title: Biopanning Phage-Display Libraries on Small Tissue Sections Captured by Laser Capture Microdissection.
abstract: Phage-display technology has been widely used for developing tumor-targeting agents. Laser capture microdissection (LCM) has proven to be an accurate method to select specific cells from histological sections. Our goal was to develop a method to combine phage-display with LCM to obtain phage-displayed ligands that bind to selected cells in human solid tumors. Two panning strategies were evaluated and optimized. The first strategy was to pan on patient tissue mounted to LCM slides before LCM occurred. The poor panning output showed that phage did not tolerate the drying conditions during LCM. The second strategy was to pan on tumor cells from the patient tumor tissue that were isolated by LCM. The catapulted tumor cells were transferred to a filter unit which retained cells but allowed rinsing of unbound phage. Six commercially available filter units were evaluated and the one with the lowest nonspecific binding to phage was selected for the panning steps. The smallest number of cells (500) in which panning could be successfully accomplished was also determined. A micropipette system was developed to further decrease background by removing catapulted cells from the filter unit after panning was complete. This left behind nearly all background binding phage in the filter unit. This strategy led to the selection of individual phage antibody clones (5 out of 79 tested) specific for tumor cells of the patient's cancer tissue. Immunofluorescence staining on tumor tissues from the same patient showed that these clones have selective signals on tumor island cells, while the scFv library only showed low nonspecific signals on tumor tissues. We established a method of panning on a small number of LCM-captured solid tumor specimens. The quick identification of specific phage-displayed antibodies in the cancer tissue of human patients will greatly enhance the therapy and diagnosis of cancer.
authors.affiliation: Department of Surgery, Vermont Comprehensive Cancer Center, University of Vermont, Burlington, VT 05405.
authors.forename: Yujing
authors.initials: Y
authors.lastname: Sun
authors.forename: Girja S
authors.initials: GS
authors.lastname: Shukla
authors.forename: Guy G
authors.initials: GG
authors.lastname: Kennedy
authors.forename: David M
authors.initials: DM
authors.lastname: Warshaw
authors.forename: Donald L
authors.initials: DL
authors.lastname: Weaver
authors.forename: Stephanie C
authors.initials: SC
authors.lastname: Pero
authors.forename: Lisa
authors.initials: L
authors.lastname: Floyd
authors.forename: David N
authors.initials: DN
authors.lastname: Krag