P50 - CA93990 - In Vivo Imaging of Neoplasia
Project #1: Imaging of Apoptosis The goal of the proposed project is to develop a transgenic rodent wherein activation of apoptosis can be imaged non-invasively. Strict coordination of proliferation and apoptosis is essential for normal physiology. An imbalance in these two opposing processes results in various diseases including AIDS, neurdegenerative disorders (Alzheimer's disease), myelodysplastic syndromes (Aplastic anemia, thalassemia), ischemia/reperfusion injury, cancer and autoimmune disease among others. Objective imaging of apoptosis will be a major advancement not only in the screening and validation of novel therapeutic molecules for the above diseases but also in the evaluation of therapeutic success or failure of current and future therapeutic treatment paradigms. We have developed a reporter cassette which when transfected into mammalian cells results in a polypeptide that has significantly attenuated levels of reporter activity. When this molecule is being expressed in cells undergoing apoptosis, a caspase (proteases activated during apoptosis) specific cleavage of the reporter gene occurs resulting in activation of the reporter thus enabling imaging of apoptosis. In the present proposal we will optimize this novel molecular construct and conduct in vitro (Specific Aim 1) and in vivo (Specific Aim 2) studies. Finally, a transgenic rodent model will be developed wherein the activation of apoptosis within the skin in response to sunlight can be imaged (Specific Aim 3). The ability to image apoptosis non-invasively and dynamically over time will be an invaluable resource to pharmaceutical industry and scientists for in vitro high throughput screening of compounds with pro- and anti- apoptotic activity and also for target validation in vivo. Top Project #2: Imaging of Carcinogenesis The development of tumor cells from normal cells requires the sequential acquisition of mutations to several genes. These genes fall into two major categories, the first can be referred to as oncogenes, genes that normally act to promote cell division and a second category that includes genes that function to arrest cell division, commonly referred to as tumor suppressor genes. Cancer can therefore arise from activation of oncogenes and/or inactivation of tumor suppression genes. The p53 tumor suppressor protein is unique in that it has the ability to detect the presence of damaged DNA wherein it blocks cell cycle progression to allow for the damaged DNA to be repaired (thus ensuring that mutations aren't propagated) but it also has the ability to activate the cellular suicide program such that cells having un-repairable DNA are eliminated from the organism. This unique role of p53 as the "Guardian of the genome" is exemplified by the fact that 60% of all tumors carry a mutation in the p53 gene and that inherited mutations in p53 strongly predispose individuals to cancer. The goal of this project is to develop a strategy wherein the activation of p53 in response to DNA damaging events (i.e. carcinogenesis) can be non-invasively imaged. The strategy is based on the fact that p53 is expressed in all normal cells but is rapidly degraded. In response to a DNA damaging event, degradation of p53 is blocked thus resulting in accumulation of p53. We hypothesize that fusion of an in-vivo reporter gene (such as luciferase) to p53 would result in degradation of the reporter with p53 however upon DNA damage, inhibition of degradation of the fusion would result in accumulation of the reporter (as well as p53) which could then be imaged as increased reporter activity (e.g. bioluminescense). This will enable rapid, non-invasive imaging of p53 levels within a tissue, which is a direct measure of the mutagenic potential of a specific environmental condition. In addition, this imaging strategy could also be used to evaluate chemopreventative agents as well as the efficacy of sunscreens. In addition, since the majority of tumor-associated, p53 inactivating mutations also result in p53 accumulation, constitutive elevation of p53 levels (and therefore bioluminescence) will be used as a reliable measure of p53 inactivation (whereas transiently increased p53 levels, and therefore bioluminescence, would reflect a recent genotoxic stress). Top
Project #3: Imaging of Oncogene Activation The primary aim of this study is to develop novel molecular imaging methodologies to visualize and measure the expression and activity of tyrosine kinase growth factor receptor in vivo. The Met tyrosine kinase growth factor receptor and its ligand hepatocyte growth factor/ scatter factor (HGF/SF), important genes in development, tumorigenicity and metastasis, will serve as a model. We propose to evaluate and image the expression and activity of Met and HGF/SF both at the cellular and organism levels by comparing different imaging modalities (confocal microscopy, MRI, PET and ultrasound) to conventional biochemical analysis. Using imaging techniques with fluorescence and/or luminescence tagged Met and HGF/SF we will directly image and measure the presence of these important molecules in normal tissues and tumors. We will, in parallel, perform direct analysis of ME and HGF/SF in normal and tumor tissue by IP and western blot. We will use this information together with molecular imaging in animal models to evaluate and quantify the efficacy of both standard therapies (e.g. drugs, radiation and hormones) and neutralizing antibodies to Met and HGF/SF for potential utilization of imaging technologies in human cancer. Specific Aims: 1). We will measure Met interaction with its ligand (HGF/SF) and its signal transduction substrates at the sub-cellular level using fluorescence tagged proteins. 2). We will develop molecular imaging techniques to determine in animal models, the presence of Met and HGF/SF, using fluorescence tagged proteins in xenograph tumors and in transgenic "knocked in" animals. These animals should allow real-time observation of Met mediated tumorigenesis. 3). We will develop non-invasive "indirect" metabolic molecular imaging methodologies for assessing Met activity at the organism level and in tissues as well as during tumor formation. We will be measure real-time in vivo using imaging modalities of MRI-BOLD, MRS, PET and Doppler ultrasound to assess their diagnostic potential. 4). We will quantify and characterize the combinatorial effect of a variety of Met signal transduction inhibitors in vivo on tumors and metastasis. The effects will be studied in animal models using direct and indirect molecular imaging modalities during therapy. Top
