In vivo microcartography and subcellular imaging of tumor angiogenesis: A novel platform for translational angiogenesis research

Purpose: To eliminate the variable of tumor heterogeneity from a novel in vivo model of tumor angiogenesis. Experimental design: We developed a method to navigate tumor neovasculature in a living tissue microenvironment, enabling relocation of a cell- or microregion-of-interest, for serial in vivo imaging. Orthotopic melanoma was grown, in immunocompetent Tie2GFP mice. Intravital multiphoton fluorescence and confocal reflectance imaging was performed, on a custom microscope with motorized stage and coordinate navigation software. A point within a Tie2GFP+ microvessel was selected for relocation. Custom software predicted target coordinates based upon reference points (tissue-embedded polystyrene beads) and baseline target coordinates. Mice were removed from the stage to make previously-obtained target coordinates invalid in subsequent imaging. Results: Coordinate predictions always relocated target points, in vivo, to within 10-200 μm (within a single 40× field-of-view). The model system provided a virtual living histology of tumor neovascularization and microenvironment, with subcellular spatial resolution and hemodynamic information. Conclusions: The navigation procedure, termed in vivo microcartography, permits control of tissue heterogeneity, as a variable. Tie2 may be the best reporter gene identified, to-date, for intravital microscopy of tumor angiogenesis. This novel model system should strengthen intravital microscopy in its historical role as a vital tool in oncology, angiogenesis research, and angiotherapeutic drug development.