Kendra M. Hasebroock

Toxicity of MRI and CT contrast agents

Anatomical and physiological imaging using CT and MRI are playing a critical role in patients’ diagnosis, disease characterization and treatment planning. CT- and MRI-based protocols increasingly require an injection of iodinated CT and gadolinium (Gd)-based MRI contrast media. Although routinely used in clinical practice, iodinated and to a less extent Gd-based contrast media possess side effects: life-threatening contrast-induced nephropathy (CIN) is associated with CT and nephrogenic systemic fibrosis (NSF) with MRI contrast agents. CIN is defined as an acute decline in renal functions (serum creatinine increase > 0.5 mg/dl) after administration of iodinated contrast media. Patients with moderate-to-severe chronic kidney disease are considered the highest risk group for development of CIN. CIN is more common with ionic high-osmolar contrast CT media. NSF is a rare condition characterized by the formation of connective tissue in the skin and systemically in the lung, liver, heart and kidney. Patients with end stage kidney disease, acute kidney injury and stage 4 – 5 chronic kidney disease are at a high risk for NSF. The nonionic linear Gd-chelates are associated with the highest risk of NSF. This review summarizes the incidence, symptoms, safety profile of various CT and MRI contrast agents based on their physiochemical properties.

Renal Inflammation: Targeted Iron Oxide Nanoparticles for Molecular MR Imaging in Mice

PURPOSE To determine the feasibility of T2-weighted magnetic resonance (MR) imaging in the noninvasive quantification of renal inflammation by using superparamagnetic iron oxide (SPIO) nanoparticles targeted to tissue-bound C3 activation fragments in a mouse model of lupus nephritis. MATERIALS AND METHODS All animal procedures were approved by the University of Colorado-Denver animal care and use committee. SPIO nanoparticles were encapsulated by using amine-functionalized phospholipids. A recombinant protein containing the C3d-binding region of complement receptor type 2 (CR2) was then conjugated to the surface of the SPIO nanoparticle. Five MRL/lpr mice (a model of lupus nephritis) and six C57BL/6 wild-type mice were assessed with T2-weighted MR imaging at baseline and after SPIO injection. The same five MRL/lpr mice and three C57BL/6 mice also underwent MR imaging after injection of CR2-targeted SPIO. A series of T2-weighted pulses with 16 echo times was used to enable precise T2 mapping and calculation of T2 relaxation times in the cortex and outer and inner medulla of the kidneys, as well as in the spleen, muscle, and fat. The effects of treatment and animal genotype on T2 relaxation times were analyzed with repeated-measures analysis of variance. RESULTS At baseline, the T2-weighted signal intensity in the kidneys of MRL/lpr mice was higher than that in the kidneys of wild-type mice. Injection of untargeted SPIO did not alter the T2-weighted signal in the kidneys in either strain of mice. Injection of CR2-targeted SPIO in MRL/lpr mice, however, caused a significant accumulation of targeted iron oxide with a subsequent decrease in T2 relaxation times in the cortex and outer and inner medulla of the kidneys. No changes in T2 relaxation time were observed in the wild-type mice after injection of targeted SPIO. CONCLUSION Injection of CR2-conjugated SPIO caused a significant reduction in T2-weighted MR imaging signal and T2 relaxation time in nephritic kidneys.

Silibinin Prevents Lung Tumorigenesis in Wild-Type but not in iNOS−/− Mice: Potential of Real-Time Micro-CT in Lung Cancer Chemoprevention Studies

Purpose: Sustained nitric oxide (NO) generation positively correlates with lung cancer development and progression. Herein, we genetically confirmed this role of iNOS and evaluated the chemopreventive efficacy of silibinin in carcinogen-treated B6/129 wild-type (WT) and iNOS−/− mice. Experimental Design: Male B6/129-Nos2tm1Lau (iNOS−/−) and B6/129PF2 WT mice were injected i.p. with 1 mg/g body weight urethane once weekly for 7 consecutive weeks, followed by silibinin gavage (742 mg/kg body weight) for 5 d/wk for 18 weeks. Results: Quantification of micro-CT data in real-time showed that silibinin significantly decreases urethane-induced tumor number and size in WT mice, consistent with measurements made ex vivo at study termination. Genetic ablation of iNOS decreased urethane-induced tumor multiplicity by 87% (P < 0.001) compared to WT mice. Silibinin decreased tumor multiplicity by 71% (P < 0.01) in WT mice, but did not show any such considerable effect in iNOS−/− mice. Tumors from WT mice expressed more iNOS (P < 0.01) but almost similar eNOS and nNOS than those in silibinin-treated mice. In these tumors, silibinin moderately (P < 0.01) inhibited cell proliferation but strongly (P < 0.01) reduced the number of newly formed nestin-positive microvessels. Silibinin decreased VEGFR2 level, and STAT3 and NF-κB activation in tumors. Conclusions: The lack of effect of silibinin in iNOS−/− mice suggests that silibinin exerts most of its chemopreventive and angiopreventive effects through its inhibition of iNOS expression in lung tumors. Our results support iNOS as a potential target for controlling lung cancer, and demonstrate the value of real-time noninvasive micro-CT imaging modality for evaluating the efficacy of lung cancer chemopreventive agents. Clin Cancer Res; 17(4); 1–9. ©2010 AACR.

