In vivo selection of phage for the optical imaging of PC-3 human prostate carcinoma in mice

Abstract

There is an increasing medical need to detect and spatially localize early and aggressive forms of prostate cancer. Affinity ligands derived from bacteriophage (phage) library screens can be developed to molecularly target prostate cancer with fluorochromes for optical imaging. Toward this goal, we used in vivo phage display and a newly described micropanning assay to select for phage that extravasate and bind human PC-3 prostate carcinoma xenografts in severe combined immune deficiency mice. One resulting phage clone (G1) displaying the peptide sequence IAGLATPGWSHWLAL was fluorescently labeled with the near-infrared fluorophore AlexaFluor 680 and was evaluated both in vitro and in vivo for its ability to bind and target PC-3 prostate carcinomas. The fluorescently labeled phage clone (G1) had a tumor-to-muscle ratio of approximately 30 in experiments. In addition, prostate tumors (PC-3) were readily detectable by optical-imaging methods. These results show proof of principle that disease-specific library-derived fluorescent probes can be rapidly developed for use in the early detection of cancers by optical means.

Figure 1

Binding of AF680-labeled phage and unlabeled phage to PC-3 human prostate carcinoma cells. PC-3 human prostate carcinoma cells were grown to 90% confluency in a 96-well plate. Phage at 5 x 10⁹, 1 x 10¹⁰, 5 x 10¹⁰, or 1 x 10¹¹ V/ml were added to wells containing a complete growth medium and were allowed to incubate for 1.5 hours at 37°C. Cells and attached phage were then washed with ice-cold PBS and fixed with 4% formaldehyde. The presence of phage was detected by a rabbit polyclonal anti-phage antibody, followed by an anti-rabbit antibody conjugated to horseradish peroxidase. Liquid peroxidase substrate was added, and the plate was then read on a µ Quant Universal Microplate Spectrophotometer at an absorbance of 405 nm.

Figure 2

Binding of AF680-labeled phage and peptides to human carcinoma cells and control normal cells. Slides containing fixed PC-3 or HEK293 cells were incubated with AF680-labeled phage solutions (1 x 10¹¹ V/ml phage in 10 mM Tris, pH7.5, 1% BSA) or biotinylated peptide solutions (20 µM peptide in 10 mM Tris, pH 7.5, 1% BSA) at room temperature for 1 hour in the dark. The binding of biotinylated peptides was detected using 10 µg/ml NeutrAvidin-Texas Red (A). Specificity of binding was determined by incubating AF680 G1 or WT phage on slides containing PC-3, PC-3M, LNCaP, DU145, RPWE-1, MB-MDA-435, and HEK293 cells (B). The presence of fluorophore was detected by laser scanning confocal microscopy.

Figure 3

Biodistribution of AF680-labeled G1 fUSE5 and WT phage in PC-3 xenografted SCID mice. SCID mice bearing human PC-3 carcinoma tumors were injected with ∼ 10⁹ TU of AF680-labeled G1 fUSE5 phage (A) and AF680-labeled WT phage (B) and were allowed to circulate for 5 minutes, 30 minutes, 2 hours, 4 hours, and 6 hours. Organs were excised, homogenized, and probed for fluorescent activity using the Xenogen IVIS 200 system.

Figure 4

Biodistribution of AF680-labeled G1 fUSE5 and WTphage in PC-3 xenografted SCID mice. Homogenized tissues from SCID mice bearing human PC-3 carcinoma tumors were probed for the fluorescent activity of AF680 label and for the presence of coat protein VIII. The presence of the phage coat protein VIII was verified by the immunoblotting of tumors, livers, fats, and muscles (A). Homogenized tissues were electrophoresed on 16% Tricine gels and transferred to 0.45-µm nitrocellulose membranes. Coat protein VIII was detected by incubating the membrane with a rabbit-polyclonal anti-coat protein VIII phage antibody followed by a goat anti-rabbit antibody conjugated to horseradish peroxidase. Signal was determined by the addition of chemiluminescent peroxidase substrate, exposure to double-emulsion Blue Lite autorad film, and development of the film using Kodak (Rochester, NY) processing chemicals. Homogenized tumors, livers, fats, and muscles were probed for fluorescent activity using the Xenogen I VIS 200 system (B).

Figure 5

In vivo imaging of a PC-3 prostate tumor using AF680-labeled G1 fUSE5 phage. SCID mice bearing PC-3 tumors were injected with AF680-labeled G1 or WTphage. Fluorescence reflectance images of anesthetized mice were obtained 0 minute, 1 hour, 4 hours, and 24 hours postinjection (A). Fluorescence signals of normal and tumor tissues were quantified using ImageJ software (B).