Ultrasound molecular imaging of tumor angiogenesis with an integrin targeted microbubble contrast agent

Abstract

Rationale and objectives: Ultrasound molecular imaging is an emerging technique for sensitive detection of intravascular targets. Molecular imaging of angiogenesis has strong potential for both clinical use and as a research tool in tumor biology and the development of antiangiogenic therapies. Our objectives are to develop a robust ultrasound contrast agent platform using microbubbles (MB) to which targeting ligands can be conjugated by biocompatible, covalent conjugation chemistry, and to develop a pure low mechanical index (MI) imaging processing method and corresponding quantification method. The MB and the imaging methods were evaluated in a mouse model of breast cancer in vivo.

Materials and methods: We used a cyclic arginine-glycine-aspartic acid (cRGD) pentapeptide containing a terminal cysteine group conjugated to the surface of MB bearing pyridyldithio-propionate (PDP) for targeting αvβ3 integrins. As negative controls, MB without a ligand or MB bearing a scrambled sequence (cRAD) were prepared. To enable characterization of peptides bound to MB surfaces, the cRGD peptide was labeled with FITC and detected by plate fluorometry, flow cytometry, and fluorescence microscopy. Targeted adhesion of cRGD-MB was demonstrated in an in vitro flow adhesion assay against recombinant murine αvβ3 integrin protein and αvβ3 integrin-expressing endothelial cells (bEnd.3). The specificity of cRGD-MB for αvβ3 integrin was demonstrated by treating bEnd.3 EC with a blocking antibody. A murine model of mammary carcinoma was used to assess targeted adhesion and ultrasound molecular imaging in vivo. The targeted MB were visualized using a low MI contrast imaging pulse sequence, and quantified by intensity normalization and 2-dimensional Fourier transform analysis.

Results: The cRGD ligand concentration on the MB surface was ∼8.2 × 10(6) molecules per MB. At a wall shear stress of 1.0 dynes/cm, cRGD-MB exhibited 5-fold higher adhesion to immobilized recombinant αvβ3 integrin relative to nontargeted MB and cRAD-MB controls. Similarly, cRGD-MB showed significantly greater adhesion to bEnd.3 EC compared with nontargeted MB and cRAD-MB. In addition, cRGD-MB, but not nontargeted MB or cRAD-MB, showed significantly enhanced contrast signals with a high tumor-to-background ratio. The adhesion of cRGD-MB to bEnd.3 was reduced by 80% after using anti-αv monoclonal antibody to treat bEnd.3. The normalized image intensity amplitude was ∼0.8, 7 minutes after the administration of cRGD-MB relative to the intensity amplitude at the time of injection, while the spatial variance in image intensity improved the detection of bound agents. The accumulation of cRGD-MB was blocked by preadministration with an anti-αv blocking antibody.

Conclusions: The results demonstrate the functionality of a novel MB contrast agent covalently coupled to an RGD peptide for ultrasound molecular imaging of αvβ3 integrin and the feasibility of quantitative molecular ultrasound imaging with a low MI.

Figure 1

Characterization of cRGD-bearing microbubbles. (A) Density of fluorescently labeled cRGD peptide on the surface of the microbubble was measured with fluorescence spectroscopy. (B) Size distribution (by number) of cRGD and cRAD control microbubbles, measured by electrozone sensing. (C) Fluorescence histogram generated by flow cytometry of unlabelled control and FITC-cRGD bearing microbubbles demonstrates significant peptide bound to microbubbles. (D) Fluorescently-labeled cRGD on the microbubble surface is shown by epifluorescence microscopy. Microbubbles are denoted by white arrows. Data are presented as mean +/− standard deviation for n=4 independent experiments. *p<0.01.

Figure 2

In vitro microbubble adhesion assays. (A) Characteristic microscopic fields of view for cRGD-MB on recombinant α_vβ₃ integrin (left) and casein (right) surfaces. (B) Functional adhesion of cRGD and control cRAD microbubbles to recombinant α_vβ₃ integrin at a wall shear stress of 1.0 dyne/cm². Adhesion of cRGD-MB was significantly reduced in the presence of an anti- α_vβ₃ integrin antibody. (C) Flow cytometry results demonstrating α_vβ₃ integrin expression on bEND.3 murine endothelial cells. (D) Functional adhesion of cRGD and control cRAD microbubbles to bend.3 cells at 1.0 dyn/cm² in the presence of blocking antibodies against various subunits of α_vβ₃ integrin. Data are presented as mean +/− standard deviation for n=5 independent experiments. *p<0.01 versus all negative control conditions.

Figure 3

Characterization of Met-1 model of mammary carcinoma in mouse. Representative ultrasound images of tumors two weeks after inoculation in (A) B-mode and (B) CPS mix mode without microbubbles. (C) H&E staining shows morphology of tumor and adjacent tissue. (D) Representative field of view showing CD31 expression by immunohistochemistry in tumor. (E)-(F) Representative confocal fluorescence images of tumor sections after administration of FITC-lectin in the absence (E) and presence (F) of DiI-labeled cRGD microbubbles. Green color (FITC-lectin) delineates endothelium, and fluorescently-labeled MB (DiI) are shown in red (arrows). (G) – (H) Confocol fluorescence images of immunohistochemical sections showing CD31 (green), α_vβ₃ integrin (red), and DAPI (blue).

Figure 4

Schematic showing contrast ultrasound imaging strategy and quantitative image analysis. (A) ten-second sequences of images were acquired in CPS mode immediately after MB administration, after a seven-minute dwell period, and after MB destruction. (B) Image sequences were processed offline to determine the average image intensity within regions of interest.

Figure 5

Analysis of contrast ultrasound imaging with cRGD-MB. (A-C) Representative single frame contrast US images (A) immediately after cRGD-MB administration. (B) after a seven-minute dwell period, and (C) after cRGD-MB destruction. Regions of interest are indicated as follows: Solid circle indicates tumor; wide dashed circle indicates adjacent tissue; and narrow dashed circle indicates the feeding blood vessel (D-F) Images corresponding to time points in A-C were averaged over 50 frames in over to suppress the echoes from circulating MB. (G-I) Images from D-F were normalized to match the spatially-integrated intensity from the time of injection with the spatial integral after the seven-minute circulation, demonstrating differences in speckle between the time of injection and after accumulation. (J-K) 2D-Fourier transform further differentiates adherent MB from circulating MB. (J) Image Fourier transform from time of injection has 0.77 cycle/mm −6 dB width. (K) Image Fourier transform after seven minutes has 2.8 cycle/mm −6 dB width due to speckle.

Figure 6

Specificity of cRGD-MB adhesion in vivo (A) Normalized image intensities of cRGD, cRAD and non-targeted MB at 7 minutes within tumor, vessel and adjacent tissue regions, respectively. *p<0.03 for cRGD-MB in tumor relative to all other conditions. (B) Normalized intensities of cRGD, cRAD and non-targeted MB are compared before and after subtraction of the post-destruction image. Representative images of (C) cRGD, (D) cRAD and (E) non-targeted microbubbles at the 7-minute time point. Normalized intensities before and after administration of a blocking antibody (F) or soluble peptide (G). Representative images for pre-administration of saline (H), pre-administration of blocking antibody (I), and pre-administration of soluble peptide (J) at 7-minute time point. Scale bars represent 5 mm * p<0.01