Development of NGR peptide-based agents for tumor imaging

Abstract

Molecular imaging allows direct visualization of targets and characterization of cellular pathways, as long as a high signal/background ratio can be achieved, which requires a sufficient amount of probes to accumulate in the imaging region. The Asn-Gly-Arg (NGR) tripeptide selected by phage display can specifically target tumor vasculature. Recognizing the aminopeptidase N (APN or CD13) receptor on the membrane of tumor cells, the peptide can be further internalized into cytoplasma by the endosomal pathway. Hence NGR can serve as an ideal candidate for tumor imaging, once it is conjugated with fluorescent or radiolabeled imaging probes. Herein, we highlight some recent developments of NGR peptide based imaging of tumors. Although still in the preliminary stage, some NGR probes have shown potential as promising agents in future clinical applications.

Keywords: Asparagine-glycine-arginine (NGR); aminopeptidase N (APN/CD13); arginine-glycine-aspartic acid (RGD); cancer; imaging; isoaspartate-glycine-arginine (isoDGR); tumor angiogenesis; vasculature.

Figure 1

Structure of common NGR peptides.

Figure 2

Imaging with NGR-OG488. Epifluorescence microscopy of (A) linear NGR-OG488 (B) cNGR-OG488 in HT-1080 cells at 37 °C after 2h incubation. Green color is from NGR-OG488, blue color is from nuclear staining agent 4'-6-Diamidino-2-phenylindole (DAPI). Scale bar = 100μm. Epifluorescence microscopy of the internalization of cNGROG488 by HT-1080 cells at (C) 4 °C (D) 37 °C after 30 min incubation. Scale bar = 100 μm. Adapted from reference [26].

Figure 3

In vivo imaging with NGR-Cy 5.5. (A) Chemical structure of Cy 5.5-labeled NGR-peptide. (B) In vivo fluorescence reflectance imaging (FRI) 24 h after the injection of NGR-Cy 5.5 to HT-1080 and MCF-7 xenografts. Arrows indicated the tumors. (C) Top part: fluorescence-mediated tomography (FMT) 24 h after injection of NGR-Cy 5.5 to mice bearing (a) HT-1080, (e) MCF-7, (c) mice bearing HT-1080 that were pre-injected with100-fold unlabeled peptide 10 min ahead of injection with NGR-Cy5.5; Bottom part: FMT 60 min after injection of NGR-Cy5.5 to (b)(d)(f) that are prepared under the same conditions to (a)(c)(e), respectively. (D) Overlay of white light and FRI images for organs of HT-1080 bearing mice 24 h after injection of NGR-Cy5.5; (1) HT-1080 tumor (2) heart (3) spleen (4) lung (5) liver (6) kidneys. Adapted from reference [18].

Figure 4

In vitro imaging of cNGR-QD. (A) Structure of QD labeled by cNGR peptide. (B) (C) Fluorescent imaging of CD13-positive murine endothelial (2F-2B) cell line (B) and CD13-negative murine emangioendothelioma (EOMA) cell line (C) incubated with cNGR-QD for two hours. Red color is from QD; blue color is from the cell-nuclei stain with Syto44. Scale bar = 25 μm. Incubations of 2F2B cells with cNGR-OG488, cNGR only or with QD-streptavidin resulted in images similar to (C). Figures (B)(C) are adapted from reference [11].

Figure 5

MRI and TPLSM images of cNGR-QD and unlabeled QD in tumor-bearing mice. (A) MRI images with merged view of ΔR₁ for tumor (T) and muscle (M) injected with cNGR-labeled or unlabeled QDs. (B) MRI images with merged view of ΔS₀ for tumor (T) and muscle (M) injected with cNGR-labeled or unlabeled QDs. (C) Representative TPLSM images of tumor showing QD signal (red) and the endothelial marker, FITC-CD31antibody signal (green) that can be co-localized. (D) Representative TPLSM images of normal muscle tissue showing QD signal (red) and FITC-CD31antibody signal (green). White arrow points out some weak cNGR-QD signal (red). Scale bar = 50 μm. The figures are adapted from reference [12].

Figure 6

In vitro imaging of FITC-HMPA-cNGR in CD13-positive/negative cells. (A) Structure of FITC-HMPA-cNGR. (B)-(G) Confocal images of (B)(E) FITC-HMPA-cNGR, (C)(F) LysoTracker Red DND-99, and their co-localization image (D)(G) in HT-1080 and A431 tumor cells, respectively. Green color is from FITC; red color is from LysoTracker Red DND-99; blue color is the nuclear staining by DAPI. The figures are adapted from reference [25].