Melanoma Cell-derived Exosomes Research Articles

Immunosuppressive functions of melanoma cell-derived exosomes in plasma of melanoma patients.

Tumor-derived exosomes (TEX) are a subset of small extracellular vesicles (sEV) present in all body fluids of patients with cancer. In plasma of patients with metastatic melanoma, numbers of exosomes produced by melanoma cells called MTEX are elevated. To study the role of MTEX in melanoma progression, immunoaffinity-based separation of MTEX from total plasma exosomes was performed. The surface of MTEX was decorated by various checkpoint inhibitory proteins, and upon coincubation with immune recipient cells, MTEX suppressed anti-tumor functions of these cells. MTEX emerge as a major mechanism of immune suppression in melanoma and thus might play a role in promoting melanoma progression.

The Role of Melanoma Cell-Derived Exosomes (MTEX) and Photodynamic Therapy (PDT) within a Tumor Microenvironment

Photodynamic Therapy (PDT), an unconventional cancer therapy with optimistic desirable effects, utilizes the delivery of a photosensitizer (PS) that is activated by light at a particular wavelength and inducing oxidative cytotoxic damage of a tumor and its surrounding vasculature. Deeper seated tumors such as internally metastasized melanomas are more difficult to treat with PDT as the penetration of laser light to those sites is less. Limitations in targeting melanomas can also be attributed to melanin pigments that hinder laser light from reaching targeted sites. Exosomes serve as naturally occurring nanoparticles that can be re-assembled with PSs, improving targeted cellular absorption of photosensitizing agents during PDT. Additionally, studies indicate that exosomes released from PDT-treated tumor cells play a critical role in mediating anti-tumor immune responses. This review collates the role of Melanoma Cell-Derived Exosomes (MTEX) in immune response mediation and metastasis. Tumor Cell-Derived Exosomes (TEX) post PDT treatment are also reviewed, as well as the effects of exosomes as carriers of photosensitizers and delivery systems for PDT. The understanding and research on the role of melanoma exosomes induced by Photodynamic Therapy and their tumor microenvironment will assist in future research in treatment prospects and implications.

Proteomic profiles of melanoma cell-derived exosomes in plasma: discovery of potential biomarkers of melanoma progression.

Cancer liquid biopsy encompassing circulating tumor cells (CTC), circulating tumor DNA (ctDNA) and/or tumor-derived exosomes (TEX) emerges as a novel approach to early detection, noninvasive monitoring of responses to therapy and predicting patient survival. TEX are a key component of liquid biopsy because they mimic tumor cells in their proteomic and genetic content. Two recent proteomic analyses of TEX released into plasma by melanoma cells confirms the potential of TEX as diagnostic and prognostic markers in melanoma.

Immunoaffinity-Based Isolation of Melanoma Cell-Derived and T Cell-Derived Exosomes from Plasma of Melanoma Patients.

Tumor-derived exosomes (TEX), a subset of small extracellular vesicles (EVs) which originate from the endocytic compartment of tumor cells, are emerging as key players in cancer progression. TEX circulate freely in patients' body fluids and transfer bioactive cargos from tumor to various recipient cells. The molecular cargo of melanoma cell-derived exosomes (MTEX) mimics that of the tumor, and MTEX serve as a liquid biopsy that provides potentially useful information for cancer diagnosis, prognosis, or responses to therapy. Plasma of melanoma patients contains a mix of MTEX and exosomes produced by nonmalignant cells (NMTEX). Isolation of these exosome subtypes from the bulk of plasma exosomes is necessary to evaluate contributions of each as potential biomarkers of melanoma progression and outcome. Here, methods for separation of MTEX from T cell-derived exosomes from a single small volume of plasma and their subsequent molecular and functional characterization are described. Following size exclusion chromatography (SEC) to isolate total plasma exosomes, immune affinity-based capture of MTEX with anti-CSPG4 antibody and then of exosomes produced by T cells with anti-CD3 antibody is used to sequentially isolate the two subsets. This immune capture method enables the recovery of MTEX and CD3+ exosomes in quantities sufficient both for molecular profiling by flow cytometry or western blotting and for functional analyses.

