Abstract 4595: Molecular imaging of urothelial cancer using EGFR-binding peptides
Authors: Pan Ying , Liu Jen-Jane , Chang Timothy C. , Hsiao Shelly , Mach Kathleen E. , Liao Joseph C. 
Published: August 2012

Abstract
INTRODUCTION White light cystoscopy (WLC), the current standard for bladder cancer diagnosis, has recognized shortcomings in differentiating non-papillary urothelial carcinoma from inflammation. Confocal laser endomicroscopy (CLE) with fluorescein (a non-specific contrast agent) can provide in vivo microscopy of the bladder as an adjunct to WLC. Targeted CLE with fluorescently labeled peptides against tumor surface markers may further enhance the utility of CLE for real time endoscopic imaging. Epidermal growth factor receptor (EGFR) is over-expressed in most bladder cancer cells, but absent in the superficial layer of normal urothelium, making it an attractive target for cancer-specific imaging. We report our efforts to identify a non-activating, EGFR-binding peptide labeled with FITC as a cancer-specific contrast agent for ex vivo bladder imaging with CLE. METHODS To screen for EGFR binding peptides, fragments of the EGFR-binding motif of human EGF (EGF1∼4) were displayed on phage surface. Binding affinity of the phage to recombinant EGFR and EGFR-expressing bladder cancer cell line was measured by ELISA. Peptides with the highest EGFR affinity were chemically synthesized and FITC labeled. Potential for activation of EGFR by the peptides was assessed in two EGFR-expressing cell lines by Western blot. For ex vivo imaging of urothelial cancer, fresh human bladder (n=1) or ureter (n=2) specimens were instilled and incubated with FITC-EGFR binding peptide, or a random sequence negative control peptide. Excess peptide was drained, specimens were opened and suspicious and normal appearing mucosa were imaged with CLE. Imaged tissue sections were excised for histopathologic comparison. The bladder and one ureter were first imaged with negative control peptide followed by imaging with the EGF peptide. RESULTS All phage displaying EGF peptides showed greater affinity to recombinant EGFR and EGFR-expressing cells compared to wild type phage. The two phage with highest affinity for EGFR in both ELISA assays were synthesized. Cell stimulation with both peptides did not induce phosphorylation of EGFR or downstream signaling molecules. In bladder and ureter specimens, greater fluorescence intensity was detected in areas of tumor compared to normal urothelium on CLE with EGF4-FITC. Histopathology confirmed that the suspicious regions of the bladder and ureter imaged were urothelial carcinoma. The random peptide showed minimal fluorescent signal on both normal and tumor areas imaged, and subsequent imaging of the same areas with EGF4-FITC demonstrated abundant fluorescent signal in tumor areas only. CONCLUSION We identified peptides derived from the EGF sequence that specifically bind, but do not activate EGFR. Ex vivo imaging of urothelial cancer with EGF4 peptide suggests that it can be used for cancer-specific molecular imaging. Future studies will be aimed at in vivo validation of EGF4 peptide on animal models for urothelial cancer.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4595. doi:1538-7445.AM2012-4595