784 A new immune evasion mechanism in melanoma

Abstract

We present a novel and highly effective immune evasion mechanism in melanoma through the downregulation of man1a1, which codes for mannosidase I. This enzyme modifies glycosylation of cell-surface proteins. Using mass spectrometry, the glycocalyx of four human melanoma cell lines and normal human melanocytes (NHEM) were analyzed. All cancer lines expressed abnormal surface carbohydrates, specifically, an elevated ratio of high mannose sugars. RNA arrays of the human cancer cells showed downregulation of man1a1 compared to NHEM. To determine if this resulted in an altered glycocalyx, mouse melanoma cell line (B16) was treated with Kifunensine (Kif), a mannosidase I inhibitor. Kif-treated cells showed more high mannose structures, similar to human melanoma cell lines, compared to untreated cells. NK cells express receptors against various carbohydrates, providing targeting mechanisms for cell-mediated killing of tumor cells. Cytotoxicity assays using isolated mouse NK cells led to a 5-fold decrease in cell-mediated lysis of Kif-treated B16 cells compared to untreated cells. B16 cells overexpressing man1a1(B16+man1a1) were generated by stable transfection of a mammalian vector containing man1a1. The glycocalyx of these cells appeared more normal by mass spectrometry. Cytotoxicity assay of B16+man1a1 resulted in a 2-fold increase in cell lysis, compared to B16 transfected with empty vector. To test our hypothesis in vivo, a control mouse group and an experimental mouse group were subcutaneously and intravenously injected with either B16+man1a1 or B16 cells carrying empty vector, respectively. After two weeks, mouse skin and lungs were harvested. Compared to control mice, skin tumor volume was 10-fold smaller in mice injected with B16+man1a1 cells. There were 7-fold (p < 0.005) fewer metastatic colonies in lung parenchyma of mice injected intravenously with B16+man1a1 cells compared to control mice. Our results show glycosylation is a major factor in cancer immune evasion and/or suppression and can be reversed by overexpressing a single gene.