PROJECT SUMMARY Given the clinical importance of radio-resistance in head and neck squamous cell cancer (HNSCC), understanding how radio-resistant tumors rewire their metabolic pathways and vascular network to escape radiotherapy (RT) is critical towards developing strategies to eliminate residual tumor cells and/or prevent subsequent recurrence. Currently, no techniques are available to provide a systems level approach to image the major axes of metabolism and the associated vasculature at a spatial resolution that can elucidate the modulation of cancer cell metabolism or vascular reprogramming in vivo. Our technological goal is to create innovative solutions in microscopy, automated algorithms and experimental strategies to image tumor metabolism, vascular function and architecture at a spatial resolution that allows for visualization of primary tumors, residual disease and recurrence following RT to facilitate the understanding of tumor biology and function, assessment of recurrence risk and design of therapies to mitigate residual disease and/or recurrence altogether in pre-clinical models. Our technological approach fills an important gap that exists between in vitro cell studies and whole- body imaging, and is complementary to metabolomics and immunohistochemistry. The Specific Aims of this proposal are to develop a portable multi-parametric microscope that combines structured illumination microscopy and dark field microscopy in a re-emission geometry to image key metabolic and vascular endpoints simultaneously (Specific Aim 1); and use the technology with in vivo HNSCC orthotropic models to test the novel hypothesis that RT-induced hypoxia-inducible factors (HIF-1α and HIF-2α) expression and subsequent changes in metabolism/vasculature underlie HNSCC radio-resistance (Specific Aim 2). This proposal will set the foundation for translating our technology to patient-derived xenograft models that have been shown to faithfully recapitulate many of the micro-environmental features of patient tumors, allowing us to move our technique forward towards translational pharmaceutical research.