2013 MIPS Molecular Imaging Seminar Series

The accelerating shift to molecularly targeted therapeutics requires parallel advances in molecular diagnostics, in vivo as well as in vitro. Antibodies provide the basis of a class of molecular imaging agents for phenotypic assessment of cells and tissues in living organisms, including patients. Engineered antibody fragments (such as minibodies and cys-diabodies) preserve high affinity binding, maintain efficient targeting in vivo, and clear promptly from the circulation, making them ideal for rapid, same-day imaging. When labeled with positron-emitting radionuclides (such as I-124, Zr-89, Cu-64, F-18), engineered antibody fragments can be employed for high resolution, sensitive, quantitative imaging by PET (positron emission tomography). ImmunoPET can also be applied to detection of immune cell subsets (such as CD8 T cells or macrophages), for monitoring immune responses following cancer therapy. ImmunoPET provides a broad approach for imaging cell surface phenotype in vivo, and stands to play an expanding role in the detection and management of cancer and other diseases, for assessing key factors such as target expression, internalization and catabolism, and response to therapy and mechanism of response.

With the recent introduction of integrated PET/MR imaging systems it is possible for the first time to investigate functional and morphological changes or different functional processes at the same time in the living human subject. This new opportunity provides a further stimulus to our ongoing imaging research program which is aimed at improving early diagnosis and therapy monitoring in Alzheimer's disease (AD) and acute ischemic stroke. With regard to AD, this talk will provide an insight into new techniques to image beta-amyloid and tau, the histopathological hallmarks to the disease. It will be discussed as to what extent these new opportunities help us to monitor the effect of cell-based and other therapies. Concerning acute ischemic stroke, our concepts to improve early diagnosis by means of simultaneous PET/MRI will be presented. Further, examples of our research to apply stroke imaging in the development of cell-based and other stroke therapies in a translational concept will be discussed. In summary, it will be concluded that cell-based therapies have a great potential to overcome the therapeutic dilemma in AD and stroke, and that PET/MRI plays an important role in the search for respective treatments.

Sigma receptor antagonists have been demonstrated in the literature to attenuate many of the effects of cocaine. Recently the sigma receptor antagonist, rimcazole, was demonstrated to selectively block cocaine self-administration and attenuate cocaine-induced dopamine elevations in the shell of the nucleus accumbens. Interestingly, rimcazole also has effects on the dopamine system that appear critical for its effects on cocaine self-administration. Highly selective sigma receptor ligands alone are not able to attenuate cocaine self-administration. Standard dopamine uptake inhibitors dosedependently shift the cocaine dose-effect curve to the left. In contrast, combinations of dopamine uptake inhibitors and sigma receptor antagonists produced dose-related decreases in cocaine selfadministration without effects on food-maintained responding. Selective sigma receptor antagonists, developed in our lab, were utilized in these studies and will be discussed. Furthermore, the identification of a novel, dual acting sigma/DAT ligand will be discussed that blocks cocaine selfadministration without effecting food intake. This may indicate a potential avenue to develop PET imaging and therapeutic approaches to monitor and treat cocaine abuse respectively.

Selective receptor-targeting radiopeptides have emerged as an important class of radiopharmaceuticals for molecular imaging and therapy of tumours that overexpress peptide receptors on the cell membrane. This presentation will give an overview of the research program of the preclinical and clinical groups of the department of Nuclear Medicin Erasmus MC, with a focus on peptide receptor-targeted imaging (PRI) and peptide receptor-targeted radionuclide therapy (PRRT) of receptor-positive cancers with radiolabelled peptides.

In the clinic we imaged over 5000 patients using radiolabelled somatostatin analogues, whereas over 1500 treatments were given. Our efforts now concentrate on widening the therapeutic window by increasing the tumour radiation dose and/or decreasing the dose to the normal, healthy, organs. To enlarge the panel of tumours to be imaged and treated, we also design and evaluate analogues of other peptides, including bombesin, CCK, and neurotensin analogues, that bind to their receptors in a variety of different tumours.

