Jessica Huamani

Molecular Imaging of Vascular Endothelial Growth Factor Receptor 2 Expression Using Targeted Contrast-Enhanced High-Frequency Ultrasonography

The aim of our study was to investigate the use of targeted contrast‐enhanced high‐frequency ultrasonography for molecular imaging of vascular endothelial growth factor receptor 2 (VEGFR2) expression on tumor vascular endothelium in murine models of breast cancer.

Relationship between retention of a vascular endothelial growth factor receptor 2 (VEGFR2)-targeted ultrasonographic contrast agent and the level of VEGFR2 expression in an in vivo breast cancer model.

Objective. The aim of this study was to characterize the relationship between retention of a vascular endothelial growth factor receptor 2 (VEGFR2)‐targeted ultrasonographic contrast agent (UCA) and VEGFR2 expression in tumor vasculature of breast cancer. Methods. 67NR breast cancer tumors implanted in mice were evaluated in vivo with both VEGFR2‐targeted and nontargeted UCAs, and a high‐frequency ultrasound system. A bolus of the UCA was injected and allowed to circulate for 4 minutes to allow binding of targeted microbubbles. After that, 2 sets of images before and after a high‐power ultrasonic destruction sequence were acquired. The average video intensity of predestruction and postdestruction images was measured and used as a relative measure of retention of the UCA in the tumor. Levels of VEGFR2 expression and tumor vascular density were quantified by immunohistochemical staining and compared with retention of the VEGFR2‐targeted UCA. Results. Retention of VEGFR2‐targeted microbubbles in tumors was significantly higher than retention of nontargeted microbubbles (mean ± SD, 47.75 ± 9.85 versus 18.5 ± 5.46 dB; P < .001). Retention of the VEGFR2‐targeted UCA was found to correlate with the level of VEGFR2 expression in the studied tumors (r2 = 0.41). In contrast, retention of the nontargeted UCA was not correlated with the level of VEGFR2 expression (r2 = 0.08). Furthermore, retention of the VEGFR2‐targeted UCA was not correlated with the level of tumor vascularity. Conclusions. The magnitude of the molecular ultrasonographic signal from a VEGFR2‐targeted UCA retained by tissue correlates with VEGFR2 expression. These results validate the use of molecular ultrasonography for in vivo detection and quantification of VEGFR2 expression in this breast cancer model.

Measuring Tumor Perfusion in Control and Treated Murine Tumors Correlation of Microbubble Contrast-Enhanced Sonography to Dynamic Contrast-Enhanced Magnetic Resonance Imaging and Fluorodeoxyglucose Positron Emission Tomography

The purpose of this study was to evaluate the ability of dynamic microbubble contrast‐enhanced sonography (MCES), in comparison with dynamic contrast‐enhanced magnetic resonance imaging (DCE‐MRI) and fluorodeoxyglucose positron emission tomography (FDG‐PET), to quantitatively characterize tumor perfusion in implanted murine tumors before and after treatment with a variety of regimens.

Correlation Between Estimates of Tumor Perfusion From Microbubble Contrast-Enhanced Sonography and Dynamic Contrast-Enhanced Magnetic Resonance Imaging

Objective. We compared measurements of tumor perfusion from microbub‐ble contrast‐enhanced sonography (MCES) and dynamic contrast‐enhanced magnetic resonance imaging (DCE‐MRI) in an animal tumor model. Methods. Seven mice were implanted with Lewis lung carcinoma cells on their hind limbs and imaged 14 days later with a Philips 5‐ to 7‐MHz sonography system (Philips Medical Systems, Andover, MA) and a Varian 7.0‐T MRI system (Varian, Inc, Palo Alto, CA). For sonographic imaging 100 μL of a perfluoropropane microbubble contrast agent (Definity; Bristol‐Myers Squibb Medical Imaging, Billerica, MA) was injected and allowed to reach a pseudo steady state, after which a high–mechanical index pulse was delivered to destroy the microbub‐bles within the field of view, and the replenishment of the microbubbles was imaged for 30 to 60 seconds. The MRI included acquisition of a T10 map and 35 serial T1‐weighted images (repetition time, 100 milliseconds; echo time, 3.1 milliseconds; α, 30°) after the injection of 100 μL of 0.2‐mmol/kg gadopente‐tate dimeglumine (Magnevist; Berlex, Wayne, NJ). Region‐of‐interest and voxel‐by‐voxel analyses of both data sets were performed; microbubble contrast‐enhanced sonography returned estimates of microvessel cross‐sectional area, microbubble velocity, and mean blood flow, whereas DCE‐MRI returned estimates of a perfusion‐permeability index and the extravascular extracellular volume fraction. Results. Comparing similar regions of tumor tissue seen on sonography and MRI, region‐of‐interest analyses revealed a strong (r 2 = 0.57) and significant relationship (P < .002) between the estimates of perfusion obtained by the two modalities. Conclusions. Microbubble contrast‐enhanced sonography can effectively depict intratumoral heterogeneity in preclinical xenograft models when voxel‐by‐voxel analysis is performed, and this analysis correlates with similar DCE‐MRI measurements.

