Molecular profiling of angiogenesis with targeted ultrasound imaging: early assessment of antiangiogenic therapy effects

Abstract

Molecular ultrasound is capable of elucidating the expression of angiogenic markers in vivo. However, the capability of the method for volumetric ''multitarget quantification'' and for the assessment of antiangiogenic therapy response has rather been investigated. Therefore, we generated cyanoacrylate microbubbles linked to vascular endothelial growth factor receptor 2 (VEGFR2) and A v B 3 integrin binding ligands and quantified their accumulation in squamous cell carcinoma xenografts (HaCaT-ras-A-5RT3) in mice with the quantitative volumetric ultrasound scanning technique, sensitive particle acoustic quantification. Specificity of VEGFR2 and A v B 3 integrin binding microbubbles was shown, and changes in marker expression during matrix metalloproteinase inhibitor treatment were investigated. In tumors, accumulation of targeted microbubbles was significantly higher compared with nonspecific ones and could be inhibited competitively by addition of the free ligand in excess. Also, multimarker imaging could successfully be done during the same imaging session. Molecular ultrasound further indicated a significant increase of VEGFR2 and A v B 3 integrin expression during tumor growth and a considerable decrease in both marker densities after matrix metalloproteinase inhibitor treatment. Histologic data suggested that the increasing VEGFR2 and A v B 3 integrin concentrations in tumors during growth are related to an up-regulation of its expression by the endothelial cells, whereas its decrease under therapy is more related to the decreasing relative vessel density. In conclusion, targeted ultrasound appears feasible for the longitudinal molecular profiling of tumor angiogenesis and for the sensitive assessment of therapy effects in vivo.

Vessel Fractions in Tumor Xenografts Depicted by Flow- or Contrast-Sensitive Three-Dimensional High-Frequency Doppler Ultrasound Respond Differently to Antiangiogenic Treatment

“…Purified by repeated flotation, airfilled microbubbles were partially hydrolyzed by exposure to sodium hydroxide solution. With this, f10% of the polymer mass was hydrolyzed and the stability of the microbubbles reduced (22), enabling them to be destroyed by sonication above a mechanical index of 0.25 (23). Size distribution of the microbubbles was measured with a particle counter (Multisizer-3, Beckman Coulter).…”

Squamous Cell Carcinoma Xenografts: Use of VEGFR2-targeted Microbubbles for Combined Functional and Molecular US to Monitor Antiangiogenic Therapy Effects

“…In addition, the use of molecularly targeted microbubbles at US has emerged as a very suitable method in preclinical settings, allowing noninvasive analysis of tumor angiogenesis and antiangiogenic therapy effects (10)(11)(12)(13). Consequently, additional preclinical studies have demonstrated the feasibility of targeting VEGFR2 for the detection of early tumor lesions and antiangiogenic therapy responses (14)(15)(16)(17)(18). The specific accumulation of targeted microbubbles at the pathologic site can be assessed by subtracting the US imaging signal after applying a destructive pulse from that before the destructive pulse (11).…”

Multiplexed imaging for diagnosis and therapy

“…However, most of these techniques are only capable of near-simultaneous multiplexing. This holds also true for multiparametric MRI (that is, T2-weighted imaging combined with diffusion-weighted (DW), dynamic contrast-enhanced (DCE) MRI and spectroscopic imaging) and contrast-enhanced ultrasound imaging using targeted microbubbles 116 . Although these methods can image multiple biological contrasts in the same imaging session, the acquisition is sequential.…”