Celecoxib-Induced Modulation of Colon Cancer CD133 Expression Occurs through AKT Inhibition and Is Monitored by 89Zr Immuno-PET

Abstract

We developed an immuno-PET technique that monitors modulation of tumor CD133 expression, which is required for the success of CD133-targeted therapies. Methods. Anti-CD133 antibodies were subjected to sulfhydryl moiety-specific 89Zr conjugation. 89Zr-CD133 IgG was evaluated for specific activity and radiolabel stability. Colon cancer cells underwent binding assays and Western blotting. Biodistribution and PET studies were performed in mice. Results. 89Zr-CD133 IgG showed excellent target specificity with $97.2\pm 0.7\%$ blocking of HT29 cell binding by an excess antibody. Intravenous 89Zr-CD133 IgG followed biexponential blood clearance and showed CD133-specific uptake in HT29 tumors. 89Zr-CD133 IgG PET/CT and biodistribution studies confirmed high HT29 tumor uptake with lower activities in the blood and normal organs. In HT29 cells, celecoxib dose-dependently decreased CD133 expression and 89Zr-CD133 IgG binding that reached $19.9\pm 2.1\%$ ( $P<0.005$ ) and $50.3\pm 10.9\%$ ( $P<0.001$ ) of baseline levels by 50 μM, respectively. Celecoxib treatment of mice significantly suppressed tumor CD133 expression to $67.5\pm 7.8\%$ of controls ( $P<0.005$ ) and reduced tumor 89Zr-CD133 IgG uptake from $15.5\pm 1.4\%$ at baseline to $12.3\pm 2.0\%\text{ID}/\text{g}$ ( $P<0.01$ ). Celecoxib-induced CD133 reduction in HT29 cells and tumors was associated with substantial suppression of AKT activation. There were also reduced HIF-1α accumulation and IκBα/NFκB phosphorylation. Conclusion. 89Zr-CD133 IgG PET provides high-contrast tumor imaging and monitors celecoxib treatment-induced modulation of tumor CD133 expression, which was found to occur through AKT inhibition. This technique may thus be useful for screening drugs that can effectively suppress colon cancer stem cells.