Fluorescent Biomarker Discovery
Figure 1 Images by confocal fluorescence microscopy demonstrate (A) the relative absence of binding of peptide to non-tumorigenic esophageal cells, HET-1A and (B) significant binding to the periphery (cell surface) of Barrett's-derived adenocarcinoma cells, TE7.
As a proof-of-concept experiment in imaging with biomarkers, our group has undertaken to isolate phage that bind preferentially to the TE7 cell line, derived from Barrett's adenocarcinoma, and fail to bind to a normal epithelial cell line HET-1A. The M13 phage library (New England Biolabs) chosen for screening in this study, is well characterized and has been used broadly. For our purposes, 10 11 phage with a density of 10 9 , representing 100 copies of each clone, were panned initially on the normal epithelial cell line HET-1A, to adsorb the phage that bind normal cells. The pre-cleared phage from the resulting supernatant was then panned with a biopsy specimen of esophagus with dysplasia to identify 10 binding clones. Phage from each of the 10 binding clones were placed into separate wells containing HET-1A and TE7 cells. Monoclonal mouse Anti-M13 phage antibody, conjugated to FITC dye, was then administered into all the wells. The binding of the antibody to the phage was evaluated by standard confocal fluorescence microscopy (Bio-Rad MRC 1024). Figure 1A shows the relative absence of binding of the antibody for HET-1A with one clone, while Figure 1B shows the extensive binding for TE7 with the same clone, in particular around the cell surface. This data supports our hypothesis that small peptides that preferentially bind to cell surface antigens of neoplastic cells can be identified by phage display methods. These will be sequenced and the peptides that they encode will be synthesized and tested as imaging agents.