Juergen Jenne

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]

Apoptosis signals in lymphoblasts induced by focused ultrasound

We investigated the effects of focused ultrasound (FUS) on specific molecular signaling and cellular response in three closely related human Tk6 lymphoblast cell lines that differed only in their p53 status. The applied ultrasound parameters fell between the physical dose range, which is safely used in medical diagnostics (peak pressure<0.1 MPa) and that used for high‐energy FUS thermal ablation therapy (peak pressure>10 MPa). Based on cDNA microarrays and protein analysis, we found that FUS at the intermediate peak pressure of 1.5 MPa induced a complex signaling cascade with upregulation of proapoptotic genes [e.g., p53, p21, Thy1 (CD 90)]. Simultaneously, FUS downregulated cellular survival components (e.g., bcl‐2, SOD). The p53 status was important for the reaction of the cells to ultrasound. Apoptosis and G1 arrest were induced primarily in p53+ cells, while p53‐ cells showed less apoptosis but exhibited G2 arrest. Likewise, the proliferation of lymphoblasts was much more strongly inhibited in p53+ than in p53‐ cells. Microarray analysis further demonstrated an upregulation of genes involved in oxidative stress (e.g., ferritin), suggesting that indirect sonochemical effects via reactive oxygen species play a causative role in the interaction of ultrasound with lymphoblasts. An important characteristic of FUS in therapeutic ultrasound applications is its ability to be administered to the human body in a targeted manner while sparing intermediate tissues. Therefore, our data indicate that this noninvasive, mechanical wave transmission, which is free of ionizing radiation, has the potential to specifically induce localized cell signals and apoptosis.

A comparison of shock wave and sinusoidal-focused ultrasound-induced localized transfection of HeLa cells

Both shock waves and sinusoidal continuous wave ultrasound can mediate DNA transfer into cells. The relative transfection efficiencies of different ultrasound modalities are unclear. The purpose of this paper is to compare the transfection efficiency of lithotripter shock waves and focused sinusoidal ultrasound in vitro. HeLa cells were transfected with beta-galactosidase and luciferase plasmid DNA reporter. Shock waves were generated by an electromagnetic sound source. Sixty to 360 pulses at 1 Hz pulse frequency were administered at 13, 16 or 19 kV capacitor voltage. Sinusoidal focused ultrasound was generated by a single focus piezoceramic air-backed disk transducer at a carrier frequency of 1.18 MHz operated in a pulsed mode. Compared to cells mixed with DNA only, shock waves induced up to eightfold more transfected cells at a cell viability of 5%, while sinusoidal-focused ultrasound induced up to 80-fold more transfected cells at a cell viability of 45%. The corresponding transfection efficiencies of the HeLa cells were 0.08% for shock waves and 3% for focused ultrasound. These results may contribute to the selection of the ultrasound modality as a localized, noninvasive and safe tool to mediate gene transfer.

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.

Focal gene induction in the liver of rats by a heat-inducible promoter using focused ultrasound hyperthermia: preliminary results.

Objective:We sought to examine high-intensity focused ultrasound (HIFU)-induced hyperthermia in the liver of a rat model to focally induce green-fluorescent protein (GFP). Materials and Methods:A total of 25 Copenhagen rats were included in this study. Rats were divided into groups treated with an adenovirus coding for green fluorescent protein (GFP) under the control of a hsp70B promoter and a CMV promoter. Ad-CMV-GFP-treated rats served as positive control. Untreated controls only subjected to MRI ± HIFU-treatment served to find out optimal power of HIFU in the target area of the liver. Temperature was noninvasively monitored by temperature sensitive magnetic resonance imaging (MRI). Results:Rats treated with Ad-hsp70B-GFP demonstrated localized gene induction within the liver parenchyma, in good correlation with MRI and histology. Applying an acoustic power of 1.92 W a relatively uniform focal temperature up to 42 ± 5°C within the liver parenchyma could be documented. 3 × 109 plaque-forming units proved to account for a very homogeneous liver infection. Number of fluorescent cells in the region of hyperthermia was similar to the control group treated with Ad-CMV-GFP. Conclusion:Using the introduced parameters spatially controlled gene induction within a parenchymal organ such as the liver in rats using HIFU under control of MRI is feasible.

Egg White Phantoms for HIFU

We used fresh egg white and polyacrylamide to create a transparent tissue mimicking phantom. Heating of phantoms by HIFU leads to egg white protein denaturation and creation of visible white lesions. We measured the acoustical and thermal properties and investigated the possibility to use such phantoms to study the lesion formation during the HIFU therapy.

Abstract B206: FG-3019, an anti-CTGF antibody, attenuates tumor invasion, delays tumor growth, and prolongs survival alone and in combination with radiation in an orthotopic glioma model.

Objective: Despite progress in understanding molecular changes in glioblastoma multiforme (GBM), it remains a cancer with poor prognosis in need of better therapy. Pathological features of GBM are explosive growth, angiogenesis and its unique diffuse, infiltrative growth pattern. There is a consensus that cell-matrix-interactions play a pivotal role for infiltrative growth. Connective tissue growth factor (CTGF) is a matricellular protein that is involved in many pathological processes including tumorigenesis and invasion. CTGF modulates cell-matrix-interactions that may allow glioma cells to invade brain parenchyma, degrade the extracellular matrix (ECM), and enhance proliferation and angiogenesis in GBM. High CTGF-expression levels have been reported to correlate with tumor grade, invasiveness and poor patient survival. Here the effects of a human monoclonal anti-CTGF-antibody (FG-3019) were investigated alone and in combination with irradiation in vitro and in vivo in an orthotopic glioma model in mice. Methods and materials: In vitro studies were performed with two established human glioma cell lines, U87MG and T98G, as well as with primary isolated human glioblastoma cancer stem-like cells (CSLCs) cells. The effects of FG-3019 alone and in combination with photon irradiation (6 MV Linac) on clonogenic survival, proliferation, migration, neurosphere formation, and limiting dilution were determined. RNA was isolated from CSLCs and gene expression changes were examined after different treatments using micro-arrays and RT-PCR. In vivo, CSLCs were subcultured as neurospheres and stereotactically implanted into brains of SCID-beige mice. Mice brains were treated with 7Gy single dose radiation +/− FG-3019. The treatment effects were assessed by MRI, survival time and histology. Results: Tumor growth delay in the orthotopic tumor model resulted from treatment with radiation or FG-3019 alone, which was significantly enhanced by combined treatment. Similarly, animal survival was prolonged by each monotherapy and significantly enhanced by the combination. Histology showed that FG-3019 significantly reduced constitutive and radiation-induced tumor invasion into the brain parenchyma. FG-3019 also reduced intracellular CTGF expression, glioma cell proliferation, deposition of different types of collagen and reduced microvessel density. FG-3019 also reduced expression of Nestin and the stem cell marker SOX-2. In vitro, FG-3019 and irradiation reduced clonogenicity, proliferation and migration of U87MG, T98G and CSLCs, which was enhanced by the combination. Gene expression analysis revealed that FG-3019 down regulated tumorigenesis- and tumor-invasion-related genes. Conclusions: The human monoclonal CTGF antibody, FG-3019, shows remarkable anti-tumorigenic and anti-invasive effects in vitro and in vivo in a primary human glioblastoma model growing orthotopically in mice. Our results indicate that FG-3019 alone and in combination with radiotherapy is a promising approach for clinical translation in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B206.