The recent definition of cellular signaling pathways has provided new insight into the means by which multi-cellular organisms coordinate a transcriptional response to environmental change. Imaging cellular signaling in mammals will provide insight into the factors that modulate these signals in vivo. Electron Paramagnetic Resonance (EPR) imaging has provided novel, high resolution oxygen images in mice. Molecular imaging with optical detection has limited depth sensitivity making measurements in larger animals difficult. Other techniques suffer from various limitations. We propose here to explore low frequency EPR imaging, with sensitivity deep in tissues, to detect, as an example of cellular signaling, the induction of hypoxia peptide signal molecules. Unique features of EPR images including low signal background, high spatial resolution (1 mm or less) in small mammals, the promise of scaling to larger animals and sensitivity deep in animal tissues distinguish this technique and encourage its exploration. Hypoxia Inducible Factor (HIF) proteins play a major role in controlling and coordinating the response of cells and organisms to hypoxia. The work will involve the construction of an EPR spin probe, to be injected into the animal, which is silent until it encounters a proteinase (MMP2). It will also involve the engineering of an MMP2 gene construct just upstream of the HIF1alpha binding protein promoter into a viral vector. The viral vector will then be used to 1) infect cells in tumors or 2) surrounding normal tissues. Tumor or tissue hypoxia will stimulate production of HIF-1alpha binding protein which will stimulate the production of MMP2 which will activate the silent spin probe. EPR oxygen images will then be obtained to correlate the levels of hypoxia at which hypoxic cell signaling is triggered in the tumors of a living animal. This will provide a measure of the in vivo threshold for hypoxic signaling. Such a technology promises order of magnitude improvements of resolution and sensitivity

R21EB005382

2005-09-15

2010-08-31