Malignant cancers, whether inherited or sporadic, can be characterized by genetic instability within highly selective local microenvironments. This combination promotes somatic evolution. The existence of a harsh environment and genotypic heterogeneity can be formally combined in "evolutionary game theory", which is summarized in a basic equation governing evolutionary rate: ∂µ/∂t = σ2(∂G/∂µ) where ∂µ/∂t is the evolutionary rate at which the strategy (phenotype) (µ) of a population varies with time (t). σ represents the heritable phenotypic diversity. ∂G/∂µ is the fitness function, and a harsh environment generally produces a high slope, meaning that even small changes in phenotype can cause large variations in fitness. Hypoxia and acidosis are commonly encountered during the process of carcinogenesis. These microenvironmental forces are not only highly selective, but also induce genetic instability through increased genotoxicity, decreased repair and chromosomal rearrangements. As a result of these process that occur throughout carcinogenesis, malignant cancers contain dynamically evolving clades of cells living in distinct microhabitats that virtually ensure the emergence of therapy-resistant populations. Cytotoxic cancer therapies also impose intense evolutionary selection pressures on the surviving cells and thus, increase the evolutionary rate. Intratumoral hypoxia, acidosis and reactive oxygen species (ROS), are strong selective pressures, leading to common metabolic phenotypes of cancers. Hypoxia is observed in in-situ cancers and in later disease, as a consequence of poor perfusion. Hypoxia leads to genomic instability via multiple mechanisms. Acidosis in tumors is caused through a combination of increased metabolism and poor perfusion clastogenic. These microenvironmental forces can work alone, or in concert with heritable mutations, that either inhibit the DDR machinery directly, reduce apoptosis, or exacerbate the hostile microenvironment through promotion of hyperplasia.

Relevance to Imaging

Induction of genomic alterations and localized selection by heritable and/or environmental factors will result in phenotypic heterogeneity. Heterogeneity can be identified with imaging, by non-­-uniform patterns of enhancement with contrast, diffusion MRI, or CT attenuation and may be associated with poor outcome. Each tumor is an ecosystem inhabited by physical factors, physiological and metabolic factors, normal cells, inflammatory cells, and the actual populations of tumor cells. An important physiological factor is tumor perfusion, which is a measureable quantity with dynamic contrast enhanced (DCE) MRI. Quantified heterogeneity represents different "niches" within tumors and hence clades. It is also a powerfully negative prognostic factor. It is our hypothesis, then, that phenotypic heterogeneity of cancers presages emergence of resistant populations and that this may be observable through appropriate analyses of MR images. We further hypothesize that such analyses should be able to inform design of therapeutic approaches to better manage cancers.

It was reported by Snyder and colleagues in 1969 that tumor cells contained an overabundance of phospholipid ethers (PLE) relative to normal cells. This was thought to be due to under expression of an alkyl cleavage enzyme. In an attempt to exploit this difference we designed and synthesized a series of radioiodinated phospholipid ether analogs as tumor imaging agents in the early 1990's. Some of these showed tumor avidity in vivo and eventually over 30 analogs were synthesized, radioiodinated and evaluated for their tumor selectivity (Pinchuk et al, J Med Chem. 49:2155-2165 (2006)). One of these PLE analogs, NM404 (18-(4-iodophenyl)-octadecylphosphocholine), has displayed remarkable in vivo tumor avidity in over 60 in vivo human xenograft and spontaneous primary and metastatic rodent tumor models. Unlike, FDG, NM404 is not taken up by inflammatory or premalignant (mammary and colon adenomas) lesions. Recent cellular confocal microscopy studies with a fluorescent bodipy analog of NM404 have shown that the agent is taken up preferentially into cancer and cancer (glioma) stem cells over normal cells in co-cultured skin fibroblasts and melanoma cells. Tumor cell uptake occurs via surface membrane lipid rafts and once inside the cell enters the PIP3 kinase/AKT pathway. Lipid rafts are known to be more prevalent in malignant cells versus normal cells. A near infrared version of the agent (CLR1502), also exhibits similar tumor uptake and retention properties and is being evaluated in surgical tumor margin illumination studies. Due to its unique prolonged tumor cell retention properties, this agent is being evaluated in radiotherapy indications. Preclinical single dose survival studies conducted with 131I-NM404 in eight mouse xenograft models (renal cell, melanoma, lung, prostate, breast, glioma, pancreatic, and ovarian) showed significant prolongation of life ranging from 40-400 percent relative to saline treated control cohorts. Moreover, the therapy version (131I) of this agent is currently being clinical evaluated in a Phase 1b MTD safety trial in 8 human cancer types.

