Mechanism of retinoblastoma tumor cell death after focal chemotherapy, radiation, and vascular targeting therapy in a mouse model

PURPOSE. To evaluate the mechanism and timing of retinal tumor cell death in the LH_BETAT_AG mouse model of retinoblastoma after treatment with vascular targeting therapies and conventional therapies (focal chemotherapy and radiation). METHODS. For vascular targeting therapy, 12- or 16-week-old mice were treated with a single subconjunctival injection of either anecortave acetate (300 μg) or combretastatin A4 (1.5 mg). Eyes were analyzed at 1 day and 1 week after treatment. Tumor cell death was evaluated using TUNEL assays or immunofluorescence analysis of activated caspase 3 to detect apoptosis. Histopathologic analysis was performed to identify areas of necrosis. For conventional therapy, LH_BETAT_AG mice were treated with six serial subconjunctival injections of focally delivered carboplatin chemotherapy (100 μg/delivery) or hyperfractionated external beam radiotherapy (EBRT; 15 Gy total dose). Cell death was analyzed by TUNEL assay. RESULTS. The highest levels of apoptotic cell death were seen 1 day after treatment in all treatment groups compared with vehicle controls. At 1 week after treatment, apoptotic cell death remained significantly elevated in the EBRT and carboplatin groups, but not after vessel targeting therapy. No significant necrosis was detected by histology in tumors of treated or of control eyes. CONCLUSIONS. Conventional therapies (focal carboplatin chemotherapy and EBRT) and vascular targeting agents significantly increase cell death through apoptosis, while not having a significant effect on necrosis in this murine model of retinoblastoma. These studies will aid in the optimization of delivery schemes of combined treatment modalities.