Fem1b antigen in the stool of ApcMin mice as a biomarker of early Wnt signaling activation in intestinal neoplasia

Background: Colorectal cancer is preventable by early detection and removal of precursor lesions. Central to early stages of colorectal neoplasia is activation of Wnt signaling, usually due to inactivation of the Apc tumor suppressor gene for which there is an established animal model, the ApcMin mouse. Immunodetection in stool of proteins up-regulated by aberrant Wnt signaling, within intestinal epithelial cells shed into the lumen, could be a rational approach to identify biomarkers of early intestinal neoplasia. Fem1b gene expression is up-regulated, following inactivation of Apc, in mouse intestinal epithelium. Methods: We initially screened pooled random stool samples by immunoblotting and found that we could detect, in ApcMin mice but not wild-type mice, a fragment of Fem1b protein with an antibody (Li-50) directed against an epitope near the middle of the protein, but not with antibodies directed against N-terminus or C-terminus epitopes. We then evaluated freshly voided individual stool samples collected on four consecutive days from four each of male and female ApcMin mice and their wild-type littermates. Results: The Fem1b antigen was detected with the Li-50 antibody in 15/16 samples from male ApcMin mice compared to 0/16 samples from male wild-type mice, and in 5/16 samples from female ApcMin mice compared to 0/16 samples from female wild-type mice. Conclusions: This study provides proof-of-principle that fragments of proteins, whose expression is increased by aberrant Wnt signaling early in intestinal neoplasia, can be immunodetected in stool. Excreted Fem1b protein fragments may be a useful biomarker for epithelial Wnt signaling and early intestinal neoplasia.