Assessment of the In vivo Antitumor Effects of ENMD-2076, a Novel Multitargeted Kinase Inhibitor, against Primary and Cell Line–Derived Human Colorectal Cancer Xenograft Models

Purpose: This in vivo study was designed to investigate the efficacy of ENMD-2076, a small-molecule kinase inhibitor with activity against the Aurora kinases A and B, and several other tyrosine kinases linked to cancer, including vascular endothelial growth factor receptor 2, cKit, and fibroblast growth factor receptor 1, against murine xenograft models of human colorectal cancer (CRC). Experimental Design: HT-29 CRC cell line xenografts were treated with either vehicle or ENMD-2076 (100 or 200 mg/kg) orally daily for 28 days. Tumor growth inhibition, dynamic contrast-enhanced magnetic resonance imaging, and 18FDG-positron emission tomography were conducted to assess the antiproliferative, antiangiogenic, and antimetabolic responses, respectively. Effects on proliferation were also analyzed by immunohistochemical methods. Additionally, three patient-derived xenografts from primary and metastatic sites were treated with ENMD-2076 (100 mg/kg) and assessed for tumor growth inhibition. Results: In the HT-29 xenograft model, ENMD-2076 induced initial tumor growth inhibition followed by regression. Treatment was associated with significant tumor blanching, indicating a loss of vascularity and substantial reductions in tumor vascular permeability and perfusion as measured by dynamic contrast-enhanced magnetic resonance imaging. Positron emission tomography scanning showed significant decreases in 18FDG uptake at days 3 and 21 of treatment, which was associated with a marked reduction in proliferation as assessed by Ki-67. All three of the patient-derived xenografts tested were sensitive to treatment with ENMD 2076 as measured by tumor growth inhibition. Conclusions: ENMD-2076 showed robust antitumor activity against cell line and patient-derived xenograft models of CRC that is detectable by functional imaging, supporting clinical investigation of this agent in CRC. Clin Cancer Res; 16(11); 2989–98. ©2010 AACR.

Detection of glomerular complement C3 fragments by magnetic resonance imaging in murine lupus nephritis

One of the challenges of treating patients with glomerulonephritis is to accurately assess disease activity. As renal biopsies are routinely stained for deposits of C3 activation fragments and glomerular C3 deposits are found in most forms of glomerulonephritis, we sought to determine whether a relatively noninvasive measure of C3 fragment deposition in the kidney can serve as a good biomarker of disease onset and severity. We recently developed a magnetic resonance imaging (MRI)-based method of detecting glomerular C3 and used this to track the progression of renal disease in the MRL/lpr mouse model of lupus nephritis using superparamagnetic iron oxide nanoparticles conjugated to complement receptor type 2 as a targeting agent. Quantitative immunofluorescence showed that glomerular C3b/iC3b/C3d deposition progressively increased with disease activity, a finding replicated by the T2-weighted MRI. The T2 relaxation times decreased with disease activity in the cortex and medulla of the MRL/lpr but not in MRL/Mpj control mice. Thus, MRI contrast agents targeted to glomerular C3 fragments can be used to noninvasively monitor disease activity in glomerulonephritis. As therapeutic complement inhibitors are used in patients with renal disease, this method, should it become feasible in humans, may identify those likely to benefit from complement inhibition.

A novel apparatus for lateral fluid percussion injury in the rat

Lateral fluid percussion injury (LFPI) is the most commonly used experimental model of human traumatic brain injury (TBI). To date, investigators using this model have produced injury using a pendulum-and-piston-based device (PPBD) to drive fluid against an intact dural surface. Two disadvantages of this method, however, are (1) the necessary reliance on operator skill to position and release the pendulum, and (2) reductions in reproducibility due to variable friction between the pistons o-rings and the cylinder. To counteract these disadvantages, we designed a low-priced, novel, fluid percussion apparatus that delivers a pressure pulse of air to a standing column of fluid, forcing it against the intact dural surface. The pressure waveforms generated by this apparatus are similar to those reported in the LFPI/PPBD literature and had little variation in appearance between trials. In addition, our apparatus produced an acute and chronic TBI syndrome similar to that in the LFPI/PPBD literature, as quantified by histological changes, MRI structural changes and chronic behavioral sequelae.

Imaging endpoints to predict response to IGF1R/IR inhibition in CRC models

This abstract is being presented as a short talk in Plenary Session 6. A full abstract is printed in the Proffered Abstracts section (PR9) of the Conference Proceedings.

