title: Phage display selection for cell-specific ligands: development of a screening procedure suitable for small tumor specimens.
abstract: Phage display technology has been widely used for developing tumor-targeting agents. Most of the efforts were directed towards identifying phage-displayed ligands against cancer-relevant purified targets and cancer cell lines. Whole cell screening procedures typically use a relatively large sample size and are not ideally suited for complex tumor tissues. We describe here a screening protocol that is suitable for non-adherent tumor cells from biopsy specimens. It requires only approximately 20,000 cells/round for biopanning and approximately 10,000 cells/well for subsequent clone binding assessment by ELISA. We standardized the newly developed protocol using erbB2-overexpressing SKBR3 breast cancer cells and compared the results with conventional protocols employing about 10-times more plate-adhered fixed or live cells. The selection rate of SKBR3-binding clones from biopanning approximately 20,000 non-adherent SKBR3 cells by our filter cup protocol was comparable to that obtained from using approximately 200,000 plate-adhered cells. Assessment of clones selected from different phage libraries showed that clones from fixed or live cells, adherent or non-adherent cells, either biopanned in filter cup or plate share specific motifs and binding properties. Some of the clones from each biopanning protocol bound to purified erbB2 and shared motifs with erbB family of receptors and their known ligands. These results demonstrated that the protocol developed in this study was capable of selecting cell-specific ligands using relatively small numbers of cells. Screening cells from a fresh human breast cancer specimen using our protocol showed enrichment of tumor binding clones at successive rounds of selection and some of the selected clones were tumor-specific in comparison to normal breast cells. These protocols have direct application to screen for tumor-binding ligands with small tumor tissue specimens.
authors.affiliation: Department of Surgery, Vermont Comprehensive Cancer Center, University of Vermont College of Medicine, Burlington, VT 05405, USA.
authors.forename: Girja S
authors.initials: GS
authors.lastname: Shukla
authors.forename: David N
authors.initials: DN
authors.lastname: Krag
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D001706:Biopsy; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D002999:Clone Cells; D016503:Drug Delivery Systems / Q000379:methods; D006801:Humans; D008024:Ligands; D008403:Mass Screening / Q000379:methods*; D019151:Peptide Library; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013048:Specimen Handling / Q000379:methods*
chemical_list: D008024:Ligands; D019151:Peptide Library; D018719:Erb-b2 Receptor Tyrosine Kinases