Expression of the tumor-associated antigen Neu5Gc-Sialyl-Tn in human carcinomas

Altered glycosylation is prevalent on cancer cells, due to aberrant expression of glycosyltransferases, and frequently involving variations in sialylation patterns. In humans, this altered sialylation can also result from metabolic incorporation of dietary N-glycolylneuraminic acid (Neu5Gc) that differs from the human sialic acid N-acetylneuraminic acid (Neu5Ac) by one oxygen atom. Neu5Gc is immunogenic in humans and anti-Neu5Gc antibodies can either promote tumor growth at low doses or cause tumor regression when administered in excess. Antibodies against Neu5Gcα2−6GalNAcα1-O-Ser/Thr (anti-Neu5Gc-Sialyl-Tn IgG) appear to be novel carcinoma biomarkers, but direct evidence for expression of this epitope on human carcinomas is lacking. This neo-sialoglycan resembles the well-known carcinoma-associated biomarker Sialyl-Tn, except that Neu5Ac is replaced with dietary Neu5Gc. Here, we characterize unique antibodies against Neu5Gc and Sialyl-Tn by sialoglycan-microarrays and use them for immunohistochemistry of human breast, prostate, ovary, colon and lung carcinomas revealing co-staining in some mucinous carcinomas. We also generate MUC1-Fc that is heavily glycosylated with Neu5Gc-Sialyl-Tn for affinity-purification of human anti-Neu5Gc-Sialyl-Tn-MUC1 IgG for further immunohistochemistry analysis. Finally, we report a mass spectrometry approach to analyze O-linked glycans from tissues to ultimately prove biomarker expression in positively-stained tissues.