Phage display peptide probes for imaging early response to bevacizumab treatment

Abstract

Early evaluation of cancer response to a therapeutic regimen can help increase the effectiveness of treatment schemes and, by enabling early termination of ineffective treatments, minimize toxicity, and reduce expenses. Biomarkers that provide early indication of tumor therapy response are urgently needed. Solid tumors require blood vessels for growth, and new anti-angiogenic agents can act by preventing the development of a suitable blood supply to sustain tumor growth. The purpose of this study is to develop a class of novel molecular imaging probes that will predict tumor early response to an anti-angiogenic regimen with the humanized vascular endothelial growth factor antibody bevacizumab. Using a bevacizumab-sensitive LS174T colorectal cancer model and a 12-mer bacteriophage (phage) display peptide library, a bevacizumab-responsive peptide (BRP) was identified after six rounds of biopanning and tested in vitro and in vivo. This 12-mer peptide was metabolically stable and had low toxicity to both endothelial cells and tumor cells. Near-infrared dye IRDye800-labeled BRP phage showed strong binding to bevacizumab-treated tumors, but not to untreated control LS174T tumors. In addition, both IRDye800- and (18)F-labeled BRP peptide had significantly higher uptake in tumors treated with bevacizumab than in controls treated with phosphate-buffered saline. Ex vivo histopathology confirmed the specificity of the BRP peptide to bevacizumab-treated tumor vasculature. In summary, a novel 12-mer peptide BRP selected using phage display techniques allowed non-invasive visualization of early responses to anti-angiogenic treatment. Suitably labeled BRP peptide may be potentially useful pre-clinically and clinically for monitoring treatment response.

Fig. 1

(A) Bevacizumab treatment (3 doses, 20 mg/kg every other day) delayed LS174T human colorectal cancer tumor growth in a nude mouse model, with the relative tumor volume (V_t/V₀) significantly lower than that of the saline control group at day 10 following initiation of treatment (n = 10/group, P < 0.05). (B) Representative near-infrared fluorescence images at 24 h after intravenous injection of IRdye800-labeled LLADTTHHRPWT phages (1 nmol dye per mouse) showed prominent tumor phage particle accumulation in bevacizumab treated, but not saline control, mice. Mice were imaged 1 day after the three doses of bevacizumab (the fifth day following the start of treatment), at which time no difference in tumor volume between the treatment and control groups was found. (C) Tumor-to-norrnal tissue (T/N) ratios of the 2D optical images at 1, 2, 4, 20, and 24 h after administration of optically labeled phage particles.

Fig. 2

(A) Colorimetric MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay showed that the bevacizumab-responsive peptide (BRP) had little effect on proliferation of the LS174T colorectal cancer cells and human umbilical endothelial (HUVEC) cells (n = 6). (B) Representative near-infrared fluorescence images at 2 h and 4 h after intravenous injection of IRdye800-labeled LLADTTHHRPWT peptide (IRDye800-BRP) (1 nmol dye/mouse). LS174T tumor mice were imaged 1 day after three doses of bevacizumab treatment. IRDye-BRP showed good tumor/background contrast in bevacizumab treated, but not in saline control mice, at which time there were no differences in tumor volume between the two groups. Photos of the same mice at 12 days after the optical imaging show that the BRP imaging successfully predicted treatment outcomes. (C) Tumor-to-normal tissue (T/N) ratios of the 2D optical images at 0.5, 1, 2 and 4 h after the administration of optically labeled BRP peptide. ** denotes P < 0.01. (D) Representative near-infrared fluorescence images at 4 h after intravenous injection of an IRdye800-labeled scrambled peptide WTLRPTLHTDHA (1 nmol dye/mouse). LS174T tumor mice were imaged 1 day after a course of three doses of bevacizumab treatment. The scrambled peptide showed rapid renal clearance and virtually no tumor contrast in either bevacizumab-treated or saline control mice. (E) T/N ratios of the 2D optical images at 4 h after the administration ofIRDye800 labeled scrambled peptide.

Fig. 3

Microscopic localization of Cy5.5-BRP (red = Cy5.5, green = fluorescein isothiocyanate (FITC), orange = overlay) in bevacizumab treated LS174T tumor mice. Animals were injected with 1 nmol of Cy5.-BRP, followed by FITC-conjugated tomato lectin for in vivo dual staining of tumor vasculature. Cy5.5 (red) fluorescence staining of tumor vasculature and FITC (green) staining of tumor vasculature were acquired separately by fluorescence microscopy and then overlaid (orange) using MetaMorph imaging processing software. BRP peptide binds specifically to bevacizumab-treated microvascular tumor endothelium cells.

Fig. 4

(A) Representative coronal PET images of LS174T tumor mice at 1 h after intravenous injection of ¹⁸F-FDG. (B) ROI analysis of ¹⁸F-FDG images showed no difference in tumor uptake (%ID/g) between the saline control and bevacizumab treatment groups. (C) Representative whole-body coronal PET images of LS174T tumor-bearing mice at 1 hand 4 h after intravenous injection of ¹⁸F-FP-BRP (100 μCi/mouse) with or without bevacizumab treatment. (D) Quantitative PET images of the tumor ROI showed significantly higher tumor uptake of ¹⁸F-FP-BRP in bevacizumab treated vs. saline control mice. Tumors are indicated by arrows, where * denotes P < 0.05, ** denotes P < 0.01 (n = 5/group).