Recent advances in cancer treatment centre around development of targeted therapies and personalisation of treatment regimes to individual tumour characterisitics. However, clinical outcomes have not improved as expected. Further development of the use of molecular imaging to predict or assess treatment response in clinic must address spatial heterogeneity of cancer within the body.

With the aim of developing molecular diagnostic tools that can stratify cancer patients accurately for treatment with anti- Human Epidermal growth factor Receptor (HER) treatment and/or chemotherapeutics such as oxaloplatin (an alkylating agent), we have developed high-content imaging-based tissue analyses that quantify in archived (paraffin) tumor samples, the relative concentrations of HER (2/3) heterodimer and the activated BRCA1(sumoylated) fraction (1). Our automated imaging platform that quantifies specific protein-protein interactions in cancer tissues is in part based on Förster resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM)(2, 3), the gold standard for the determination of FRET (as reviewed recently (4-6)), which has been used to directly monitor, validated protein-protein interactions and post-translational modifications (PTMs).

Our current goal is to combine molecular imaging (whole body with PET and MRI, coupled to tissue nanoscopic imaging) with genomics to stratify medicines to cancer patients amd predict clinical outcome.Treatment regimes could therefore be individually tailored both at diagnosis and throughout treatment, through monitoring of drug pharmacodynamics providing early readout of response or resistance.

Cerenkov radiation is the low level of blue-light produced by particles traveling faster than the speed of a light through a diaelectric medium such as tissue. While this phenomenon has been described originally in the early 20th century and rewarded with the Nobel Price for Physics in 1958 it was only 2009 that this phenomenon was recognized as utilizable tool for optical in vivo imaging of nuclides. In this context Cerenkov Luminescence Imaging (CLI) is a new, emerging modality that merges nuclear and optical imaging since it allows for optical imaging of radio-tracers used for Positron Emission Tomography (PET) and radiotherapy. It requires highly sensitive optical equipment to detect the low amount of photons emitted compared to other optical imaging modalities. However, it offers several compelling advantages. CLI utilizes clinical approved tracers, thus avoiding significant hurdles for approval of the imaging agent. It is able to image radionuclides that cannot be imaged otherwise such as the [90)Y or 225[Ac]. By reverting to PET of the very same agent an internal standard is provided that allows for quantification as well as true multimodality imaging from the same imaging label. The imaging equipment remains still cheaper than a PET scanner; in fact, already present equipment such as a bioluminescence scanner can be used. Several animals can be imaged in parallel within few minutes, allowing for a higher throughput. We have demonstrated Cerenkov imaging of prostate as well as of breast tumor models using the radiolabeled antibodies J591 for prostate and herceptin for breast cancer. More recently, Quantum Dots (QDs) and fluorochromes have been used to shift the light from the blue to greater penetrating red. We have developed an sensor system quantifying enzymatic activity with radiotracers and thus creating the first switchable agent based on a radionuclide. Recently, clinical applications for this new entity emerged and first clinical images have been obtained, among them through an ongoing study at MSKCC to evaluate clinical Cerenkov imaging.

