4551 Vascular endothelial and epidermal growth factor receptors (VEGFR, EGFR) are critical regulators of tumor and endothelial cells. To further understand the role of VEGFR and EGFR signaling in human lung cancer progression, we investigated the antiangiogenic and antimetastatic activity of ZD6474 (a selective small molecule inhibitor of VEGFR-2 tyrosine kinase with additional activity against the EGFR tyrosine kinase) using invitro and invivo systems. In the human lung cancer cell line NCI-H441 (which we determined to express VEGF, TGF-alpha, VEGFR-2 and EGFR) and mouse lung endothelial cells (MLECs), ZD6474 inhibited EGFR phosphorylation, VEGFR-2 phosphorylation and subsequently downstream Akt phosphorylation. ZD6474 treatment of both NCI-H441 and MLECs decreased proliferation and colony formation, induced apoptosis and inhibited migration and invasion. These in vitro data suggest that ZD6474 targets multiple events that are important for both angiogenesis and metastasis. ZD6474 also inhibited in vivo angiogenesis formation, demonstrated by a decrease in microvessel density (MVD) in subcutaneously implanted gelfoam–agarose sponges. To better characterize the effect of ZD6474 in human lung cancer, an orthotopic model using NCI-H441 cells was developed that closely mimics the patterns of growth and metastasis observed in the clinic. Treatment was evaluated at different stages of tumor progression (early, middle and late) with ZD6474 initiated on day 5, 10 or 15 after tumor injection, respectively. ZD6474 (25 mg/kg and 50 mg/kg) impaired pleural effusion formation and pleural invasion at every stage. Daily oral dosing with 25 or 50 mg/kg ZD6474 (initiated on day 5 following tumor inoculation) resulted in the almost complete suppression of the growth, invasion and metastasis of established lung tumors, when compared with controls (P 300% relative to the control (25 days vs 80 days, the time of experiment termination, P