Project Summary In response to the RFA for a Cellular Cancer Biology Imaging Program we propose a program focused on imaging and molecularly probing the cell biological events that drive the formation of new metastatic tumors. Specifically, we will address two questions: 1) How does the intersection of shifts in cell-intrinsic and cell- extrinsic signals associated with shifts in expression of the membrane adaptor protein Caveolin-1 affect the metastatic propensity of pediatric sarcoma (Research Testbed Unit 1)? 2) What are the effects of cell-intrinsic and cell-extrinsic variation in lipid metabolism on melanoma metastasis patterns (Research Testbed Unit 2)? Answers to both questions depend on technology to capture the molecular, metabolic, and morphological states of individual metastatic cells as they colonize the distant site: In the Technology Development Unit-1 we will develop a multi-modal, multi-scale live imaging platform to investigate the effects of intersecting microenvironmental variation across an organism and cell intrinsic heterogeneity on metastatic spreading. The platform will leverage the exquisite optical and physiological properties of the zebrafish embryos to ‘watch’ at once how cells form human tumor xenografts spread to multiple distant sites where they form metastatic tumors. The microscope will allow seamless switching between a high-throughput screening mode observing the metastatic patterns in tens to hundreds of embryos in one experiment and a high-resolution imaging mode with fully isotropic resolution of 300 nm in XYZ that allows detailed analysis of the molecular, metabolic, morphologic, and proliferation/survival states of individual cells within an emerging metastatic niche. In the Technology Development Unit-2 we will develop a multi-scale imaging platform to investigate by hyper-spectral analysis the molecular, metabolic, morphological, and functional states of metastatic cells across entire mouse organs. The platform will leverage advances in tissue clearing, fully automated high-speed and high-resolution light-sheet fluorescence imaging, and computer vision, to integrate a mesoscopic imaging mode for fast acquisition of volumes of up to 20 x 20 x 20 mm at a ~5-10 micron isotropic resolution with a nanoscopic imaging mode providing 300 nm XYZ-resolution throughout a 300 micron field of view anywhere in the organ. Biological features can thus be rapidly identified and immediately interrogated with high subcellular resolution. We will then develop physically and chemically accelerated 60-plex cyclic immunofluorescence assays to comprehensively characterize the molecular, metabolic and architectural states of colonizing cells and their surroundings in the metastatic niche in thick (~200 microns) tissue sections. To accurately describe metastatic heterogeneity, the entire system, including sample handling, labeling, and imaging, will be fully automated and operated in a high-throughput fashion. Our goal with this system is to enable comprehensive profiling of heterogeneous cell metastatic cell behavior in 100’s of intact tissue specimens. Together, these platforms will generate versatile imaging tools for a new era of in situ cancer cell biology.

Project Narrative We propose a cancer cell imaging program to probe the mechanisms of metastatic tumor formation in the context of the host environment. This critical step in the development of metastatic disease is likely to be accompanied by vulnerabilities of the cancer cells that could be exploited for clinical treatment. The proposed work will establish novel microscopy with unprecedented resolution while maintaining sufficient experimental throughput to discover the relevant metastasis-driving processes at the single cell level.