Eva C. Woenne

Angiogenesis is essential for the development of malignant tumors and provides important targets for tumor diagnosis and therapy. To noninvasively assess the angiogenic profile of tumors, novel alpha(v)beta(3) integrin-targeted ultrasmall superparamagnetic iron oxide particles (USPIOs) were designed and their specific uptake by endothelial cells was evaluated in vitro and in vivo. USPIOs were coated with 3-aminopropyltrimethoxysilane (APTMS) and conjugated with Arg-Gly-Asp (RGD) peptides. Accumulation in human umbilical vein endothelial cells (HUVECs) was evaluated using Prussian blue staining, transmission electron microscopy, magnetic resonance (MR) imaging, and inductively coupled plasma mass spectrometry. Uptake of RGD-USPIO by HUVECs was significantly increased when compared with unlabeled USPIO and could be competitively inhibited by addition of unbound RGD. The ability of the RGD-USPIO to noninvasively distinguish tumors with high (HaCaT-ras-A-5RT3) and lower (A431) area fractions of alpha(v)beta(3) integrin-positive vessels was evaluated using a 1.5-T MR scanner. Indeed, after RGD-USPIO injection, there was a more pronounced decrease in T(2) relaxation times in HaCaT-ras-A-5RT3 tumors than in A431 tumors. Furthermore, T(2)*-weighted images clearly identified the heterogeneous arrangement of vessels with alpha(v)beta(3) integrins in HaCaT-ras-A-5RT3 tumors by an irregular signal intensity decrease. In contrast, in A431 tumors with predominantly small and uniformly distributed vessels, the signal intensity decreased more homogeneously. In summary, RGD-coupled, APTMS-coated USPIOs efficiently label alpha(v)beta(3) integrins expressed on endothelial cells. Furthermore, these molecular MR imaging probes are capable of distinguishing tumors differing in the degree of alpha(v)beta(3) integrin expression and in their angiogenesis profile even when using a clinical 1.5-T MR scanner.

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 alphavbeta3 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 alphavbeta3 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 alphavbeta3 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 alphavbeta3 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.

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.

PURPOSE: 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. MATERIALS AND METHODS: In two independent experiments, nude mice bearing human skin squamous cell carcinoma xenografts were treated with a vascular endothelial growth factor (VEGF) inhibitor (bevacizumab) or a multitargeted tyrosine kinase inhibitor (SU11248). Changes in tumor vascularity were assessed by DCE-MRI and vessel size imaging. DCE-MRI data were analyzed applying a two-compartment model (Brix), calculating the parameters Amplitude and k(ep). RESULTS: For both experiments Amplitude decreased significantly in treated tumors while k(ep) did not change significantly. VSI showed controversial results. VSI was significantly increased in SU11248-treated A431 tumors, whereas no changes were found in bevacizumab-treated HaCaT-ras-A-5RT3 tumors. Immunohistology confirmed these results and suggest differences in the maturation of tumor vascularization as a possible explanation. CONCLUSION: DCE-MRI and vessel size imaging provide reliable and supplementing biomarkers of antiangiogenic therapy response. The results of both methods are in excellent agreement with histology. Nevertheless, our results also indicate that vascular remodeling is complex and that a uniform response cannot be expected for different tumors and therapies.