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Comparative Study
. 2006 Dec;8(12):1011-8.
doi: 10.1593/neo.06610.

In vivo imaging of molecularly targeted phage

Affiliations
Comparative Study

In vivo imaging of molecularly targeted phage

Kimberly A Kelly et al. Neoplasia. 2006 Dec.

Abstract

Rapid identification of in vivo affinity ligands would have far-reaching applications for imaging specific molecular targets, in vivo systems imaging, and medical use. We have developed a high-throughput method for identifying and optimizing ligands to map and image biologic targets of interest in vivo. We directly labeled viable phage clones with far-red fluorochromes and comparatively imaged them in vivo by multichannel fluorescence ratio imaging. Using Secreted Protein Acidic and Rich in Cysteine (osteonectin) and vascular cell adhesion molecule-1 as model targets, we show that: 1) fluorescently labeled phage retains target specificity on labeling; 2) in vivo distribution can be quantitated (detection thresholds of approximately 300 phage/mm(3) tissue) throughout the entire depth of the tumor using fluorescent tomographic imaging; and 3) fluorescently labeled phage itself can serve as a replenishable molecular imaging agent. The described method should find widespread application in the rapid in vivo discovery and validation of affinity ligands and, importantly, in the use of fluorochrome-labeled phage clones as in vivo imaging agents.

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Figures

Figure 1
Figure 1
Phage labeling. (A) Fluorochromes used for phage labeling. Listed are the mean numbers of fluorochromes from a single comparative experiment. The relative fluorescence signal at a 6-mm tissue depth of different fluorochrome-labeled phages was calculated from known spectral tissue absorption. Note the much higher fluorescence of 680- to 750-nm fluorochrome-labeled phages in tissues. (B) Emission spectra of fluorescently labeled phages (excitation and color of data points as listed in A).
Figure 2
Figure 2
Properties of labeled phage. (A) Effect of increasing fluorochrome labeling (VT680) on phage affinity determined by ELISA (blue region: error range). (B) Effect of increasing labeling on phage fluorescence. Beyond 800 fluorochromes per M13 phage, there is little increase in fluorescence. (C) VT680-labeled phage clone (800 fluorochromes/phage) binds specifically to SPARC-expressing fibroblasts, but not to fibroblasts derived from SPARC-/- mice (scale bar: 10 µm). (D) Effect of successive phage labeling on viability (ability of the phage to infect and amplify in E. coli).
Figure 3
Figure 3
In vivo behavior of labeled phage. (A) Time course of tumor homing. Mice bearing subcutaneous bilateral LLC-derived tumors were coinjected through the tail vein with VT680-labeled SPARC-targeted phage and AF750-labeled wild-type phage (no insert) and imaged at 0, 2, 4, 6, and 24 hours after injection. Blue line: SPARC-targeted phage clone 23. Brown line: wild-type phage (no insert). (B) Detection threshold. Tumor-bearing mice were injected with increasing log doses of labeled phage and imaged 4 hours after injection. The line indicates detection threshold. (C) Reflectance imaging. Mice bearing subcutaneous bilateral tumors (LLC cells) were injected with either VT680-labeled wild-type phage (right) or VT680-labeled SPARC-targeted phage. Note the brightly fluorescent tumors in the near-infrared fluorescence channel of the SPARC-targeted phage clone [identical white light (WL) settings].
Figure 4
Figure 4
In vivo imaging of labeled phage. Mice bearing subcutaneous bilateral tumors (LLC cells) were injected with fluorescent phage. (A) Serial coronal tomographic images at different depths of a representative mouse injected with the SPARC-targeted phage clone. Note the distribution of phages throughout the tumors. The scale bar is in nanomolars of fluorescence. (B) Wild-type control phage (identical white light settings as in A).
Figure 5
Figure 5
Screening of SPARC-targeted phage clones. (A) Comparison of the target affinity of different SPARC clones by in vitro (left) or in vivo (middle and right) imaging (control ELISA: BSA; control FMT: wild-type phage). Note the high target-to-background ratio of clone 23 in vivo, whereas most clones are similar by in vitro screening. (B) FMT imaging of mice bearing bilateral LLC tumors injected with different phage clones. Maximum intensity projections are shown for the five phage constructs investigated. All images have been equally leveled. The scale bar is presented as the concentration of fluorochromes in nanomolars. (C) Comparative immunofluorescence of indicated phage clones' tumoral accumulation (red). Nuclei are counterstained with DAPI (blue). Scale bar = 10 µm.
Figure 6
Figure 6
In vivo microscopic imaging of phage. (A) Microscopic imaging of bilateral ear vessels of a live mouse injected with labeled VCAM-1-targeted phage (CVHSPNKKC, green) and vascular marker (Angiosense-750, red). Shown are confocal projections of Z stacks from mTNFα-treated ear or control ear at 4 hours after phage injection. Images are representative of different animals in each case and five different fields per ear. (B) Correlative histology: adjacent sections of ears taken from in vivo intravital experiments were stained for CD31, VCAM-1, or phage protein pVIII (M13).

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