Consequences of NeuGc expression in mice melanoma tumor biology and their usefulness as vaccine target

MARIANO R GABRI

La Habana

Workshop; 9th International Workshop immunotherapy 2010: rupture and restoration of self tolerance; 2010

Centro de Inmunología Molecular

The most common sialic acids in mammals are N-acetylneuraminic acid and N-glycolylneuraminic acid, usually found as terminal constituents of different membrane glycoconjugates such as the GM3 ganglioside. The only structural difference between them consists in a single oxygen atom at the C-5 position of N-glycolylneuraminic acid, catalyzed by the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). In humans, NeuGc is a proposed tumor specific antigen in breast carcinoma and melanoma, being an accepted target for tumor vaccines such as the NeuGcGM3/VSSP. This vaccine was obtained by hydrophobic conjugation of NeuGcGM3 ganglioside with outer membrane proteins from N. Meningitidis to form very small size proteoliposomes. Up till now, there are no murine melanoma models expressing NeuGcGM3 able to be used for preclinical evaluation of NeuGcGM3-targeted cancer vaccines. While most of mice somatic cells express the CMAH enzyme, this expression in malignant mice cells seems to be silenced. To develop a NeuGc-positive murine melanoma model, we isolated and amplified the CMAH sequence from normal mouse liver and transfected it into B16 cells (B16-H). B16-H cells were cultured in standard DMEM medium with 10% fetal calf serum with 1:6 split for subculture. Transfection resulted in a positive expression of CMAH mRNA and NeuGc antigen in tumor cells. Interestingly, although B16-H cells were able to develop solid tumors when injected in syngeneic mice, it was needed to challenge mice with four-times more B16-H than B16 cells to obtain the same tumor incidence, showing a lower tumorigenicity. Using this B16-H model, antitumor activity of NeuGcGM3/VSSP vaccine (kindly provided by the Center of Molecular Immunology, La Habana, Cuba) was assessed. Mice were inoculated with 2x104 cells in the subcutis of the flank. Immunization with 4 intramuscular doses of 200ug at 2-week intervals significantly reduced tumor incidence and prolonged mice survival in B16-H group. No vaccine effects were obtained in animals bearing control B16 tumors. In B16-H, we observed that NeuGc expression level is passage-dependent, levels falling in relation with the time in culture. After around 30 passages, NeuGc expression decreases. Therefore, when using aged B16-H cells, vaccine antitumor activity was ineffective and showed no differences with the control group. Results indicate that NeuGc modulates tumor-cell behaviour and that NeuGcGM3/VSSP vaccine antitumor activity should depend on NeuGc expression.