Ablation of Tumor Microvessels Upon Dimerization of iCaspase-9

Anti-angiogenic therapies based on the disruption of tumor microvascular network might be beneficial for patients with head and neck cancer. We have recently demonstrated that activation of the Caspase-9 apoptotic pathway is sufficient to induce endothelial cell apoptosis in vitro and to disrupt microvessels in vivo. Objective: The purpose of this project is to examine if direct activation of Caspase-9 has an effect on tumor microvessel density. Methods: The catalytic domain of Caspase-9 was fused to a dimerizer domain (FKBP) to express an inducible Caspase-9 (iCaspase-9) that is activated upon exposure to the dimerizer drug AP20187. The ability of AP20187 to activate Caspase-9 and downstream protease Caspase-3 was tested with a fluorometric assay in human dermal microvascular endothelial cells (HDMEC) expressing iCaspase-9 (HDMEC-iCasp9) or empty vector (HDMEC-LXSN) exposed to 0-100 nM AP20187 (ARIAD). HDMEC-iCasp9 or HDMEC-LXSN cells were co-implanted with oral squamous cell carcinoma cells (SCC-17B) in highly porous poly-L (lactic) acid scaffolds, implanted in SCID mice. Fourteen days after implantation, we began daily intraperitoneal injections of 2 mg/kg AP20187 for 3 days. Tumor microvessel density was evaluated after immunostaining with Factor VIII antibody. Results: HDMEC-iCasp9 exposed to 100 nM AP20187 presented a 2,825-fold induction of Caspase-3 activity as compared to HDMEC-LXSN exposed to AP20187 (p<0.05). Tumors engineered with HDMEC-iCasp9 and SCC-17B had a 2.6-fold reduction in microvessel density as compared to tumors containing HDMEC-LXSN and SCC-17B in mice injected with AP20187. Conclusions: Activation of the Caspase-9 apoptotic pathway in neovascular endothelial cells is sufficient to decrease tumor microvessel density. Supported in part by NIDCR short-term training grant #DE07101 (MP), and NIDCR grant #R01DE/CA14601-01 (JEN).