Overall, Cerenkov imaging provides a paradigm shift, providing the first truly multimodal imaging system and transgressing conventional borders between imaging systems, thus allowing for completely new imaging approaches and applications, which will be discussed.

RNA interference is an innate cellular mechanism for post-transcriptional regulation of gene expression in which double-stranded ribonucleic acid inhibits the expression of genes with complementary nucleotide sequences. Its therapeutic potential is indisputable, considering that one can use this mechanism to silence virtually any gene with single-nucleotide specificity. Recently described phenomenon of miRNA silencing has been attributed to targeting about 60% of mammalian genes and is an important modulator in various pathologies. We applied RNAi therapy in conjunction with imaging to studies in cancer and diabetes. Theranostic agents that we develop enable delivery of nucleic acid-based and combination therapies, optimization of targeted delivery, and assessment of efficacy through non-invasive imaging, which provides the necessary set of tools to accomplish this in authentic physiologic environments and across time. This presentation will focus on application of nucleic acid-based theranostics for cancer therapy and in diabetes.

Dr. Cai is the director of the The Molecular Imaging and Nanotechnology laboratory at the University of Wisconsin - Madison is primarily focused on three areas:

1. development of multimodality molecular imaging agents;
2. nanotechnology and its biomedical applications; and
3. molecular therapy of cancer.

In this talk, Dr. Cai will present his recent work on molecular imaging (e.g. positron emission tomography and near-infrared fluorescence) of angiogenesis in cancer and various cardiovascular diseases, as well as the development of protein-based and various nanomaterial-based agents that target (tumor) angiogenesis. The three main molecular targets that he is interested in are: CD105 (also called endoglin), vascular endothelial growth factor receptor, and integrin αvβ3. The nanomaterials that will be discussed in this presentation include nanographene oxide, zinc oxide, unimolecular micelles, iron oxide nanoparticles, silica nanoparticles, among others.

Cerenkov radiation is the low level of blue-light produced by particles traveling faster than the speed of a light through a diaelectric medium such as tissue. While this phenomenon has been described originally in the early 20th century and rewarded with the Nobel Price for Physics in 1958 it was only 2009 that this phenomenon was recognized as utilizable tool for optical in vivo imaging of nuclides. In this context Cerenkov Luminescence Imaging (CLI) is a new, emerging modality that merges nuclear and optical imaging since it allows for optical imaging of radio-tracers used for Positron Emission Tomography (PET) and radiotherapy. It requires highly sensitive optical equipment to detect the low amount of photons emitted compared to other optical imaging modalities. However, it offers several compelling advantages. CLI utilizes clinical approved tracers, thus avoiding significant hurdles for approval of the imaging agent. It is able to image radionuclides that cannot be imaged otherwise such as the [90)Y or 225[Ac]. By reverting to PET of the very same agent an internal standard is provided that allows for quantification as well as true multimodality imaging from the same imaging label. The imaging equipment remains still cheaper than a PET scanner; in fact, already present equipment such as a bioluminescence scanner can be used. Several animals can be imaged in parallel within few minutes, allowing for a higher throughput. We have demonstrated Cerenkov imaging of prostate as well as of breast tumor models using the radiolabeled antibodies J591 for prostate and herceptin for breast cancer. More recently, Quantum Dots (QDs) and fluorochromes have been used to shift the light from the blue to greater penetrating red. We have developed an sensor system quantifying enzymatic activity with radiotracers and thus creating the first switchable agent based on a radionuclide. Recently, clinical applications for this new entity emerged and first clinical images have been obtained, among them through an ongoing study at MSKCC to evaluate clinical Cerenkov imaging.

Overall, Cerenkov imaging provides a paradigm shift, providing the first truly multimodal imaging system and transgressing conventional borders between imaging systems, thus allowing for completely new imaging approaches and applications, which will be discussed.