Targeted therapy against VEGFR and EGFR signaling with ZD6474 enhances the therapeutic efficacy of irradiation in an orthotopic mouse model of human non-small cell lung cancer

Abstract

Chemotherapy and radiation therapy have had limited therapeutic success in the treatment of advanced lung cancer. ZD6474 is a potent inhibitor of the VEGFR tyrosine kinase activity with additional activity against the EGFR tyrosine kinase. The purpose of the current study is to evaluate the feasibility of combining targeted therapy against VEGFR and EGFR (using ZD6474) with radiation therapy, as compared with chemoradiation, for human non-small lung cancer, in vitro and in vivo using an orthotopic nude mouse model that closely mimics patterns of growth observed in the clinic. The human lung adenocarcinoma NCI-H441 cell line was used for all studies. After irradiation, cell proliferation with or without ZD6474 treatment was tested using an MTT assay. Surviving fractions of cells after irradiation and inhibition of sublethal damage repair by ZD6474 were determined using a clonogenic assay. For in vivo studies, 5 × 105 lung adenocarcinoma cells were injected into the left lung of nude mice. The mice were then randomized to treatment with vehicle (p.o. daily), ZD6474 (15mg/kg orally once daily), radiation (4 Gy fractions 3 times weekly to a total dose of 20Gy), ZD6474 and radiation, paclitaxel (200μg weekly i.p.), or paclitaxel and radiation. Therapy was initiated on day 8 and mice were sacrificed on day 24–26 after tumor injection, when control mice showed signs of disease-associated morbidity. Tumor burden was assessed by lung and tumor weight, pleural effusion volume, and total weight of disseminated thoracic lesions. Tumor and adjacent lung tissues were then subjected to immunohistochemical analyses. In the MTT assay, marked repopulation of the H441 cells was observed 72–96h after 4Gy irradiation. Cell proliferation was inhibited by 55 ± 4.7% (p = 0.007) of control by 2.5μM ZD6474 treatment for 4 h before and 72 h after irradiation. Radioresponse in the clonogenic assay was enhanced by a factor of 1.37. Sublethal damage repair was completely inhibited by incubation with 5μM ZD6474 for 4h split time. Recovery ratios between 4Gy single dose and 2 + 2Gy split dose were 1.34 in untreated control and 1.04 in ZD6474 treated group. In vivo, the suppression of both tumor growth and metastasis in the orthotopic lung model was greatest in mice treated with combined radiation and ZD6474 compared with radiation with paclitaxel or monotherapy with each modality. The lung weight of control, radiation, ZD6474, radiation with paclitaxel, and radiation with ZD6474 treated mice increased by 330%, 200%, 155%, and 147%, and 110%, respectively, as compared with normal lung weight and the total weight of disseminated lesions was 197 ± 38.1, 160 ± 49.7, 53.9 ± 14.5, 47.5 ± 16.6, and 17.2 ± 8.00, respectively. Radiation therapy had no effect on the formation of pleural effusion (565 ± 99.4μl) as compared with control (745 ± 112μl). Combined therapy with ZD6474 and radiation blocked pleural effusion (14.2 ± 4.84μl) more effectively than radiation and paclitaxel (246 ± 69.4μl). By immunohistochemical analysis, tumor cell apoptosis in the lung primary tumors was substantially increased in mice treated with combined radiation and ZD6474. Microvessel density of lung primary tumors was 55 ± 3.3, 52 ± 7.3, 13 ± 3.3, 42 ± 4.6, 31 ± 3.5, and 16 ± 1.9 in control, radiation, ZD6474, paclitaxel, radiation with paclitaxel, and radiation with ZD6474 treated groups, respectively. When radiation therapy is combined with ZD6474 as targeted therapy against VEGFR and EGFR a significant enhancement of antiangiogenic, anti-vascular and anti-tumor effects are seen in an orthotopic model of lung cancer. These data provide support for clinical trials combining biologically targeted therapies and conventional therapies in human lung cancer