Abstract
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.
Highlights
Melanoma patients’ plasma contains exosomes produced by malignant and normal cells
Specificity of the immunocapture for melanoma exosomes was verified by showing that: (i) consistently, non-MTEX were CSPG4(−); only MTEX were CSPG4(+) (SFig. 2a,b); (ii) exosomes from healthy donors (HDs)’ plasma were negative for CSPG4 (SFig. 2c); (iii) only MTEX were highly enriched in melanoma-associated antigens (MAAs) (SFig. 4a); (iv) MTEX were CSPG4 (+) but CD3(−); only non-MTEX carried CD3 (SFig. 2d); (v) in spiking experiments, where melanoma exosomes were added to exosomes obtained from HDs (1:1), the captured fraction contained all CSPG4(+) exosomes, while the non-captured fraction was CSPG4(−)
Evidence suggested that extracellular vesicles (EVs) isolated from supernatants of murine or human melanoma cell lines were decorated by death ligands such as FasL, TRAIL or biologically-active TGF-β1, and directly or indirectly suppressed functions of various immune cell subsets[8,9,28,29,30,31,32]
Summary
Melanoma patients’ plasma contains exosomes produced by malignant and normal cells. Circulating melanoma cell-derived exosomes (MTEX) enriched in suppressive proteins might represent such an immunoinhibitory mechanism[8,9]. Melanoma cells produce more exosomes than normal melanocytes, and melanoma patients’ plasma contains increased levels of exosomes carrying melanoma-associated antigens (MAAs), immunosuppressive proteins (FasL, TGF-β) and oncoproteins, including Myc[8,9,12,13]. We recently reported a method for immune capture of melanoma-cell derived exosomes (MTEX) from patients’ plasma[17], and have successfully separated MTEX from normal cell-derived exosomes (non-MTEX)[17,18,19,20]. While MTEX carry an abundance of immunosuppressive proteins and inhibit functions of human primary immune cells ex vivo, non-MTEX stimulate immune cell activity. The phenotypic and functional characterization of MTEX emphasizes their potential role as biomarkers of melanoma progression
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