Histone deacetylase inhibitor NVP-LAQ824 sensitizes human nonsmall cell lung cancer to the cytotoxic effects of ionizing radiation.

Stage III nonsmall cell lung cancer is primarily treated with combined chemotherapy and radiation therapy. Relapses for progression of disease within irradiated sites remains a primary pattern of failure. To evaluate the interaction between histone deacetylase inhibitors and irradiation in nonsmall cell lung cancer, we studied NVP-LAQ824 in mouse models of human lung cancer. Colony formation assays were performed to determine whether LAQ824 sensitized nonsmall cell lung cancer to the cytotoxic effects of ionizing radiation. LAQ824 reduced clonogenic survival of the H23 and H460 cell lines five-fold compared with controls and four-fold compared with either agent alone (P<0.001). Western blot analysis of caspase cleavage, microscopic analysis of nuclei and Annexin–fluorescein isothiocyanate/propidium iodide flow cytometry assays showed that LAQ824 enhanced radiation-induced apoptosis and attenuated mitosis (P<0.001). Immunostaining for &ggr;-H2AX nuclear foci was performed to determine the effect of LAQ824 on radiation-induced DNA double-strand breaks. Combined modality treatment delayed the resolution of &ggr;-H2AX foci with over 30% of cells staining positive 6 h after treatment versus approximately 5 and 3% in cells treated with LAQ824 or radiation alone (P<0.001). Additionally, an in-vivo xenograft model was utilized to study the effects of fractioned irradiation and LAQ824 on tumor growth. Fractioned irradiation and LAQ824 delayed tumor growth by 19 days versus 7 and 4 days for treatment with LAQ824 and radiation alone. This study shows the effectiveness of histone deacetylase inhibitors to enhance the cytotoxic effects of radiation by attenuating DNA repair and inducing apoptosis in human nonsmall cell lung cancer.

A Method for Assessing the Microvasculature in a Murine Tumor Model Using Contrast-Enhanced Ultrasonography

Objective. The purpose of this study was to develop a method for assessing tumor vascularity in a preclinical model of breast cancer using contrast‐enhanced ultrasonography. Methods. Eight mice were injected with 67NR breast cancer cells on their hind limbs and imaged with ultrasonography 8 days later. Mice were injected with an ultrasound contrast agent (UCA), and a sequence of images of the resultant backscattered echoes was recorded before and after high‐power “destruction” pulses for each of multiple parallel planes. From these, data maps of the maximum contrast enhancement (within each time course) were constructed for each pixel, which enabled reconstruction of high‐resolution coregistered sections into a 3‐dimensional (3D) volume reflecting tumor vascularity. Additional studies were performed to determine the duration and repeatability of image enhancement, and images were correlated with conventional 3D power Doppler measurements. Results. The lifetime of the UCA in vivo was found to be 4.3 ± 1.09 minutes (mean ± SD). The 3D contrast‐enhanced ultrasonographic technique produced images that correlated well with power Doppler images in specific regions but also depicted additional regions of flow surrounding the power Doppler signal. The mean correlation coefficient between voxel measurements of the central slice for each animal was 0.64 ± 0.07 (P < .01). In addition, sequential studies in each animal were reproducible. Conclusions. A method producing high‐resolution volumetric assessments of tumor vascularity in a preclinical model of breast cancer is shown that correlates with other ultrasonographic measures of blood flow, which may provide greater sensitivity to the microvasculature.

