Developing branch stress microscopy for the mechanobiology of 3D morphogenesis and invasive diseases

项目概要/摘要 分支结构对于发育过程中许多器官和腺体的形成至关重要。此外， 许多侵袭性疾病，包括异常血管生成和集体癌症侵袭，也采取以下形式 分支机构。因此，了解分支的图案形成和形态发生的机制至关重要 在发育的基础生物学和人类疾病的治疗中都至关重要。分支 过程，包括分支的伸长、分叉和终止，可以通过 生化信号，例如成纤维细胞生长因子和激素。最近的工作还表明，机械 来自细胞外基质和邻近细胞的信号也会影响分支动力学。然而， 可能是由于缺乏可以测量机械力在内部的分布的定量工具 分支，力学如何调节分支过程仍不清楚。在这个项目中，我们建议 开发一种新型定量工具，称为分支应力显微镜（BSM），可以精确地绘制 分支过程中细胞间机械应力的时空分布。即使与 过去几十年细胞和组织力学的重大发展，量化了细胞间的力学 三维空间内的应力仍然是一项具有挑战性的任务。因此，为了管理风险，建议 项目的设计有两个逐渐风险更大和回报更高的目标。在目标 1 中，我们将开发一个相对 BSM 的简单一维版本，可量化沿形态发生分支的横截面应力。共焦 显微镜将与三维牵引应力计算相结合，以获得总力和 通过力平衡方程施加在横截面上的平均应力。然后我们将验证压力 使用 3D 癌症集体迁移作为现有技术水平，根据 1D BSM 计算得出 生物模型。在目标 2 中，我们将更进一步开发 3D 版本的 BSM，以解决完整的问题 分支段内细胞间应力的 3D 分布。我们将进行必要的测量并 关于分支材料属性和边界处的应力或位移值的假设 分支分段并将任务转化为固体力学中的边值问题。然后我们将计算 使用有限元分析计算侵袭性癌症分支内的应力分布并验证假设 以及通过与当前技术水平测量的应力进行比较来评估工具的鲁棒性。总而言之， 该项目将结合计算机和体外工程和生物方法来开发一种新型 定量工具可广泛适用于体外、离体甚至体内的任何分支过程，因此 为发育和疾病领域的机械生物学分支提供通用技术。

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.