Peter Hauff

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]

Pulsatile shear and Gja5 modulate arterial identity and remodeling events during flow-driven arteriogenesis

In the developing chicken embryo yolk sac vasculature, the expression of arterial identity genes requires arterial hemodynamic conditions. We hypothesize that arterial flow must provide a unique signal that is relevant for supporting arterial identity gene expression and is absent in veins. We analyzed factors related to flow, pressure and oxygenation in the chicken embryo vitelline vasculature in vivo. The best discrimination between arteries and veins was obtained by calculating the maximal pulsatile increase in shear rate relative to the time-averaged shear rate in the same vessel: the relative pulse slope index (RPSI). RPSI was significantly higher in arteries than veins. Arterial endothelial cells exposed to pulsatile shear in vitro augmented arterial marker expression as compared with exposure to constant shear. The expression of Gja5 correlated with arterial flow patterns: the redistribution of arterial flow provoked by vitelline artery ligation resulted in flow-driven collateral arterial network formation and was associated with increased expression of Gja5. In situ hybridization in normal and ligation embryos confirmed that Gja5 expression is confined to arteries and regulated by flow. In mice, Gja5 (connexin 40) was also expressed in arteries. In the adult, increased flow drives arteriogenesis and the formation of collateral arterial networks in peripheral occlusive diseases. Genetic ablation of Gja5 function in mice resulted in reduced arteriogenesis in two occlusion models. We conclude that pulsatile shear patterns may be central for supporting arterial identity, and that arterial Gja5 expression plays a functional role in flow-driven arteriogenesis.

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.

Development of pH-responsive core–shell nanocarriers for delivery of therapeutic and diagnostic agents

In this paper new dendritic core-shell architectures with pH-labile linkers based on hyperbranched polyglycerol cores and biocompatible poly(ethylene glycol) shells were synthesized which encapsulate the anticancer agent doxorubicin and a dye for near-infrared imaging, an indotricarbocyanine. Acid-sensitive properties of the new nanocarriers and in vitro cytotoxicity of the doxorubicin-nanocarrier are presented as well as preliminary data regarding their toxicity and tumor targeting potential in nude mice.

Dendritic multishell architectures for drug and dye transport

Here we present the efficiency and versatility of newly developed core-multishell nanoparticles (CMS NPs), to encapsulate and transport the antitumor drugs doxorubicin hydrochloride (Dox), methotrexate (Mtx) and sodium ibandronate (Ibn) as well as dye molecules, i.e., a tetrasulfonated indotricarbocyanine (ITCC) and nile red. Structurally, the CMS NPs are composed of hyperbranched poly(ethylene imine) core functionalized by alkyl diacids connected to monomethyl poly(ethylene glycol). In order to evaluate their transport in aqueous media in vitro, we have used and compared SEC, UV, ITC, and NMR techniques. We observed that the CMS NPs were able to spontaneously encapsulate and transport Dox, Mtx and nile red in both organic and aqueous media as determined by SEC and UV-VIS spectroscopy. For the VIS transparent Ibn Isothermal Titration Calorimetric (ITC) experiments show an exothermic interaction with the CMS NPs. The enthalpic stabilization (DeltaH) upon encapsulation was in the order of approximately 7 kcals/mol which indicates stable interaction between Ibn and nanoparticles. A T(1) inversion recovery NMR experiment was carried out for 31P and 1H nuclei of Ibn and an increment of spin-lattice relaxation time for respective nuclei was observed upon encapsulation. CMS NPs were also found to encapsulate ITCC dye with stoichiometry of 6-8 molecules/nanocarrier. For in vivo imaging studies the dye loaded CMS NPs were injected to F9 teratocarcinoma bearing mice and a strong contrast was observed in the tumor tissues compared to free dye after 6 h of administration.

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.

Angiopoietin-2 drives lymphatic metastasis of pancreatic cancer

Lymphatic metastasis constitutes a critical route of disease dissemination, which limits the prognosis of patients with pancreatic ductal adenocarcinoma (PDAC). As lymphangiogenesis has been implicated in stimulation of lymphatic metastasis by vascular endothelial growth factor‐C (VEGF‐C) and VEGF‐D, we studied the effect of the angioregulatory growth factor angiopoietin‐2 (Ang‐2) on PDAC progression. Ang‐2 was found to be expressed in transformed cells of human PDAC specimens, with corresponding Tie‐2 receptors present on blood and lymphatic endothelium. In vitro in PDAC cells, Ang‐2 was subject to autocrine/paracrine TGF‐β stimulation (2‐fold induction, P= 0.0106) acting on the –61‐ to +476‐bp element of the human Ang‐2 promoter. In turn, Ang‐2 regulated the expression of genes involved in cell motility and tumor suppression. Orthotopic PDAC xenografts with forced expression of Ang‐2, but not Ang‐1, displayed increased blood and lymphatic vessel density, and an enhanced rate of lymphatic metastasis (6.7‐ to 9.1‐fold, P<0.01), which was prevented by sequestration of Ang‐2 via coexpression of soluble Tie‐2. Notably, elevated circulating Ang‐2 in patients with PDAC correlated with the extent of lymphatic metastasis. Furthermore, median survival was reduced from 28.4 to 7.7 mo in patients with circulating Ang‐2 ≥ 75th percentile (P=0.0005). These findings indicate that Ang‐2 participates in the control of lymphatic metastasis, constitutes a noninvasive prognostic biomarker, and may provide an accessible therapeutic target in PDAC.—Schulz, P., Fischer, C., Detjen, K. M., Rieke, S., Hilfenhaus, G., von Marschall, Z., Böhmig, M., Koch, I., Kehrberger, J., Hauff, P., Thierauch, K.‐H., Alves, F., Wiedenmann, B., Scholz, A. Angiopoietin‐2 drives lymphatic metastasis of pancreatic cancer. FASEB J. 25, 3325–3335 (2011). www.fasebj.org