Abstract B19: Suppression of CD8+ T-cell functions by melanoma cell-derived exosomes captured from plasma of patients with melanoma

Abstract Exosomes, virus-sized (30-150nm) vesicles, derived from the endocytic compartment of parent cells, are produced by all cells and are present in all body fluids. Melanoma patients’ plasma contains exosomes produced by normal and malignant cells. To study the impact of melanoma cell-derived exosomes (MTEX) on human immune cells and melanoma progression, we isolated MTEX from total exosomes in plasma by immune capture with anti-CSPG-4 mAbs on beads. 19 proteins carried by MTEX and by non-MTEX of 12 patients with metastatic melanoma were quantified by on-bead flowcytometry. Immunoregulatory functions of these exosomes were measured in co-incubation assays with human immune cell subsets. Plasma exosomes of 6 normal donors served as controls. Melanoma-associated antigens (MAA) were present on MTEX only. MTEX carried more inhibitory ligands than non-MTEX (FasL, p<0.007; TRAIL, p<0.02), inhibited CD69 expression (p<0.002) on CD8+ T cells, suppressed T-cell proliferation (p<0.0005), and induced apoptosis of CD8+ T cells (p< 0.0005). MTEX-mediated suppression accounted for suppressive activities of total plasma exosomes. Although non-MTEX were enriched in immunostimulatory proteins (p<0.002), they induced greater suppression of NKG2D on NK cells than MTEX (p<0.001). This is the first study to show that MTEX, a subset of circulating exosomes derived from melanoma cells, are largely responsible for immuno-suppressive effects that potentially translate into systemic immune suppression and tumor promotion in melanoma patients. Citation Format: Priyanka Sharma, Brenda Diergaarde, Soldano Ferrone, John M Kirkwood, Theresa L. Whiteside. Suppression of CD8+ T-cell functions by melanoma cell-derived exosomes captured from plasma of patients with melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B19.

Signaling of Tumor-Derived sEV Impacts Melanoma Progression.

Small extracellular vesicles (sEV or exosomes) are nanovesicles (30–150 nm) released both in vivo and in vitro by most cell types. Tumor cells produce sEV called TEX and disperse them throughout all body fluids. TEX contain a cargo of proteins, lipids, and RNA that is similar but not identical to that of the “parent” producer cell (i.e., the cargo of exosomes released by melanoma cells is similar but not identical to exosomes released by melanocytes), possibly due to selective endosomal packaging. TEX and their role in cancer biology have been intensively investigated largely due to the possibility that TEX might serve as key component of a “liquid tumor biopsy.” TEX are also involved in the crosstalk between cancer and immune cells and play a key role in the suppression of anti-tumor immune responses, thus contributing to the tumor progression. Most of the available information about the TEX molecular composition and functions has been gained using sEV isolated from supernatants of cancer cell lines. However, newer data linking plasma levels of TEX with cancer progression have focused attention on TEX in the patients’ peripheral circulation as potential biomarkers of cancer diagnosis, development, activity, and response to therapy. Here, we consider the molecular cargo and functions of TEX as potential biomarkers of one of the most fatal malignancies—melanoma. Studies of TEX in plasma of patients with melanoma offer the possibility of an in-depth understanding of the melanoma biology and response to immune therapies. This review features melanoma cell-derived exosomes (MTEX) with special emphasis on exosome-mediated signaling between melanoma cells and the host immune system.

Targeting CSPG4 for isolation of melanoma cell-derived exosomes from body fluids.

This manuscript describes the functional properties of the exosomes released from melanoma cells. It details the characteristics of the tumor antigen chondroitin sulfate proteoglycan4 (CSPG4), which is used as amarker to separate exosomes released by melanoma cells from exosomes released by nonmalignant cells. The results are discussed in view of the potential role of melanoma cell-derived exosomes in the escape of malignant cells from the host's immune system.

