BackgroundMetastatic melanoma is an untreatable cancer lacking reliable and non-invasive markers of disease progression. Exosomes are small vesicles secreted by normal as well as tumor cells. Human tumor-derived exosomes are involved in malignant progression and we evaluated the presence of exosomes in plasma of melanoma patients as a potential tool for cancer screening and follow-up.Methodology/Principal FindingsWe designed an in-house sandwich ELISA (Exotest) to capture and quantify exosomes in plasma based on expression of housekeeping proteins (CD63 and Rab-5b) and a tumor-associated marker (caveolin-1). Western blot and flow cytometry analysis of exosomes were used to confirm the Exotest-based findings. The Exotest allowed sensitive detection and quantification of exosomes purified from human tumor cell culture supernatants and plasma from SCID mice engrafted with human melanoma. Plasma levels of exosomes in melanoma-engrafted SCID mice correlated to tumor size. We evaluated the levels of plasma exosomes expressing CD63 and caveolin-1 in melanoma patients (n = 90) and healthy donors (n = 58). Consistently, plasma exosomes expressing CD63 (504±315) or caveolin-1 (619±310) were significantly increased in melanoma patients as compared to healthy donors (223±125 and 228±102, respectively). While the Exotest for CD63+ plasma exosomes had limited sensitivity (43%) the Exotest for detection of caveolin-1+ plasma exosomes showed a higher sensitivity (68%). Moreover, caveolin-1+ plasma exosomes were significantly increased with respect to CD63+ exosomes in the patients group.Conclusions/SignificanceWe describe a new non-invasive assay allowing detection and quantification of human exosomes in plasma of melanoma patients. Our results suggest that the Exotest for detection of plasma exosomes carrying tumor-associated antigens may represent a novel tool for clinical management of cancer patients.

The existence of lymphocytes within melanoma deposits that, when isolated, are capable ofrecognizing specific tumor antigens on autologous and allogeneic melanomas in a major histocompatibility complex (MHC) restricted fashion provides strong evidence that an immune response to cancer exists in humans (1-8). The ability of these tumor-infiltrating lymphocytes (TILs) to mediate the regression ofcancer when adoptively transferred into patients with metastatic melanoma attests to the clinical importance of the antigens recognized (9, 10). Characterization of these antigens may thus be important for development of strategies for cancer immunotherapy. ) have characterized two genes coding for melanoma antigens, MAGE-1 and tyrosinase, by using T-cell clones established from the peripheral blood of patients who were repetitively immunized in vivo with mutagenized tumor cells or whose peripheral blood lymphocytes were sensitized by repetitive in vitro stimulation with tumor. The strategy used in the present study attempted to identify the genes coding for tumor antigens that were recognized by TILs from tumor-bearing patients in the absence of immunization to enhance the possibility that genes would be identified that coded for antigens involved in the natural immune response against the growing cancer. Anti-melanoma T cells appear to be enriched in TILs probably as a consequence of clonal expansion and accumulation at the tumor site in vivo (14). We have now cloned and sequenced a gene coding for a shared, commonly expressed melanoma antigen, restricted by HLA-A2.1. § MATERIALS AND METHODS Generation of Cytotoxic T Lymphocytes (CTLs) and Culture of Cell Lines. CTLs were generated from excised tumor specimens by culturing a suspension of cells with interleukin 2 (IL-2) (6000 units/ml) (Chiron) for

CD14+HLA-DR-/lo cells exerting TGF-beta-mediated immune suppression represent a new subset of MSC potentially expandable by the administration of GM-CSF-based vaccines in metastatic melanoma patients.

The hypothesis that FasL expression by tumor cells may impair the in vivo efficacy of antitumor immune responses, through a mechanism known as ‘Fas tumor counterattack,’ has been recently questioned, becoming the object of an intense debate based on conflicting results. Here we definitely show that FasL is indeed detectable in the cytoplasm of melanoma cells and its expression is confined to multivesicular bodies that contain melanosomes. In these structures FasL colocalizes with both melanosomal (i.e., gp100) and lysosomal (i.e., CD63) antigens. Isolated melanosomes express FasL, as detected by Western blot and cytofluorimetry, and they can exert Fas-mediated apoptosis in Jurkat cells. We additionally show that melanosome-containing multivesicular bodies degranulate extracellularly and release FasL-bearing microvesicles, that coexpress both gp100 and CD63 and retain their functional activity in triggering Fas-dependent apoptosis of lymphoid cells. Hence our data provide evidence for a novel mechanism potentially operating in Fas tumor counterattack through the secretion of subcellular particles expressing functional FasL. Such vesicles may form a sort of front line hindering lymphocytes and other immunocompetent cells from entering neoplastic lesions and exert their antitumor activity.