Career Development Projects: The ICMIC will provide for training support in the amount of $100,000 per each year which will allow for the enrollment of two people per year into this Program. This funding will be provided for either pre- or post-doctoral trainees or for visiting scientists wishing to gain expertise and training in the field of molecular imaging. Viable candidates will have a commitment to pursuing high-level molecular imaging research with a strong foundation/background in a biological, engineering or imaging field. The ICMIC will provide a multidisciplinary venue in order to promote the trainees imagination and understanding of the myriad of scientific possibilities provided by molecular imaging. A key component of this training will be accessibility to both state-of-the-art imaging equipment, transgenic animal expertise, and creative scientific exchange between all participants in the ICMIC. Trainees will be required to attend various teaching sessions including the 1) the ICMIC monthly journal club meeting and will present an in depth analysis of at least 2 papers per year; 2) Attend specific lectures which will be selected for each trainee from chemistry, engineering, medical school, etc. courses in order to provide a "taylor-made" curriculum to advance the understanding of the trainee in their area of interest/focus; 3) Attend regular molecular imaging seminars in which either internal or external speakers highlight special high-impact work relevant to ICMIC members; 4) Present and defend their research work at on of the ICMIC seminars each year; 5) Attend laboratory meetings and meet regularly with ICMIC sponsor. These Trainees will also be informed of the various opportunities to attend the UM Comprehensive Cancer Center's numerous seminars including Grand Rounds, special invited speakers, special seminar series and conferences which occur throughout the year, and attend the Cancer Biology courses being taught by Dr. Rehemtulla and the imaging courses taught by Drs. Chenevert and Meyer. All trainees will have the opportunity to practice what they learn by conducting their own research project. This will provide motivation to learn how to run the various imaging systems, conduct post-processing of acquired data, make and test molecular biology molecular imaging reporter constructs and learn specifics related to transgenic animal production and maintenance. These opportunities will allow each trainee the overall experience and knowledge to be competitive and contribute long term to the field of molecular imaging and to be an independent investigator. Top
Development Project: Endothelial cells as well as smooth muscle cells within angiogenic vasculature express unique cell surface markers which we propose to target with fusion molecule. A recombinant purified luciferase fused to a targeting peptide will be evaluated for imaging angiogenic vasculature using bioluminescence imaging (BLI). Preliminary results have been obtained revealing that a fusion protein containing luciferase and the integrin ligand CDCRGDCFC was able to target to angiogenic vasculature within a tumor in vitro and in vivo. We propose to further develop and optimize this molecular targeted approach using an integrin specific ligand conjugated luciferase reporter. The successful development of this imaging approach would provide for noninvasive imaging of angiogenic activity within tumor vasculature and would provide a significant new imaging approach for the detection and targeting of antineoplastic agents. This approach could easily be translated to a radiotracer approach for SPECT or PET imaging. Top
P01 - CA85878 - Brain Tumor Therapeutic Efficacy by Quantitative MR Project #1: Development of Imaging Biomarkers for Treatment Response Among the causes of death due to cancer, brain tumors are ranked second in the pediatric age group and fourth in middle-aged men. Malignant gliomas have remained uniformly fatal; only 50% of patients survive one year from the time of diagnosis. These stark statistics underscore the urgent need for an early assessment of therapeutic efficacy in these patients. Sensitive and early surrogate predictors of therapeutic outcome for patients would improve care and potentially improve prognosis due to the opportunity to individualize and adjust the treatment to each patient. The central hypothesis of this project is that the effectiveness of therapeutic interventions can be determined prior to tumor shrinkage using appropriate imaging surrogate response markers and that the optimal surrogate for outcome prediction will be dependent upon the therapeutic target. A genetically engineered PDGF-driven transgenic glioma model will be utilized with specific molecular imaging reporters developed in Project #2 and used in this Project (#1) to evaluate cytotoxic, as well as molecularly targeted therapeutic interventions. Therapeutic effects on tumor 1H MR metabolites, tumor perfusion and diffusion will be evaluated against cytotoxic agents (chemotherapy, radiation and chemoradiation) and molecularly targeted agents (a PDGFR tyrosine kinase inhibitor, an Akt inhibitor, an mTOR inhibitor and a MEK inhibitor) to determine the optimal surrogate marker for assessment of treatment response for each class of therapy. The effectiveness of MR for detection of treatment response in brain tumors will be evaluated in two Specific Aims. MR data from tumors will be compared with efficacy determined using animal survival, changes in tumor volume/growth rates, and histology to determine which specific or combined imaging-observable parameter can be utilized as a surrogate marker for predicting therapeutic outcome. These studies will provide the rationale for pairing a specific imaging surrogate marker with a therapeutic intervention in order to obtain the most effective readout of early treatment response. These rodent imaging studies will serve as the basis for translating and evaluating these approaches in the clinic as proposed in Project #3. Top