Comparison of conventional time-intensity curves vs. maximum intensity over time for post-processing of dynamic contrast-enhanced ultrasound

Our aim was to prospectively compare two post-processing techniques for dynamic contrast-enhanced ultrasound and to evaluate their impact for monitoring antiangiogenic therapy. Thus, mice with epidermoid carcinoma xenografts were examined during administration of polybutylcyanoacrylate-microbubbles using a small animal ultrasound system (40 MHz). Cine loops were acquired and analyzed using time-intensity (TI) and maximum intensity over time (MIOT) curves. Influences of fast (50 microl/2s) vs. slow (50 microl/10s) injection of microbubbles on both types of curves were investigated. Sensitivities of both methods for assessing effects of antiangiogenic treatment (SU11248) were examined. Correlative histological analysis was performed for vessel-density. Mann-Whitney test was used for statistical analysis. Microbubble injection rates significantly influenced upslope, time-to-peak and peak enhancement of conventional TI curves (p<0.05) but had almost no impact on maximum enhancement of MIOT curves (representing relative blood volume). Additionally, maximum enhancement of MIOT curves captured antiangiogenic therapy effects more reliably and earlier (already after 1 day of therapy; p<0.05) than peak enhancement of TI curves. Immunohistochemistry validated the significantly (p<0.01) lower vessel densities in treated tumors and high correlation (R(2)=0.95) between vessel-density and maximum enhancement of MIOT curves was observed. In conclusion, MIOT is less susceptible to variations of the injections speed. It enables to assess changes of the relative blood volume earlier and with lower standard deviations than conventional TI curves. It can easily be translated into clinical practice and thus may provide a promising tool for cancer therapy monitoring.

High-Resolution Ultrasound Imaging: A Novel Technique for the Noninvasive in Vivo Analysis of Endometriotic Lesion and Cyst Formation in Small Animal Models

Endometriosis, the presence of endometrial tissue at ectopic sites, is a highly prevalent gynecological disease severely affecting a patients quality of life. To analyze the mechanisms involved in the disease and to identify new molecular targets for effective therapies, small animal models are an important approach. Herein, we report the first use of high-resolution ultrasound imaging for the in vivo analysis of intraperitoneal endometriotic lesions in mice. This noninvasive technology allows for the repetitive quantitative analysis of growth, cyst development, and adhesion formation of endometriotic lesions with a low intra- and interobserver variability. Moreover, it enables one to easily differentiate between endometrial cysts and stroma. Accordingly, volume measurements of both endometrial cysts and stroma indicated that the initial establishment of endometriotic lesions is associated with enhanced cellular proliferation, followed by a phase of increased secretory activity of endometrial glands. Results of ultrasound analysis correlated well with measurements of lesion volumes by caliper and histology. Importantly, ultrasound imaging could be performed repetitively and noninvasively and reflected best the in vivo situation. The technique could further be demonstrated to successfully monitor the significant inhibition of growth of endometriotic lesions after specific estrogen receptor destabilizator treatment. Thus, high-resolution ultrasound imaging represents an important tool for future preclinical small animal studies, which address the pathophysiology of endometriosis and the development of new treatment strategies.

Sagopilone Inhibits Breast Cancer Bone Metastasis and Bone Destruction Due to Simultaneous Inhibition of Both Tumor Growth and Bone Resorption

Purpose: Bone metastases have a considerable impact on quality of life in patients with breast and other cancers. Tumors produce osteoclast-activating factors, whereas bone resorption promotes the growth of tumor cells, thus leading to a “vicious cycle” of bone metastasis. Sagopilone, a novel, fully synthetic epothilone, inhibits the growth of breast cancer cells in vitro and in vivo, and here we report its activity in the MDA-MB-231(SA) breast cancer bone metastasis mouse model. Experimental Design: The potency of sagopilone was determined in treatment models simulating the adjuvant (preventive) and metastatic (therapeutic) settings in the clinic. Results: We showed that sagopilone inhibited tumor burden and bone destruction, in addition to reducing tumor-induced cachexia and paraplegia. The reduction in osteolytic lesions, tumor growth in bone, and weight loss was statistically significant in the preventive model compared with the vehicle group. In the therapeutic model, sagopilone treatment significantly lowered the number of activated osteoclasts and significantly reduced the osteolytic lesion area, bone volume loss, and bone resorption compared with vehicle treatment while simultaneously inhibiting tumor burden. An in vitro assay confirmed that sagopilone inhibited osteoclast activation without cytotoxic effects, whereas paclitaxel resulted in lower inhibition and high levels of cytotoxicity. Conclusions: Sagopilone seems to inhibit the vicious cycle at both the tumor growth and bone resorption stages, suggesting the possibility for substantial benefit in the treatment of patients with breast cancer at risk from bone metastases or with bone lesions already present. Phase II clinical trials with sagopilone in patients with breast cancer are ongoing.