High levels of exosomes expressing CD63 and caveolin-1 in plasma of melanoma patients.
BACKGROUND
Metastatic 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.


RESULTS
We 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
We 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.
Logozzi
Mariantonia
M
Unit of Antitumor Drugs, Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Rome, Italy.
De Milito
Angelo
A
Lugini
Luana
L
Borghi
Martina
M
Calabrò
Luana
L
Spada
Massimo
M
Perdicchio
Maurizio
M
Marino
Maria Lucia
ML
Federici
Cristina
C
Iessi
Elisabetta
E
Brambilla
Daria
D
Venturi
Giulietta
G
Lozupone
Francesco
F
Santinami
Mario
M
Huber
Veronica
V
Maio
Michele
M
Rivoltini
Licia
L
Fais
Stefano
S
D000818:Animals; D015703:Antigens, CD / Q000097:blood*; D051242:Caveolin 1 / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay; D055354:Exosomes; D006801:Humans; D008545:Melanoma / Q000097:blood*; D051379:Mice; D016513:Mice, SCID; D010980:Platelet Membrane Glycoproteins; D012680:Sensitivity and Specificity; D060149:Tetraspanin 30
D015703:Antigens, CD; C554586:CD63 protein, human; D051242:Caveolin 1; C554587:Cd63 protein, mouse; D010980:Platelet Membrane Glycoproteins; D060149:Tetraspanin 30