Targeted Molecular Imaging: A Guide to Combination Therapy
Recombinant adenovirus is widely used to deliver genes for cancer gene therapy. Coxsackie and Adenovirus' Receptor (CAR) is the primary receptor for recombinant adenovirus. Little attention has been paid to determine CAR protein expression and promoter activity in vivo. This study tested the hypothesis that targeted molecular imaging of CAR could predict the tissue receptivity to viral infection and the response to treatment with histone deacetylases inhibitor (HDACi). To image CAR protein expression, human prostate cancer xenografts were established in nu/nu mice. The ability of iodinated anti-CAR intact, F(ab')2 fragments and control F(ab')2 fragments to distinguish CAR (+) tumors was tested by biodistribution, gamma camera scintigraphy and validated by western blot. Tumor susceptibility to infection was tested with adenoviruses carrying the reporter β-galactosidase. To assess CAR promoter activity, a sodium iodide symporter (NIS) reporter construct containing the NIS open reading frame driven by the CAR promoter (CAR-NIS), was constructed by directional cloning. Tumor cell lines stably expressing CAR-NIS or empty vector were established. NIS protein function was assessed by intracellular accumulation of 99mTcO4- and mRNA level was tested by RT-PCR. The inductivity of the CAR promoter by HDACi in vivo was tested by imaging CAR-NIS tumors after administration of 99mTcO4- using a gamma camera. A replication-deficient recombinant adenovirus coding CAR-NIS was constructed to deliver the reporter construct to cells and tumors to permit radionuclide imaging. Radiolabeled anti-CAR F(ab')2 fragments more effectively distinguished CAR(+) from CAR(-) tumors at early time points. Tumors with greater retention of radiolabeled anti-CAR showed higher levels of CAR protein expression by western blot and ß-galactosidase activity after adenoviral infection. Stable CAR-NIS transfectants showed 16-fold to180-fold increase of 99mTcO4- accumulation after HDACi treatment in PC3 prostate cancer cells or TCC bladder cancer cells, respectively (p<0.001). Ad-CAR-NIS effectively delivered the HDACi inducible CAR-NIS into target cells in a dose dependent manner. The transgene CAR-NIS expression was imaged in vivo using gamma camera scintigraphy. Molecular imaging approaches to image CAR protein expression and assess CAR promoter activity in vivo can predict tissue receptivity to adenoviral infection and have potential to direct combination of gene delivery and chemotherapy.