PROJECT SUMMARY/ABSTRACT Branched structures are essential for the formation of many organs and glands during development. In addition, many invasive diseases including abnormal angiogenesis and collective cancer invasion also take the form of branches. Hence, understanding the mechanism underlying the patterning and morphogenesis of branches is of critical importance in both fundamental biology of development and treatment of human diseases. Branching processes, including the elongation, bifurcation, and termination of the branches, can be regulated by biochemical signals, such as fibroblast growth factors and hormones. Recent work also suggests that mechanical signals from the extracellular matrix and from neighboring cells also influence branching dynamics. However, likely due to the lack of quantitative tools that can measure the distribution of mechanical forces within the branches, how mechanics regulates the branching process is still not well understood. In this project, we propose to develop a novel quantitative tool, termed branch stress microscopy (BSM), that can precisely map the spatiotemporal distribution of intercellular mechanical stresses during the branching process. Even with significant developments in cell and tissue mechanics over the past decades, quantifying intercellular mechanical stresses within a three-dimensional space remains a challenging task. Hence, to manage the risk, the proposed project is designed with two progressively riskier and more rewarding aims. In Aim 1, we will develop a relatively simple 1D version of BSM that quantifies the cross-sectional stress along a morphogenetic branch. Confocal microscopy will be combined with a three-dimensional traction stress calculation to obtain the total force and average stress exerted at the cross section via force balance equations. We will then validate the stress calculated from 1D BSM against that from the current state of the art using 3D cancer collective migration as a biological model. In Aim 2, we will take one step further to develop a 3D version of BSM to resolve the complete 3D distribution of intercellular stresses within a branch segment. We will make necessary measurements and assumptions regarding the branch material properties and stress or displacement values at the boundary of the branch segment and turn the task into a boundary value problem in solid mechanics. We will then calculate the stress distribution within invading cancer branches using finite element analysis and validate the assumptions and the robustness of the tool by comparing with the stresses measured by the current state of the art. In sum, this project will combine in silico and in vitro engineering and biological approaches to develop a novel quantitative tool that may be widely applicable to any branching processes in vitro, ex vivo and even in vivo, thus providing a versatile technology for branching mechanobiology in development and diseases.