Abstract 950: Energy deprivation by Inositol Hexaphosphate inhibits prostate tumor growth and progression in TRAMP mice

Prostate cancer (PCa) is the second leading cause of cancer-associated deaths in elderly males in Western world, suggesting that newer approaches are needed to prevent/control PCa. Herein, for the first time, by employing anatomical and dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI), we evaluated non-invasively, the in vivo chemopreventive efficacy of inositol hexaphosphate (IP6), a major constituent of high fiber diets, against prostate tumor growth and progression in transgenic adenocarcinoma of the mouse prostate (TRAMP) model. The study was conducted on a longitudinal basis to simultaneously assess both prostate sizes and changes in tumor vascularization (perfusion and permeability) due to IP6 feeding. Male TRAMP mice, beginning 4 weeks of age, were fed with 1, 2 or 4% (w/v) IP6 in drinking water or only drinking water till 28 weeks of age and monitored using MRI over the course of treatment. Longitudinal assessment of volumes of prostate and seminal vesicles showed profound effect of 2% and 4% IP6 doses on selectively reducing prostate volume without noticeable changes in seminal vesicles. Further, gadolinium-based DCE-MRI showed decreased tumor perfusion and permeability, indicative of anti-angiogenic effects of IP6 treatment. Histopathological analysis at the end of the study corroborated the MRI results, and showed that IP6-fed mice had less severe prostatic lesions compared to positive controls. Specifically, the mice in IP6-fed groups showed higher incidence of prostatic intraepithelial neoplasia (PIN) at the expense of a strong decrease in adenocarcinoma incidence. Importantly, the mice in IP6-fed groups also had prostatic regions with typical normal histology, which was totally absent in positive controls. IHC and WB results indicated anti-proliferative, pro-apoptotic, and anti-angiogenic effect of IP6 feeding, as evidenced by decreased number of PCNA-, PECAM-1/CD-31-, VEGF- and iNOS- positive cells, together with increased number of TUNEL positive cells. As potential mechanisms of IP6 efficacy, decrease in the expression of glucose transporter (GLUT) proteins together with an increase in phosphorylated levels of AMP-activated kinase (AMPK Th172 ) and acetyl-CoA carboxylase (ACC Ser79 ) were observed in the prostatic tissue of mice from IP6 fed-groups. Since phosphorylation results in decreased activity of ACC which is an essential enzyme for fatty acid synthesis, our findings suggest that IP6 is interfering with the metabolic events occurring in TRAMP prostate tumor tissue. Together, these findings show that oral IP6 feeding blocks PCa growth and progression in TRAMP mouse model via modulation of metabolic events involved in tumor sustenance and thereby results in energy deprivation within the tumor, suggesting its practical and translational potential in suppressing growth and progression of PCa in humans (NCI RO1 grant CA116636). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 950.

Abstract 948: Silibinin selectively inhibits growth and progression of urethane-induced lung tumors in wild-type but not inducible nitric oxide synthase (-/-) mice: potential usefulness of micro-computed tomography scanning in lung cancer chemoprevention studies

Lung cancer is the second most common cancer but accounts for highest mortality in both men and women in the United States and Europe. The overall 5 year survival rate is still 16% underscoring the importance of new approaches to prevention and treatment of this deadly malignancy. Since sustained and excessive nitric oxide (NO) generation is positively correlated with lung cancer development and progression, recent studies have also focused on the involvement of inducible NO synthase (iNOS) and its inhibition by natural and synthetic agents for both prevention and therapy of lung cancer. Here we evaluated the role of iNOS in lung tumor occurrence and progression as well as the angiopreventive efficacy of silibinin employing B6/129J wild-type (WT) and iNOS (−/−) mice. Mice were injected with 1 mg/g body weight (i.p.) urethane once weekly for seven consecutive weeks, and thereafter were divided into two groups and gavaged with vehicle (control) or silibinin (742 mg/kg body weight) 5 days/week for 18 weeks. Using micro-computed tomography (micro-CT), we monitored the growth and progression of lung tumors from 8 through 20 weeks in WT mice. At 25 weeks, mice were sacrificed and their lung tumor multiplicity and diameters recorded. Quantification of micro-CT data showed that silibinin treatment significantly decreases urethane-induced tumor number and size in WT mice. This correlated with the decreases in tumor number and size directly measured at the termination of the study. Genetic ablation of iNOS decreased urethane-induced tumor multiplicity by 89% (P (-/-) mice. Tumors in WT control mice expressed more iNOS than those in silibinin-treated mice. Further analyses of tumors from WT mice showed that compared to controls silibinin treatment significantly decreased newly formed microvessels as measured by nestin immunostaining, activation of STAT3 and NF-κB as determined by pSTAT3 (ser727) and p65NF-κB (ser276) levels, and proliferation as measured by PCNA expression. Whereas a decrease in lung tumor number in iNOS (-/-) mice compared to WT confirms previous studies that iNOS is a critical regulator of lung tumorigenesis; this study identified an essential role of iNOS in lung tumor angiogenesis. The lack of effect of silibinin in iNOS null mice suggests that silibinin exerts its angiopreventive effects at least in part through its inhibition of iNOS expression in lung tumors. Together, our results suggest iNOS as a potential target for treating lung cancer and support the value of micro-CT as a non-invasive imaging modality for evaluating the efficacy of lung cancer chemopreventive agents. (RO1 CA113876) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 948.