Abstract
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.
Highlights
Exosomes are small endosome-derived vesicles (50–100 nm in size), actively secreted through an exocytosis pathway normally used for receptor discharge and intercellular cross-talk [1,2,3]
Our results suggest that the Exotest for detection of plasma exosomes carrying tumorassociated antigens may represent a novel tool for clinical management of cancer patients
The ELISA we developed is based on the presence on exosome of proteins shared with cytoplasmic organelles such as endosomes and lysosomes (Rab-5b and CD63), whose membranes are not shed or recycled as for plasma membrane structures, excluding the possible presence of structures deriving from membranes shedding and disruption [23]
Summary
Exosomes are small endosome-derived vesicles (50–100 nm in size), actively secreted through an exocytosis pathway normally used for receptor discharge and intercellular cross-talk [1,2,3]. We and others have recently shown that exosomes secreted by human tumor cells of various origins are able to induce apoptosis in activated T cells, through the expression of death ligands (e.g. FasL, TRAIL) [9,10,13], inhibit NK functions [14,15] and promote the generation of myeloid-derived suppressor cells from normal monocytes [10] These data, together with the reproducible evidence that exosomes of likely tumor origin can be abundantly found in plasma and neoplastic effusions of cancer patients [16,17,18] support a role of tumor exosomes in molding host microenvironment to allow tumor growth and progression [19,20]. 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
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