MAb L9E10 to blood group H2 antigen binds to colon cancer stem cells and inhibits tumor cell migration and invasion

Abstract

The functions of the precursor H antigen for ABO blood group antigens are still not fully understood, particularly in cancer cells. In this study, we used hybridoma technology and NSY human colon cancer cells as an immunogen to generate a monoclonal antibody designated as MAb L9E10. The binding antigen of MAb L9E10 was identified as blood group (BG) H2 antigen using carbohydrate array and erythrocyte agglutination assays. In immunofluorescence study, we found that BG-H2 was expressed on the surfaces of both colon cancer stem cells and their differentiated progeny. In a functional study, we observed that MAb L9E10 inhibited tumor cell migration and invasion at a concentration of 10 microg/mL in vitro. This result suggests that MAb L9E10 could be used to study cancer biology, particularly cancer stem cell biology. In addition, it is potentially useful for studying gastric diseases caused by Helicobacter pylori bacteria, with attachment to human gastric epithelial cells mediated by blood group antigens Lewis b and H2. Finally, MAb L9E10 is an ideal biological reagent for identifying Bombay blood type in which erythrocytes have no BG-H2 antigen expression.

FIG. 1.

Identification of MAb L9E10 binding antigen. (A) Carbohydrate array slide stained with MAb L9E10 mouse ascites at 800 × dilution. (B) Quantitative data of carbohydrate array slide stained with MAb L9E10 at different concentrations. (C) Agglutination assay of blood type O erythrocytes with MAb L9E10. Upper panel, control by adding equal volume (50 μL) of PBS to 5% blood type O erythrocytes; lower panel, agglutinated red blood cells after adding equal volume (50 μL) of 4000 × diluted MAb L9E10 mouse ascites and incubated at room temperature for 20 min.

FIG. 2.

Expression of BG-H2 antigen in colon CSCs and biological effect of MAb L9E10 on tumor cell migration and invasion. (A) Immunofluorescence staining of human colon cancer NSY cancer stem (CD133+) cells and differentiated (CD133-) cells with 2000 × diluted MAb L9E10 mouse ascites. (B) Images of human colon cancer NSY cell migration and invasion assay. Left panel control, 10 μL PBS added to equivalent amount of antibodies; middle panel control, 10 μg/mL normal mouse IgG; right panel, MAb L9E10 (10 μg/mL). The larger pink dots are migration and invasion tumor cells. Tiny black dots are 8 micron membrane pores. (C) Quantitative analysis of MAb L9E10 on colon cancer NSY cell migration and invasion.