Using New Optical Techniques to Study Cell Signaling in 3D Matrices

描述（由申请人提供）：乳腺上皮细胞仅在柔顺的3D细胞外基质（漂浮胶原凝胶或Matrigel）中发生导管形态发生，而在其他相同的坚硬基质（附着的3D胶原凝胶）或胶原密度增加的基质中则不发生导管形态发生。了解癌细胞如何与局部组织环境相互作用需要能够观察信号分子，亚细胞结构和ECM组件在3D环境中的关系。很少有研究调查了3D信号事件，据我们所知，还没有人使用活细胞成像或FRET方法进行过研究。3D成像增加了这些荧光研究的复杂性，包括3D体积相对于2D图像的固有挑战、重叠光谱、低信号和光散射的问题。该项目的目标是在3D培养系统的背景下开发成像技术，以直接可视化与乳腺肿瘤细胞行为相关的信号通路。我们的基本假设是Rho信号通路是一种感知机制，通过这种机制，微环境的物理特性被传递给细胞骨架内的信号效应器。我们提出以下目标：目标一：表征我们希望研究信号事件的3D基质和组织环境的内源性信号A)表征不同密度和成分的3D胶原基质内细胞内内源性荧光团的多维信号内源性荧光团的“指纹”(NADH， FAD，将利用多光子激光扫描显微镜（MPLSM）和光谱和荧光寿命显微镜（SLIM和FLIM）对胶原基质中的胶原蛋白和胶原蛋白进行空间映射。B)表征小鼠乳腺肿瘤发生过程中的内源性信号（NADH、FAD和胶原蛋白）将通过MPLSM、SLIM和FLIM在小鼠乳腺肿瘤发生模型中进行表征，从而获得关于肿瘤发生过程中代谢变化的新数据。目标2：利用多光子激光扫描显微镜（MPLSM）和荧光寿命显微镜（FLIM）开发直接可视化3D矩阵中Rho/ROCK信号通路的策略A)研究Rho GTPase的基质依赖性激活。荧光探针耦合到Rho和Rho结合域将被用作FRET对，以确定Rho在三维背景下在胶原凝胶和可定义的微流体通道内的空间和时间上的激活。B)应用FRET/FLIM技术表征Rho与其效应物ROCK对胶原密度的耦合。活化的Rho与ROCK的耦合，以及肌球蛋白介导的收缩性的调节将在三维胶原基质中确定空间和时间。

DESCRIPTION (provided by applicant): Mammary epithelial cells undergo ductal morphogenesis only in compliant 3D extracellular matrices (floating collagen gels or Matrigel), but not in otherwise identical matrices that are stiff (attached 3D collagen gels) or have increased collagen density. An understanding of how cancer cells interact with local tissue environments requires the ability to observe the relationship between signaling molecules, subcellular structures, and components of the ECM within a 3D environment. Few studies have investigated signaling events in 3D, and to our knowledge none have done so using live cell imaging or FRET approaches. 3D imaging adds complexity to these fluorescence studies, including the inherent challenge of a 3D volume as opposed to a 2D image, problems of overlapping spectra, low signals, and light scatter. The goal of this project is to develop imaging techniques in the context of 3D culture systems to directly visualize signaling pathways relevant to breast tumor cell behavior. Our underlying hypothesis is that the Rho signaling pathway is the sensing mechanism by which the physical properties of the microenvironment are conveyed to signaling effectors within the cytoskeleton. We propose the following Aims: Aim 1: Characterize endogenous signals of the 3D matrix and tissue environment within which we wish to investigate signaling events A) Characterize the multidimensional signals of endogenous fluorophores within cells within a 3D collagen matrix of various densities and compositions The "fingerprint" of endogenous fluorophores (NADH, FAD, and collagen) within collagen matrices will be mapped spatially using multiphoton laser-scanning microscopy (MPLSM) and Spectral and Fluorescent Lifetime Microscopy (SLIM and FLIM). B) Characterize the endogenous signals in the mouse mammary gland during tumorigenesis Endogenous signals (NADH, FAD, and collagen) will be characterized by MPLSM, SLIM, and FLIM in models of mouse mammary tumorigenesis, generating novel data regarding metabolic changes during tumorigenesis. Aim 2: Develop strategies for directly visualizing Rho/ROCK signaling pathways within 3D matrices using Multiphoton Laser-Scanning microscopy (MPLSM) and Fluorescent Lifetime Microscopy (FLIM) A) Investigate the matrix-dependent activation of Rho GTPase. Fluorescent probes coupled to Rho and a Rho binding domain will be used as FRET pairs to determine Rho activation spatially and temporally in a 3D context within collagen gels and definable microfluidic channels. B) Apply FRET/FLIM techniques to characterize the coupling of Rho to its effector ROCK in response to collagen density. Coupling of activated Rho to ROCK, and regulation of myosin-mediated contractility will be determined spatially and temporally within 3D collagen matrices.