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

Christopher R. Anderson, Xiaowen Hu, Hua Zhang, Jose Tlaxca, Anne Emilie Declèves, Robert Houghtaling, Kumar Sharma, Michael Lawrence, Katherine W. Ferrara, Joshua J. Rychak

Research output: Contribution to journalArticle

168 Scopus citations

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 α vβ 3 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 α vβ 3 integrin protein and α vβ 3 integrin-expressing endothelial cells (bEnd.3). The specificity of cRGD-MB for α vβ 3 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 molecules per MB. At a wall shear stress of 1.0 dynes/cm, cRGD-MB exhibited 5-fold higher adhesion to immobilized recombinant α vβ 3 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-α v 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 α vβ 3 integrin and the feasibility of quantitative molecular ultrasound imaging with a low MI.

Original languageEnglish (US)
Pages (from-to)215-224
Number of pages10
JournalInvestigative Radiology
Volume46
Issue number4
DOIs
StatePublished - Apr 2011

Keywords

  • alphavbeta3 integrin
  • microbubble
  • molecular imaging
  • RGD
  • tumor angiogenesis
  • ultrasound contrast

ASJC Scopus subject areas

  • Radiology Nuclear Medicine and imaging

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    Anderson, C. R., Hu, X., Zhang, H., Tlaxca, J., Declèves, A. E., Houghtaling, R., Sharma, K., Lawrence, M., Ferrara, K. W., & Rychak, J. J. (2011). Ultrasound molecular imaging of tumor angiogenesis with an integrin targeted microbubble contrast agent. Investigative Radiology, 46(4), 215-224. https://doi.org/10.1097/RLI.0b013e3182034fed