Melanoma cell-derived exosomes in plasma of melanoma patients suppress functions of immune effector cells

Melanoma patients’ plasma contains exosomes produced by malignant and normal cells. Plasma exosomes were isolated and separated by immunocapture into two fractions: melanoma cell-derived exosomes (MTEX) and normal cell-derived exosomes (non-MTEX). Immunosuppressive effects of MTEX on primary human immune cells were evaluated. Exosomes were isolated from plasma of 12 melanoma patients and six healthy donors (HDs). Expression levels of 19 immunoregulatory proteins in MTEX, non-MTEX and HDs exosomes were evaluated by on-bead flow cytometry. Functional/phenotypic changes induced in CD8+ T or natural killer (NK) cells by MTEX or non-MTEX were compared. Plasma protein levels were higher in patients than HDs (P < 0.0009). In patients, MTEX accounted for 23–66% of total exosomes. MTEX were enriched in immunosuppressive proteins (P = 0.03). MTEX, but not HDs exosomes, inhibited CD69 expression (P ≤ 0.0008), induced apoptosis (P ≤ 0.0009) and suppressed proliferation (P ≤ 0.002) in CD8+ T cells and downregulated NKG2D expression in NK cells (P = 0.001). Non-MTEX were enriched in immunostimulatory proteins (P = 0.002) and were only weakly immunosuppressive. Elevated MTEX/total exosome ratios and, surprisingly, non-MTEX ability to induce apoptosis of CD8+ T cells emerged as positive correlates of disease stage. MTEX emerge as the major mechanism of tumor-induced immune suppression and as an underestimated barrier to successful melanoma immunotherapy.

Abstract 3091: Effects of melanoma cell-derived exosomes in melanoma patients’ plasma on immune cell functions

Abstract Background: Exosomes are a subset of small (30-150nm) extracellular vesicles (EVs) of the endocytic origin that mediate intercellular communication in health and disease. Exosomes are actively produced by tumor cells, circulate freely in body fluids and carry messages that alter functions of normal cells in the tumor microenvironment (TME), including immune cell functions. The ability of melanoma exosomes to reprogram immune cell functions might contribute to disease progression. Methods: To evaluate effects of melanoma cell-derived exosomes (MTEX) on human immune cell subsets, we isolated exosomes from plasma of 12 patients with melanoma and separated MTEX from non-MTEX by immune capture using anti-CSPG4 mAb on beads. These exosome fractions, and exosomes from plasma of 6 normal donors used as controls (NC), were studied by on-bead flow cytometry to quantify their surface protein profiles and in co-incubation assays with immune cell subsets for functional attributes. Phenotypic and functional profiles of MTEX and non-MTEX were compared. Results &amp; Conclusion: MTEX were enriched (p&amp;lt;0.005)( in FasL and TRAIL, induced apoptosis of CD8+T cells, down-regulated CD69 expression levels on T cells and inhibited their proliferation (all p&amp;lt;0.0005). MTEX also interfered with NK cell functions (p&amp;lt;0.001). Non-MTEX were enriched (P&amp;lt;0.005) in co-stimulatory proteins, CD40L, OX40L, OX40 and, similar to exosomes of NC, were not immunosuppressive. Thus, MTEX, present in excess in plasma of patients with metastatic melanoma, mediate immune suppression and contribute to immune escape of the tumor. Citation Format: Priyanka Sharma, Brenda Diergaarde, Soldano Ferrone, John M. Kirkwood, Theresa L. Whiteside. Effects of melanoma cell-derived exosomes in melanoma patients’ plasma on immune cell functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3091.

