Abstract Project 2: Physical Mechanisms and Clinical Implications of Mechano-transduction in Hepatocellular Carcinoma Tumor Microenvironment. In Project 2, a team of investigators from the physical sciences, engineering, and cell biology will interact closely with hepatologists and liver oncologists through the clinical-core, and theorists through the theory-core. We will advance and test a new hypothesis for mechano-transduction in the hepatocellular carcinoma (HCC) microenvironment. We hypothesize that an entire membrane signalosome will translate changes in the physical microenvironment into alterations in membrane-mediated regulatory processes such as receptor trafficking and membrane-cortex interactions. This in turn will alter the specificity of signaling pathways and influence cell fate. Theory/membrane modeling will advance hypotheses on how physical mechanisms govern biological (cellular) behavior, and will direct design of physical parameters tunable in experiments. Super resolution microscopy will be used to track nanoscale assemblies, and force spectroscopy and microrheology will be used to determine static/dynamic responses of the cell membrane and membrane cortex interactions. In parallel, high-dimensional kinome profiling and single-cell gene expression will link these nanoscopic mechanisms with cellular decisions. Outcomes of these experiments will quantitatively and mechanistically relate the physical microenvironment in HCC to dictation of cell fate in cancer progression, as well as providing iterative feedback to the computational models for refinement of mechanisms, formulate new hypotheses. Specifically, the Aims of project 2 will establish quantitative and mechanistic relationships between the physical characteristics of the HCC microenvironment (namely membrane tension, matrix stiffness, substrate stiffness, and uniaxial compressive stress) and membrane-mediated signaling mechanisms, namely how receptor trafficking and membrane cortex interactions alter specificity of downstream of growth factor and G-protein mediated signals to regulate gene expression and cell fate. Our studies in Project 2 will also probe how static and dynamic responses of the cell membrane and membrane-cortex interactions in normal hepatocytes and stromal cells are altered by changes in the physical microenvironment variables relevant for HCC. Our project on mechano-transduction in HCC at the cellular scale is closely aligned with the goals of Project 1, namely HCC disease progression at the tissue scale, and those of Project 3, namely nuclear mechanics and HCC oncogenesis at the subcellular scale. We expect that the new physical-chemical paradigms governing HCC emerging from this project will inform and impact future HCC therapies. In particular, our results provide multidimensional, multiphysics characterization of subcellular (membrane, cortex, signals, gene-expression) alterations in response to changes in the microenvironment variables, some at single-cell resolution. The findings in Project 2 compliment those in Project 1, but extend the analyses and outcomes at the cellular to sub-cellular scale, molecular scale, and help identify physical biomarkers at this finer length-scale. Kinome profiling in Project 2 should also link our physical perspective to possible combinations of therapeutic targets.