127]. 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 [128]. 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 [128]. 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 [129]. Another trial is monitoring the level of exosomes in the circulation to determine if it could be utilized clinically in colorectal cancer 14

as high levels of exosomes correlated with shorter survival and poor prognosis [130]. 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. 6. 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 [131, 132]. 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 [133135]. In contrast, an exosome has membrane proteins, nucleic acids, and other cellular components, including cytoskeletal proteins that alter the properties of the exosome [42, 66]. 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 [136]. 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 [136]. By modulating the membrane composition, they can selectively target certain cells. Exosomes can be loaded with therapeutic cargos using electroporation [137], chemical based transfection using Lipofectamine 2000 [138], incubation of exosomes with a particular cargo [139], transfection of exosome producing cells and cell activation [140]. Also, methods have been developed to load both lipophilic and hydrophilic substances into exosomes [141]. The methods for using exosomes as targeted delivery vehicles are illustrated in Figure 3. This emerging area will likely see extensive development in the near future. 6.1 Exosomal delivery of RNA Exosomes have been studied as delivery vehicles for RNA [142]. Therapeutic efficacy of exosomes loaded with siRNA can lead to selective gene silencing of the MAPK-1 gene [143]. Similar methodology was used to induce knockdown of RAD51-, RAD52- and BACE protein levels in cancer cells to decrease cell viability [138]. Exosome-delivered tumor suppressor miRNAs, miR143 and let- 7a, let-7b inhibited the growth of skin, prostate and breast cancer cells, respectively [44, 144, 145]. For neurodegenerative diseases, siRNA loaded into exosomes has mediated knockdown of target genes in neural cell types for treatment of these conditions [146]. Cells were engineered to express Lamp2b, an exosomal membrane protein, fused with homing peptides to facilitate active targeting 15

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 [146]. 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 [147]. 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 [148]. Therefore, exosomes can be both synthetic and natural RNA and could potentially be used to gain therapeutic advantage. 6.2 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 [137, 149]. 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 [150-153]. Exosomes loaded with the anti-inflammatory drug curcumin showed increased bioavailability and the formulation-protected mice from LPS (lipopolysaccharide) induced shock [154]. These particles have also been used for the delivery of doxorubicin and paclitaxel [137, 149]. 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 [137]. 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 bloodbrain barrier [149]. 6.3 Exosomal delivery of proteins Exosomes naturally function in the transport and transfer of proteins and are capable of inducing physiological responses [155]. 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 [156]. 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. 16

7. 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 [157]. 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 [157]. 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 [116, 158]. Exosomes can function as therapeutic cell-free vaccines for the treatment of various cancers [116]. Clinical trials have been completed using dendritic cell-based exosomes (Dex) injected intradermally [116]. 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 [116]. 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 [158]. 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) [158]. 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 [159]. Seven out of 22 (32%) patients experienced stable disease for at least 4 months [159]. 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 [159]. 8. Needed areas for future melanoma exosome research Exosomes have been called the “messengers of metastasis” [6]. Exosomes play a critical role in the metastatic process from invasion to preparation of the metastatic niche [89]. 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.

17

Figure legends. Figure 1. Biogenesis 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. Figure 2. Melanoma-derived exosomes are messengers for metastatic invasion. Melanomaderived 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. Figure 3. Methods 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.

18

Table1: Clinical trials using exosomes for cancer treatment Trial

Loaded agent

Indication/Effects

Disease type

References

Exosomes with antisense molecule

IGF-1R/AS ODN

Activation of immune system

Glioma of Brain

NCT01550523

Plasma derived exosomes

____

Prognostic biomarker

Cutaneous ulcers

NCT02565264

Exosomes comparing drug sensitive vs resistant patients

____

Prognostic markers

Melanoma

NCT02310451

Exosomes from blood and tissue

____

Prognostic markers

Pancreatic cancer

NCT02393703

Grape extract

Chemotherapy and radiotherapy

Head and Neck cancer

NCT01668849

Effect on immune response on the patient

Melanoma stage III/IV and Non-small cell lung cancer III/IV

[116, 158]

Immunomodulatory responses

Unrespectable Nonsmall lung cancer

NCT01159288

Cancer treatment

Pancreatic cancer

NCT03608631

Grape derived exosomes

Patients’ Whitcomb, (loaded with MAGE3)

Chemotherapeutic agent metronomic cyclophosphamide with Patient’s dexosomes Mesenchymal Stromal Cells-derived Exosomes with KRAS G12D siRNA

MAGE3

metronomic cyclophosphamide

siRNA

Curcumin loaded exosomes

Curcumin

Cancer treatment

Colorectal cancer

NCT01294072

Patient’s exosomex combined with GM-CSF

GM-CSF

Specific CD8+ response

Colorectal cancer

[160]

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Review article highlights 1.

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.

2.

Exosomes are become attractive new biomarkers for the diagnosis and prognosis of cancer.

3.

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.

4.

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.

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