Immunoaffinity-based isolation of melanoma cell-derived exosomes from plasma of patients with melanoma

ABSTRACTTumour-derived exosomes (TEX) are a subset of extracellular vesicles (EVs) present in body fluids of patients with cancer. The role of this exosome subset in melanoma progression has been of interest ever since ex vivo studies of exosomes produced by melanoma cell lines were shown to suppress anti-melanoma immune responses. To study the impact of melanoma-derived exosomes (MTEX) present in patients’ plasma on melanoma progression, isolation of MTEX from total plasma exosomes is necessary. We have developed an immunoaffinity-based method for MTEX capture from plasma of melanoma patients. Using mAb 763.74 specific for the CSPG4 epitope uniquely expressed on melanoma cells, we separated MTEX from non-tumour cell-derived exosomes and evaluated the protein cargo of both fractions by quantitative flow cytometry. Melanoma-associated antigens were carried by MTEX but were not detectable in exosomes produced by normal cells. Separation of plasma-derived MTEX from non-MTEX provides an opportunity for future evaluation of MTEX as potential biomarkers of melanoma progression and as surrogates of melanoma in tumour liquid biopsy studies.

Abstract 3673: High-density lipoprotein-like nanoparticles target SR-B1 and inhibit the cellular uptake of melanoma-cell derived exosomes

Abstract Exosomes play a crucial role in the progression of cancer through the transport of a variety molecular cargo, including proteins, lipids, and nucleic acids, to and from cells as a means of intercellular communication. Unraveling mechanisms of exosome-cell interactions may open avenues for studying cellular communication and lead to new therapies. Cellular exosome uptake depends on cholesterol-rich membrane microdomains called lipid rafts. Non-specific depletion of lipid raft cholesterol reduces cellular exosome uptake; however, to our knowledge, no targeted mechanism of inhibiting cellular exosome uptake has been reported. Scavenger receptor type B-1 (SR-B1) localizes to lipid rafts, and is a high-affinity receptor for cholesterol-rich high-density lipoproteins (HDL). SR-B1 is an intriguing therapeutic target because it is upregulated in many different cancers due to the high need for cholesterol of rapidly dividing cancer cells. Therefore, we hypothesized that specific targeting of SR-B1 and modulation of cholesterol flux through this receptor with biomimetic HDL-like nanoparticles (HDL NPs) would disrupt cellular exosome uptake. As a model, we explored exosomes derived from melanoma cells as they have been shown to promote angiogenesis and immunosuppression both crucial events in melanoma progression. Melanoma exosomes have also been shown to actively prepare metastatic sites, creating a suitable microenvironment allowing for the development of metastasis. Because of this, targeting exosomes and intercellular signaling could be beneficial for the treatment of metastatic melanoma. Using a variety of techniques including confocal microscopy, flow cytometry and automated image analysis, data demonstrate that HDL NPs specifically target SR-B1 in lipid rafts in melanoma cells and modulate cholesterol flux through this receptor. This leads to a clustering of SR-B1 at the cell membrane and potent inhibition of the cellular uptake of melanoma cell-derived exosomes. Citation Format: Michael P. Plebanek, Alexandre Matov, Kannan Mautharasan, Jesse Gatlin, C. Shad Thaxton. High-density lipoprotein-like nanoparticles target SR-B1 and inhibit the cellular uptake of melanoma-cell derived exosomes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3673. doi:10.1158/1538-7445.AM2015-3673

Melanoma cell-derived exosomes alter macrophage and dendritic cell functions in vitro

To clarify controversies in the literature of the field, we have purified and characterized B16F1 melanoma cell derived exosomes (mcd-exosomes) then we attempted to dissect their immunological activities. We tested how mcd-exosomes influence CD4+ T cell proliferation induced by bone marrow derived dendritic cells; we quantified NF-κB activation in mature macrophages stimulated with mcd-exosomes, and we compared the cytokine profile of LPS-stimulated, IL-4 induced, and mcd-exosome treated macrophages. We observed that mcd-exosomes helped the maturation of dendritic cells, enhancing T cell proliferation induced by the treated dendritic cells. The exosomes also activated macrophages, as measured by NF-κB activation. The cytokine and chemokine profile of macrophages treated with tumor cell derived exosomes showed marked differences from those induced by either LPS or IL-4, and it suggested that exosomes may play a role in the tumor progression and metastasis formation through supporting tumor immune escape mechanisms.