Jochen Huppert

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

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 αvβ3 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 αvβ3 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 αvβ3 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 αvβ3 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. [Mol Cancer Ther 2008;7(1):101–9]

Functional and molecular ultrasound imaging: concepts and contrast agents.

Contrast-enhanced ultrasound has shown convincing results for monitoring vessel morphology, surrogate markers of vascularization and changes in molecular marker expression in oncological and cardiovascular diseases. Ultrasound contrast agents have the ability to increase the backscattering signal intensity of an ultrasound pulse. An interesting class of ultrasound contrast agents are gas filled microbubbles, which can be synthesized by external bubble encapsulation using sugar matrices or microspheres consisting of lipids or polymers with or without surfactant and by selecting gases with low blood solubility and diffusion coefficient such as perfluorocarbons or sulphur hexafluoride. Ultrasound contrast agents can be classified according to the rigidity of their shell. Soft-shell microbubbles are coated with a thin monolayer of surfactant molecules such as palmitic acid or phospholipids and are very sensitive to pressure changes. Hard-shell microbubbles have a rigid shell made of polymers such as polycyanoacrylate, which dramatically increases their stability. Depending on the acoustic properties of the microbubbles and on the purpose of the examination either destructive or non destructive methods are preferred for their detection. Microbubbles can be detected by destructive and non-destructive methods. Both soft- and hard-shell microbubbles coated with target-specific molecules can also be used for molecular imaging. Using target-specific approaches, the expression of several angiogenic markers such as VEGFR2, alphavbeta3 Integrins, ICAM, and E-selectin has been investigated in neoplastic and vascular diseases. This article summarizes the synthesis and properties of contrast agents as well as the indications, limitations and future potential of contrast-enhanced functional and molecular ultrasound.

RGD-labeled USPIO Inhibits Adhesion and Endocytotic Activity of αvβ3-Integrin–expressing Glioma Cells and Only Accumulates in the Vascular Tumor Compartment

PURPOSE To investigate the biologic effect of arginine-glycine-aspartic acid (RGD)-labeled ultrasmall superparamagnetic iron oxide (USPIO) (referred to as RGD-USPIO) on human umbilical vein endothelial cells (HUVECs), ovarian carcinoma (MLS) cells, and glioblastoma (U87MG) cells and on U87MG xenografts in vivo. MATERIALS AND METHODS All experiments were approved by the governmental review committee on animal care.USPIOs were coated with integrin-specific (RGD) or unspecific (arginine-alanine-aspartic acid [RAD]) peptides. USPIO uptake in HUVECs, MLS cells, and U87MG cells and in U87MG tumor xenografts was determined with T2 magnetic resonance (MR) relaxometry in 16 nude mice. Cells and tumors were characterized by using immunofluorescence microscopy. Trypan blue staining and lactate dehydrogenase assay were used to assess cytotoxicity. Statistical evaluation was performed by using a Mann-Whitney test or a linear mixed model with random intercept for the comparison of data from different experiments. Post hoc pairwise comparisons were adjusted according to a Tukey test. RESULTS HUVECs and MLS cells internalized RGD-USPIOs significantly more than unspecific probes. Controversially, U87MG cells accumulated RGD-USPIOs to a lesser extent than USPIO. Furthermore, only in U87MG cells, free RGD and alpha(v)beta(3) integrin-blocking antibodies strongly reduced endocytosis of nonspecific USPIOs. This was accompanied by a loss of cadherin-dependent intercellular contacts, which could not be attributed to cell damage. In U87MG tumors, RGD-USPIO accumulated exclusively at the neovasculature but not within tumor cells. The vascular accumulation of RGD-USPIO caused significantly higher changes of the R2 relaxation rate of tumors than observed for USPIO. CONCLUSION In glioma cells with unstable intercellular contacts, inhibition of alpha(v)beta(3) integrins by antibodies and RGD and RGD-USPIO disintegrated intercellular contacts and reduced endocytotic activity, illustrating the risk of inducing biologic effects by using molecular MR probes.

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

High-frequency volumetric Power Doppler ultrasound (HF-VPDU) captures flow-dependent signals in blood vessels and can be used to assess antiangiogenic therapy effects in rodent tumors. However, the sensitivity is limited to vessels larger than capillaries. Contrast-enhanced HF-VPDU reveals all perfused vessels by assessing stimulated acoustic emissions from disintegrating microbubbles. Thus, we investigated whether flow-sensitive and contrast-enhanced HF-VPDU can depict different vessel fractions and assess their early response to antiangiogenic therapy. Mice with A431 tumors were scanned before and after administration of polybutylcyanoacrylate microbubbles by HF-VPDU. Animals received either antiangiogenic treatment (SU11248) or a control substance and were imaged repeatedly over 9 days. At each time point, tumors were removed for immunohistochemical analysis. During growth of untreated tumors, vascularization decreased correspondingly on flow-sensitive and contrast-enhanced scans. Treated tumors showed a significantly (P < 0.05) stronger decline in vascularization than controls, which was more pronounced in contrast-enhanced scans. Surprisingly, whereas vascularization remained low in contrast-enhanced scans, flow-sensitive ultrasound indicated a reincrease after day 6 with a higher vascularization than the controls at day 9. Histologic evaluation indicated that immature vessels degraded markedly on therapy, whereas large mature vessels on the tumor periphery were more therapy resistant and drew closer due to tumor shrinkage. In conclusion, contrast-enhanced HF-VPDU and flow-sensitive HF-VPDU are both capable of assessing the effects of antiangiogenic therapy. Because contrast-sensitive ultrasound is more sensitive for small immature vessels and flow-sensitive ultrasound mostly captures large vessels at the tumor periphery, the combination of both methods can provide evidence of vascular maturity in tumors.

Assessment of vascular remodeling under antiangiogenic therapy using DCE-MRI and vessel size imaging†

To assess vascular remodeling in tumors during two different antiangiogenic therapies with dynamic contrast‐enhanced magnetic resonance imaging (DCE‐MRI) and vessel size imaging and to evaluate the vessel size index (VSI) as a novel biomarker of therapy response.

Pharmacodynamics of streptavidin-coated cyanoacrylate microbubbles designed for molecular ultrasound imaging

Objectives:To assess the pharmacodynamic behavior of cyanoacrylate, streptavidin-coated microbubbles (MBs) and to investigate their suitability for molecular ultrasound imaging. Materials and Methods:Biodistribution of MBs was analyzed in tumor-bearing mice using &ggr;-counting, immunohistochemistry, flow cytometry, and ultrasound. Further, vascular endothelial growth factor receptor 2-antibody coupled MBs were used to image tumor neovasculature. Results:After 1 minute >90% of MBs were cleared from the blood and pooled in the lungs, liver, and spleen. Subsequently, within 1 hour a decent reincrease of MB-concentration was observed in the blood. The remaining MBs were removed by liver and spleen macrophages. About 30% of the phagocytosed MBs were intact after 48 hours. Shell fragments were found in the kidneys only. No relevant MB-accumulation was observed in tumors. In contrast, vascular endothelial growth factor receptor 2-specific MBs accumulated significantly within the tumor vasculature (P < 0.05). Conclusions:The pharmacokinetic behavior of streptavidin-coated cyanoacrylate MBs has been studied. In this context, the low amount of MBs in tumors after >5 minutes is beneficial for specific targeting of angiogenesis.