) Imaging reporter gene expression in living animals provides critical spatiotemporal information about changes in cell growth, cell trafficking and gene expression during normal and disease processes. In addition to enabling non-invasive in vivo assays for cell migration and function, these bioluminescence-based methods permit real time analyses of gene expression patterns in neoplastic and normal cells. We have already shown that marking and transferring tumor cells with informative reporter gene constructs and creating transgenic mice expressing these constructs provides a clear, time-ordered view of in vivo tumor growth and of normal cell responses to stimuli. We propose here to add another dimension to this powerful in vivo assay technology by coupling it with equally powerful fluorescence methods for ex vivo identification of cells in suspension or tissue sections. Together, these technologies enable development of wholly new and vastly more effective methods for evaluating and improving cell-based and other anti-cancer therapies. In our first steps towards development of this dual methodology, we generated an unparalleled description of tumor-host immune interaction based on real time observations of trafficking and proliferation of immune and tumor cells in intact animals. Now, continuing along this path, we plan to capitalize on the ex vivo subset-discriminating capabilities of the multiparameter fluorescence-activated cell sorter (FACS) by developing multifunctional reporter genes (fusion proteins comprised of luciferases and fluorescent proteins) that are detectable in vivo by bioluminescence and ex vivo by fluorescence. By creating tumor cells and transgenic mice expressing these constructs, we will be able to monitor migration of cells into a tumor site (by imaging) and to characterize the cells at the site (by FACS). In addition, by creating appropriate constructs, we plan use this dual methodology in a novel """"""""gene-trap"""""""" strategy that will enable isolation and identification of genes that are turned on or turned off in tumors and responding host cells. We will perfect and demonstrate these technologies here in three systems, one in which a tumor is killed by co-transferred natural killer cells, a second in which B cells naturally give rise to CLL-like tumors, and a third in which a radiation-sensitive B cell tumor is induced to give rise to radiation-resistant variants. The overall technology that we develop through these applications will enable high throughput analyses of immune cell and tumor interactions, rapid localization and genetic characterization of defined subsets of transferred immune cells active against tumors, and the facile identification of molecular determinants involved in potential tumor evasion from immune surveillance. 1 R33 CA88303-01 -3- Christopher Contag, Ph.D.
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