Noninvasive Assessment of Tumor Vasculature Response to Radiation-Mediated, Vasculature-Targeted Therapy Using Quantified Power Doppler Sonography Implications for Improvement of Therapy Schedules

Objective. Stereotactic radiotherapy (ablative radiation) is a modality that holds considerable promise for effective treatment of intracranial and extracranial malignancies. Although tumor vasculature is relatively resistant to small fractionated doses of ionizing radiation, large ablative doses of ionizing radiation lead to effective demise of the tumor vasculature. The purpose of this study was (1) to noninvasively monitor and compare tumor physiologic parameters in response to ablative radiation treatments and (2) to use these noninvasive parameters to optimize the schedule of administration of radiation therapy. Methods. Lewis lung carcinoma tumors were implanted into C57BL/6 mice and treated with ablative radiation. The kinetics of change in physiologic parameters of a response to single‐dose 20‐Gy treatments was measured. Parameters studied included tumor blood flow, apoptosis, and proliferation rates. Serial tumor sections were stained to correlate noninvasive Doppler assessment of tumor blood flow with microvasculature histologic findings. Results. A single administration of 20 Gy led to an incomplete tumor vascular response, with subsequent recovery of tumor blood flow within 4 days after treatment. Sustained reduction of tumor blood flow by administering the successive ablative radiation treatment before tumor blood flow recovery led to a 3‐fold tumor growth delay. The difference in tumor volumes at each measurement time point (every 2 days) was statistically significant (P = .016). Conclusions. This study suggests a rational design of schedule optimization for radiation‐mediated, vasculature‐directed treatments guided by noninvasive assessment of tumor blood flow levels to ultimately improve the tumor response.

DIFFERENTIAL EFFICACY OF COMBINED THERAPY WITH RADIATION AND AEE788 IN HIGH AND LOW EGFR-EXPRESSING ANDROGEN-INDEPENDENT PROSTATE TUMOR MODELS

PURPOSE To determine the efficacy of combining radiation (XRT) with a dual epidermal growth factor receptor (EGFR)/vascular endothelial growth factor receptor inhibitor, AEE788, in prostate cancer models with different levels of EGFR expression. METHODS AND MATERIALS Immunoblotting was performed for EGFR, phosphorylated-EGFR, and phosphorylated-AKT in prostate cancer cells. Clonogenic assays were performed on DU145, PC-3, and human umbilical vein endothelial cells treated with XRT +/- AEE788. Tumor xenografts were established for DU145 and PC-3 on hind limbs of athymic nude mice assigned to four treatment groups: (1) control, (2) AEE788, (3) XRT, and (4) AEE788 + XRT. Tumor blood flow and growth measurements were performed using immunohistochemistry and imaging. RESULTS AEE788 effectively decreased phosphorylated-EGFR and phosphorylated-AKT levels in DU145 and PC-3 cells. Clonogenic assays showed no radiosensitization for DU145 and PC-3 colonies treated with AEE788 + XRT. However, AEE788 caused decreased proliferation in DU145 cells. AEE788 showed a radiosensitization effect in human umbilical vein endothelial cells and increased apoptosis susceptibility. Concurrent AEE788 + XRT compared with either alone led to significant tumor growth delay in DU145 tumors. Conversely, PC-3 tumors derived no added benefit from combined-modality therapy. In DU145 tumors, a significant decrease in tumor blood flow with combination therapy was shown by using power Doppler sonography and tumor blood vessel destruction on immunohistochemistry. Maldi-spectrometry (MS) imaging showed that AEE788 is bioavailable and heterogeneously distributed in DU145 tumors undergoing therapy. CONCLUSIONS AEE788 + XRT showed efficacy in vitro/in vivo with DU145-based cell models, whereas PC-3-based models were adequately treated with XRT alone without added benefit from combination therapy. These findings correlated with differences in EGFR expression and showed effects on both tumor cell proliferation and vascular destruction.