) 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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants Phase II (R33)
Project #
5R33CA088303-02
Application #
6378160
Study Section
Special Emphasis Panel (ZCA1-SRRB-C (M1))
Project Start
2000-07-24
Project End
2003-06-30
Budget Start
2001-07-01
Budget End
2002-06-30
Support Year
2
Fiscal Year
2001
Total Cost
$449,995
Indirect Cost
Name
Stanford University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
Beilhack, Andreas; Schulz, Stephan; Baker, Jeanette et al. (2008) Prevention of acute graft-versus-host disease by blocking T-cell entry to secondary lymphoid organs. Blood 111:2919-28
Hassibi, Arjang; Contag, Christopher; Vlad, Marcel O et al. (2005) Bioluminescence regenerative cycle (BRC) system: theoretical considerations for nucleic acid quantification assays. Biophys Chem 116:175-85
Beilhack, Andreas; Schulz, Stephan; Baker, Jeanette et al. (2005) In vivo analyses of early events in acute graft-versus-host disease reveal sequential infiltration of T-cell subsets. Blood 106:1113-22
Zhao, Hui; Doyle, Timothy C; Wong, Ronald J et al. (2004) Characterization of coelenterazine analogs for measurements of Renilla luciferase activity in live cells and living animals. Mol Imaging 3:43-54
Yang, Yang; Contag, Christopher H; Felsher, Dean et al. (2004) The E47 transcription factor negatively regulates CD5 expression during thymocyte development. Proc Natl Acad Sci U S A 101:3898-902
Mandl, Stefanie J; Mari, Carina; Edinger, Matthias et al. (2004) Multi-modality imaging identifies key times for annexin V imaging as an early predictor of therapeutic outcome. Mol Imaging 3:1-8
Wang, Thomas D; Contag, Christopher H; Mandella, Michael J et al. (2004) Confocal fluorescence microscope with dual-axis architecture and biaxial postobjective scanning. J Biomed Opt 9:735-42
Cao, Yu-An; Wagers, Amy J; Beilhack, Andreas et al. (2004) Shifting foci of hematopoiesis during reconstitution from single stem cells. Proc Natl Acad Sci U S A 101:221-6
McCaffrey, Anton; Kay, Mark A; Contag, Christopher H (2003) Advancing molecular therapies through in vivo bioluminescent imaging. Mol Imaging 2:75-86
Wang, Thomas D; Mandella, Michael J; Contag, Christopher H et al. (2003) Dual-axis confocal microscope for high-resolution in vivo imaging. Opt Lett 28:414-6

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