Project #2: Development of Molecular Imaging Tools for Non-Invasive Monitoring of Drug-Target Interaction The emerging fields of genomics and proteomics have led to a better comprehension of the pathophysiology of cancer and the identification of novel signaling pathways. These pathways offer novel 'targets' (e.g. Akt, MEK, mTOR and Receptor Tyrosine Kinases) which has led to the development of 'lead molecules' designed to inhibit the signaling derived from these pathways. However, this poses a tremendous challenge for selecting and/or validating these targets and for broad profiling of lead molecules for candidate selection. Molecular imaging technologies have the potential to address these scientific and technological challenges. The overall goal of Project 2 is to develop strategies wherein activation or inhibition of key pathways in tumor formation as well as in the response of tumors to therapies can be non-invasive imaged. Since targeted therapies often lead to tumor cytostasis (GI arrest), we will in Aim 1 develop and test a non-invasive reporter for proliferation (entry of cells to S-phase of the cell cycle from G1). This reporter will be used to investigate the efficacy of four targeted therapeutic agents (PTK 787, a receptor Tyrosine Kinase (PDGFR) inhibitor; Perifosine, an AKT inhibitor; CCI 779, an mTOR inhibitor and CI 1040, a MEK inhibitor). In Aim 2 we will use a non-invasive reporter for apoptosis to test the hypothesis that while targeted therapies may not induce apoptosis as single agents, in combination with other targeted therapies or with traditional therapies induction of apoptosis will correlate with efficacy and enhanced tumor control. In Aim 3, we will develop a reporter for Akt function and use it to test the ability of PTK 787, CI 779, CI 1040 and Perifosine to inhibit Akt activity. Each of the three molecular imaging approaches will be validated using traditional "gold standard" measures of function of these pathways (e.g. Western blots, immunohistochemistry, kinase assays). We believe that studies proposed in Project 2, will result in the development of tools that will be invaluable in testing the efficacy of targeted therapeutic agents as well as in optimization of their dose, schedule and development of the most efficacious combination therapies. Top
Project #3: Quantitation of Human Brain Tumor Therapy Response by MR Malignant gliomas have a high mortality rate, short median length of survival and impart devastating consequences to both patients and family. The early identification of tumors responsive to therapy, versus those that are not, would greatly facilitate modifying an ineffective treatment regimen in a more timely fashion. During the initial funding period, we have translated preclinical studies on the utility of quantitative MRI techniques as early predictors of therapeutic efficacy in brain tumor patients. Our current clinical study is providing further evidence on the predictive value of diffusion MRI, particularly in terms of depiction of the spatial heterogeneity of response. In the proposed study we will intensify the scope of imaging to more ideally sample the evolution of multiple tissue properties over the treatment interval. Quantitative response indicators under investigation include serial measures of tumor water diffusion, blood volume and flow, and microvessel permeability. Pre-treatment proton metabolite status will also be assessed. Each indicator, in its own way, depicts biophysical properties which are relevant to tumor viability, cellular alteration by therapy, as well as accessibility of the tumor to therapy. In Specific Aim 1, we will perform detailed imaging measures on well defined patient populations enrolled in tree UMCC treatment protocols in order to obtain definitive validation of the clinical utility of quantitative MRI as an early predictor of treatment response measured a later date by standard criteria. In Specific Aim 2, we will investigate the ability of these measures to predict local therapeutic response by analyses of sub-tumor regions which exhibit clearly different patterns of local response apparent at a later date. Our overarching objective in pursuit of these aims is to demonstrate that biophysical tissue properties measurable by noninvasive imaging are highly relevant to therapeutic outcome. Moreover, spatial heterogeneity of these properties are predictive of local failure, thus may form the basis for a rational design in spatially-directed treatment planning. Top
