Abstract A183: Ex vivo imaging of EGF receptor-mediated signal transduction for prediction of anti-EGFR treatment efficacy

Abstract

Abstract Objectives: EGFR is a promising treatment target in solid tumors. Two major types of anti-EGFR agents have entered the clinical setting: antibodies and small-molecule EGFR TKIs. EGFR-TKI inhibits autophosphorylation of EGFR by binding to ATP binding site of tyrosine kinase domain of EGFR and interrupts the signal transduction. On the other hand, anti-EGER therapeutic antibodies interrupt EGFR signaling by blocking ligand binding. In addition, as for antibodies, the effects of the ADCC are also expected. In this study, we aimed at developing a method to estimate the effectiveness of anti-EGFR agents by analyzing these mechanisms using ex vivo imaging technique. Methods: To establish a method that confirms the inhibition effect of the EGFR-signal transduction, we prepared fluorescent-labeled ligand and developed the imaging strategy to visualize membrane binding and subsequent internalization of labeled EGF. In addition, fluorescent-labeled NK-based effector cells that have been established by introducing fcgr3a gene into a NK leukemia cell line were used for characterization of ADCC susceptibility of individual tumor cells. In our strategy, a continuous confocal laser scanning microscopy was used for tracing fluorescent-labeled EGF or effector cells as well as tumor cell death. Results: Fluorescent probe bearing EGF was highly specific and potent in the binding and activation of the EGF receptor, being rapidly internalized into endosomes. Pretreatment with therapeutic anti-EGFR antibody completely inhibited the ligand-binding and subsequent events. Meanwhile, EGFR TKIs partially prevented endocytotic step. K-ras mutation status did not significantly affect the ligand binding and internalization. The imaging analysis of the ADCC using primary colorectal cancer specimens revealed that cetuximab-coated tumor cells were gradually damaged by direct interaction of effector cells. ADCC susceptibility of individual tumor cells has varied to some extent. Conclusion: We have established imaging methods of the early events of EGFR signaling pathway and ADCC reaction. This imaging method soffer the rapid reliable means that analyze the effect of the EGFR targeted agent to the individual tumor cells. By combining with already established prognostic factors such as K-ras mutation status or Fc receptor polymorphism, our imaging analysis will propose novel strategy to predict an individual treatment prognosis more precisely. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A183.