N-glycolyl sialic acids as a cancer vaccine target: developing of a mouse B16 melanoma model by transient or stable expression

MARIANO ROLANDO GABRI; LAURA LIRIA OTERO; VALERIA INÉS SEGATORI; DANIEL EDUARDO GOMEZ; DANIEL FERNANDO ALONSO

Barcelona, España

Congreso; 14th European Cancer Conference; 2007

European Cancer Conference

Sialic acids are normal components of the glycocalyx in most normal cells that participate in biological processes such as migration, adhesion and specific receptor recognition. N-glycolyl sialic acids (NeuGc) are a subset of these molecules synthesized by the enzyme CMP-NeuAc hydroxilase in murine cells. Although normal human cells do not express NeuGc, it has been described that the antigen can be detected in the cell membrane in melanoma and breast cancer. These facts support the idea to use NeuGc as a target for cancer vaccines in human beings. On the contrary, mouse B16 melanoma cells, as well as most murine tumors, do not express NeuGc, making difficult the development of appropriate preclinical tumor models. Our aim was to obtain B16 melanoma cells with transient expression of NeuGc by in vitro antigen incubation or by stable overexpression of CMP-NeuAc hydroxilase. Incubation of B16 cells with mucin, a NeuGc-rich compound, induced the presence of this antigen in B16 cell membrane during 48 hours. Preincubation with mucin caused an enhancement in tumor cell adhesion on plastic surfaces. In vivo, mucin-incubated B16 cells showed a rapid subcutaneous primary tumor formation and an increase in the metastatic ability after endovenous injection in syngeneic C57Bl6 mice. Using molecular techniques we were able to isolate and amplify the CMP-NeuAc hydroxilase sequence. Transfected B16 cells showed the presence of CMP-NeuAc hydroxilase mRNA and the presence of the NeuGc antigen in tumor cell membrane. We observed an increase of in vitro proliferation and cell adhesion in transfected cells as compared with control non-transfected B16 cells. Interestingly, stable NeuGc expression was associated with a weak tumorigenicity in syngeneic mice after subcutaneous implantation of transfected B16 cells and a decrease of lung metastasis. Taken together, the results indicate that the presence of NeuGc modulates positively in vitro proliferation and adhesion of mouse melanoma cells, but stable expression of the antigen may induce a negative selection during tumor progression in immunocompetent mice.