Core A: Administration Core The Administrative Core will be responsible for supporting the individual projects, including their integration into the overall Program. In addition, this Core will also coordinate general administrative and fiscal management activities of the Program under the direction of the principal investigator. These activities include budget analysis and reporting, supervision of expenditures, personnel management, coordination of consultants and provision of secretarial support. The Core Director will also oversee the internal and external Scientific Advisory Meetings and follow-up discussions. Support for the internal advisory committee and the external advisory committee will be provided, as the internal advisory committee will meet quarterly to provide continuity and guidance to the Program Director. The external advisory committee was carefully selected to provide external peer review, assessment of progress and quality assurance for all elements of this program. The external advisory committee will meet annually with all members of the program faculty and staff at an annual retreat. This Core will ensure compliance with all general, governmental and NCI regulations and requirements, communicate with NCI project officer and other NCI staff, prepare all reports and publications and work with the University of Michigan Comprehensive Cancer Center related to core service requirements and patient accrual issues. Top
Core B: Animal Imaging Core The Animal Imaging Core will perform all MRI/S and bioluminescence studies on mouse tumor models for Projects 1 and 2. Core personnel will provide these services: (a) define and implement data acquisition/imaging sequences; (b) assist with animal preparation for imaging; (c) perform all data collection on the 9.4 Tesla MRI, 7 Tesla MRI and Xenogen bioluminescence systems; (d) preliminary data processing/image reconstruction; (e) security archival of raw data and network transfer of reconstructed data to the Digital Image Processing Core (Core C); (f) scheduling of imaging experiments; and (g) provide support to Program Investigators in technical design of imaging experiments. These services designed, in part, after a "clinical MRI service" model since continual, high-volume animal scanning is essential for success of each project. More then 8000 sessions of MRI/S and 7000 bioluminescence scans will be performed over five years. Specific animal MRI/S protocols will vary with each project, but will primarily include serial studies using: standard T2-weighted; T1-weighted dynamic contrast enhanced; multislice quantitative water diffusion mapping; arterial spin labeling perfusion mapping; single-voxel proton spectroscopy. Dr. B.A. Moffat will serve as Core B Director. Dr. Moffat has extensive experience in small animal imaging research. A Co-Investigator and a Post-Doc will assist Dr. Moffat with day-to-day operations and technique development. A full time MR research technician will perform the remainder of Core B services. Top
Core C: Digital Imaging Processing Core The goals of the Digital Image Processing Core are to continue provide analytic tools for all projects. These unique image analysis tools are necessary for unbiased, accurate, quantitative analysis of temporal volumetric changes in single and multimodal MRI data sets of lab animals and human patients. Currently all MRI acquisitions independent of weightings for all interval exams of both animal and human studies are fully automatically registered to a "reference" data set in the first exam typically using a rotate-translate geometry model; this is a multimodality registration problem using existing software tools. Typically a T1-weighted, post-Gad sequence, is used as the reference for registering all successive interval exam sets. B1-field corrections have been found to be unnecessary thus far. In cases where the acquisitions involve high gradient fields such as those used for diffusion or perfusion weighted imaging, registrations are accomplished using a full affine model to support correction of shears caused by the gradients. Currently all human scan data including both original acquisitions and registered datasets, as well as animal backups, are stored on the Core's disk system. In the future all animal scans will be likewise stored on the Core's disks which can be accessed via TCP/IP or SAMBA protocols over 100 Mb ethernet. Of course the Core is responsible for maintaining data integrity and backup. The core will also develop the ability to track the positions of voxels within treated lesions across interval exams using high degree of freedom warpings. This facility for both animal and human imaging will support further investigation of the role of the apparent diffusion coefficient (ADC) as well as other parameters, e.g. perfusion, as potentially early indicators of therapeutic response. Additionally in the A1 amendment this Core demonstrated the ability to map histology back to in vivo MRI voxels for animals (where whole head ex vivo MRI acquisitions are possible) using only intrinsic scan information (i.e. no implanted fiducials of any kind). Top
Core D: Biostatistics Core The staff of the Biostastics Core will be responsible for providing biostatistical support to the research of this program. The Biostatistical Core is under the supervision of Dr. Timothy D. Johnson of the Biostatistics Department in the University of Michigan School of Public Health. The core provides assistance in the design, analysis and interpretation of preclinical and clinical experiments of the program project. Core personnel will interact with project investigators to ensure that appropriate designs and methods of analysis are used. Design issues involve selection of dose, randomization, timing of measurements, number of animals or patients. For analysis of data, the core will ensure that efficient methods are used. Standard graphical, group comparison and correlation methods of analysis will be used for initial investigation of the experimental data. Mixed model methods will be used for efficient use of the data in experiments involving repeated measures. Core personnel are experienced in the design and analysis of both animal and clinical data. This will ensure that all data obtained from MR measurements (Projects 1-3), tumor histology (Projects 1-2), cell kill and growth changes associated with therapy (Projects 1-2) and patient outcome (Project 3) will be collected efficiently and analyzed appropriately. Top
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