Targeted contrast-enhanced ultrasound imaging of tumor angiogenesis with contrast microbubbles conjugated to integrin-binding knottin peptides

Abstract

Targeted contrast-enhanced ultrasound imaging is increasingly being recognized as a powerful imaging tool for the detection and quantification of tumor angiogenesis at the molecular level. The purpose of this study was to develop and test a new class of targeting ligands for targeted contrast-enhanced ultrasound imaging of tumor angiogenesis with small, conformationally constrained peptides that can be coupled to the surface of ultrasound contrast agents.

Methods: Directed evolution was used to engineer a small, disulfide-constrained cystine knot (knottin) peptide that bound to alpha(v)beta(3) integrins with a low nanomolar affinity (Knottin(Integrin)). A targeted contrast-enhanced ultrasound imaging contrast agent was created by attaching Knottin(Integrin) to the shell of perfluorocarbon-filled microbubbles (MB-Knottin(Integrin)). A knottin peptide with a scrambled sequence was used to create control microbubbles (MB-Knottin(Scrambled)). The binding of MB-Knottin(Integrin) and MB-Knottin(Scrambled) to alpha(v)beta(3) integrin-positive cells and control cells was assessed in cell culture binding experiments and compared with that of microbubbles coupled to an anti-alpha(v)beta(3) integrin monoclonal antibody (MB(alphavbeta3)) and microbubbles coupled to the peptidomimetic agent c(RGDfK) (MB(cRGD)). The in vivo imaging signals of contrast-enhanced ultrasound with the different types of microbubbles were quantified in 42 mice bearing human ovarian adenocarcinoma xenograft tumors by use of a high-resolution 40-MHz ultrasound system.

Results: MB-Knottin(Integrin) attached significantly more to alpha(v)beta(3) integrin-positive cells (1.76 +/- 0.49 [mean +/- SD] microbubbles per cell) than to control cells (0.07 +/- 0.006). Control MB-Knottin(Scrambled) adhered less to alpha(v)beta(3) integrin-positive cells (0.15 +/- 0.12) than MB-Knottin(Integrin). After blocking of integrins, the attachment of MB-Knottin(Integrin) to alpha(v)beta(3) integrin-positive cells decreased significantly. The in vivo ultrasound imaging signal was significantly higher after the administration of MB-Knottin(Integrin) than after the administration of MB(alphavbeta3) or control MB-Knottin(Scrambled). After in vivo blocking of integrin receptors, the imaging signal after the administration of MB-Knottin(Integrin) decreased significantly (by 64%). The imaging signals after the administration of MB-Knottin(Integrin) were not significantly different in the groups of tumor-bearing mice imaged with MB-Knottin(Integrin) and with MB(cRGD). Ex vivo immunofluorescence confirmed integrin expression on endothelial cells of human ovarian adenocarcinoma xenograft tumors.

Conclusion: Integrin-binding knottin peptides can be conjugated to the surface of microbubbles and used for in vivo targeted contrast-enhanced ultrasound imaging of tumor angiogenesis. Our results demonstrate that microbubbles conjugated to small peptide-targeting ligands provide imaging signals higher than those provided by a large antibody molecule.

FIGURE 1

Representative images of integrin-positive (A–D) and integrin-negative (E–H) cells exposed to MB-Knottin_Integrin (A and E), MB_cRGD (B and F), MB_αvβ3 (C and G), and MB-Knottin_Scrambled (D and H) under flow shear stress conditions in flow chamber cell culture attachment studies. Microbubbles are small, rounded white structures.

FIGURE 2

Targeted contrast-enhanced ultrasound imaging with MB-Knottin_Integrin (A) and MB_αvβ3 (B) in nude mouse with subcutaneous human ovarian adenocarcinoma xenograft tumor (arrows). Two-dimensional ultrasound imaging was performed in same session, with 30 min between injections to allow clearance of previously injected microbubbles. Molecular imaging signal from attached microbubbles is color coded as green signal overlaid on gray scale ultrasound image and was higher after MB-Knottin_Integrin injection than after MB_αvβ3 injection in this tumor. Because subcutaneous human ovarian cancer xenograft tumors are poorly vascularized, only low, but substantial, targeted contrast-enhanced ultrasound imaging signal can be measured. In addition, low signal is visible at tumor boundaries, most likely from microbubbles attached to angiogenic vessels in tissue surrounding tumor.

FIGURE 3

Subcutaneous human ovarian adenocarcinoma xenograft tumor (arrows) imaged with targeted contrast-enhanced ultrasound imaging after intra-venous injection of MB-Knottin_Integrin (A) and MB-Knottin_Scrambled (control) (B). Imaging signal obtained after MB-Knot-tin_Integrin injection was substantially higher than that obtained after MB-Knottin_Scrambled (control) injection.

FIGURE 4

Transverse color-coded ultrasonic images of subcutaneous human ovarian adenocarcinoma xenograft tumor (arrows) in nude mouse. Imaging with MB-Knottin_Integrin was performed before (A) and 30 min after (B) intravenous administration of blocking c(RGDyK). Targeted contrast-enhanced ultrasound imaging signal, shown as green signal overlaid on gray scale image, was substantially reduced after administration of blocking peptide. Low signal is visible at tumor boundaries, most likely from microbubbles attached to angiogenic vessels in tissue surrounding tumor.

FIGURE 5

Micrograph of frozen human ovarian adenocarcinoma xenograft tumor tissue slices after immunofluorescence staining for both β₃ integrin subunit (A) and CD31 (B). CD31 was used as marker of tumor endothelial cells. Immunofluorescence images show tissue staining of mouse β₃ integrin subunit (red color) and mouse CD31 (green color). Image resulting from merging of those images (staining of β₃ integrin subunit and CD31) (C) demonstrates that β₃ integrin subunit is localized on tumor vessel endothelial cells (arrows; yellow on image resulting from merging confirms colocalization). β₃ integrin was visualized with AlexaFluor 594 (red), and CD31 was visualized with AlexaFluor 488 (green). Original magnification, ×400.