Project Summary This proposal aims to apply an imaging technique for three-dimensional (3D) visualization and mapping of the tumor and microenvironment including cancer cells, stromal cells, immune cells, and vascular cells. The capability to determine the location, density, and functional orientation of different cell populations throughout a tumor would be a powerful tool not only for cancer biology but also for improving diagnosis and advancing the development of effective therapies. Although immunohistochemistry (IHC)/immunofluorescence (IF) staining has long been used in cancer diagnostics, conventional IHC/IF methodology is based on localization of antigens in single thin sections. Considering that most cells in the field of view may be only fragments, interpretation of thin section staining is subject to multiple artifacts. Significantly, IHC/IF may fail to demonstrate important features such as pushing margin, lymphocytic infiltrate, or squamous change, leaving room for diagnostic and therapeutic uncertainties. Recent progress in tissue optical clearing and light microscopy enable 3D fluorescent imaging of normally opaque, thick tissues and organs. Dramatic examples include progress mapping neuronal connectivity in whole rodent brains at subcellular resolution. I sought to adapt these strategies to advance analysis of inflammatory infiltrates, drug delivery, and therapeutic responses in whole mouse tumors and human tumor biopsies. In order to advance 3D tumor imaging, I optimized optical clearing, cell staining, high resolution imaging, and computational reconstruction. Eventually, I developed a 3D tumor imaging method, Transparent Tumor Tomography (T3), as a tool to visualize and map tumors at single cell resolution. In this project, I will apply T3 for spatial analysis of tumor immunology and immunotherapy. T3-mediated 3D tumor imaging will provide integrated spatial information regarding antibody drug distribution and immune contexts in whole tumors which will be useful as a new assay tool for cancer immunotherapy research.