Intravital Imaging, Cell Isolation and Fate Mapping

The aim of Core A is to provide the technology necessary to image breast cancer progression and tumor cell dissemination and metastatic seeding in the most biologically relevant manner possible, ensuring that imaged events are truly in vivo, match natural biology and are not artifacts of the imaging method. The aim of Core A is to combine this capability with the collection and identification of migratory cells in the primary tumor using the in vivo invasion assay. This has required a comprehensive multiphoton- intravital approach with noninvasive optical technology (no stick or endoscope probes) that can reliability capture cell behavior in deep undisturbed tissue (10-100 cell diameters deep) with high temporal and spatial sensitivity and fidelity, but also with good preservation of viability of cells being imaged. These requirements preclude the use of confocal microscopes which image only the most superficial layer of cells (one cell deep) and have significant photo-toxicity. The multiphoton technology we developed has included imaging strategies designed to expand the spectral range and number of fluorescent proteins that may be simultaneously imaged and has been used to characterize novel fluorescent proteins. Our custom built microscope systems employ broadly tunable (optical parametric) laser sources required for multispectral photo-conversion. An important application arising from this development work is the use of photo-conversion fate mapping of single cells in live mice. Hence, Core A will provide the following services: 1) Intravital Imaging at single cell resolution in vivo and with the mammary imaging window. 2) Photo-conversion for phenotype analysis and correlated FACs recovery of cells in vivo. 3) Intravital Imaging of intravasation TEM and extravasation TEM in vivo and the corresponding TEM assays in vitro. 4) Ex vivo whole lung imaging and cell fate mapping. 5) In vivo invasion assay cell collection and typing. Core A Is essential to the overall goals of the program project since the behaviors and interactions of tumor cells and stromal cells must be defined at high resolution in vivo in mice for all of the projects. RELEVANCE (See instructions): Without the services provided by Core A, the primary and secondary tumors would be treated as black boxes, and all measures of invasion and metastasis would be relegated to indirect and/or end stage assays (e.g. histology of dead tissue) which do not give information about mechanism or behavior at the single cell level(19). The multiphoton microscope developed in Core A is the result of collaborations of the investigators of this PPG, and its capabilities, including real-time single-cell fate mapping in live mice using photo- conversion, are unique.