Nanoplasmonics-enabled Quantitative Spatiotemporal Activity Mapping in Engineered

描述（由申请人提供）：分支形态发生是一个动态过程，用于构建各种器官，如肾脏，唾液腺和乳腺。为了研究分支形态发生，我们已经能够在三维（3D）工程组织阵列中概括体内组织结构，这些组织结构类似于体内分支乳腺的结构。显示适当的细胞空间组织和组织结构的功能特性的工程分支组织结构可能潜在地用于替换缺陷组织。了解细胞分泌的信号如何直接分支形成对于工程替代组织结构以及修复缺陷组织结构非常重要。基质金属蛋白酶（MMPs）已被确定为分支形态发生的关键介质，可能通过降解脂肪垫中的细胞外基质（ECM）。然而，目前尚不清楚这些蛋白酶的信号如何随时间向分支组织提供空间依赖的模式信息。我假设分支模式是蛋白酶在局部微环境中的时空活动概况的结果，至少部分是这样。MMPs还通过提供肿瘤细胞侵入ECM所需的蛋白水解活性，在肿瘤进展中发挥重要作用。我们有理由怀疑功能正常组织分支的模式信息与恶性组织分支的模式信息不同，这些差异将反映在蛋白酶信号的时空活动谱上。为了回答这个问题，需要长期，实时和定量测量局部微环境中分泌的蛋白酶的方法，但目前缺乏。纳米等离子体尺由肽链贵金属纳米粒子组成，通过光学监测其随时间的光散射光谱，可以定量测量蛋白酶活性，具有单分子灵敏度。纳米等离子体尺子不会遭受光漂白或闪烁(即。波动强度)，因此，纳米等离子体尺子能够长期和连续的光学测量分泌的蛋白酶。我建议使用纳米等离子体尺子，能够长期、实时和定量地测量分泌的蛋白酶，以了解分支过程和工程分支结构。以下具体目标支持实现这一目标：(1)验证纳米等离子体尺子适用于长期、实时和定量测量蛋白酶活性，使用先前表征的纳米等离子体尺子作为性能的积极模型。(2)与恶性器官型培养相比，确定非恶性器官型培养中分支形成过程中分泌蛋白酶的时空活性谱。(3)确定蛋白酶的调节是否会改变时空活动概况和由此产生的分支模式。

DESCRIPTION (provided by applicant): Branching morphogenesis is a dynamic process used to construct a variety of organs, such as kidneys, salivary glands, and the mammary glands. To study branching morphogenesis, we have been able to recapitulate in vivo tissue structures in three-dimensional (3D) engineered tissue arrays that resemble the structure of the branching mammary glands in vivo. Engineering branched tissue structures that display appropriate spatial organization of cells and functional properties of a tissue structure may be potentially useful for replacing defective tissues. Understanding how secreted signals from cells direct branching formation is important for engineering replacement tissue structures as well as repairing defective tissue structures. Matrix metalloproteinases (MMPs) have been identified previously as key mediators of branching morphogenesis presumably by degrading the extracellular matrix (ECM) in the fat pad. However, it is still unclear how these proteinases' signals provide spatially dependent patterning information to the branching tissue over time. I hypothesize that branching patterns are the result, at least in part, of proteinases' spatiotemporal activity profiles in the local microenvironment. MMPs have also been shown to play an important role in tumor progression by providing the proteolytic activity necessary for tumor cells to invade the ECM. It is reasonable to suspect that patterning information differs for branching in functionally normal tissues from that in malignant tissues, and these differences would be reflected in the proteinase signals' spatiotemporal activity profiles. To answer this question, methods for long term, real time, and quantitative measurement of secreted proteinases in the local microenvironment are needed but are currently lacking. Nanoplasmonic rulers consist of peptide- linked noble metal nanoparticles which can be used to quantitatively measure proteinase activity with single molecule sensitivity by optically monitoring their light scattering spectra over time. Nanoplasmonic rulers do not suffer from photobleaching or blinking (ie. fluctuating intensities), and therefore, nanoplasmonic rulers are capable of long term and continuous optical measurements of secreted proteinases. I propose to employ nanoplasmonic rulers, capable of long term, real time, and quantitative measurements of secreted proteinases, for the purpose of understanding the branching process and engineering branched structures. The following specific aims support the attainment of this goal: (1) Verify nanoplasmonic rulers are appropriate for long term, real time and quantitative measurements of proteinase activity using previously characterized nanoplasmonic rulers as a positive model of performance. (2) Identify spatiotemporal activity profiles of secreted proteinases during branching formation in non-malignant as compared to malignant organotypic cultures. (3) Determine whether modulation of proteases modifies spatiotemporal activity profiles and resulting branching patterns. PUBLIC HEALTH RELEVANCE: The gap between the number of available tissue/organ donations and the number of patients in need of tissue/organ transplants continues to grow, creating an urgent need for alternative therapeutic strategies. Engineered tissues - displaying appropriate spatial organization of cells and functional properties of a tissue structure - present a promising, future alternative to tissue transplants. Whereas there has been some initial success, engineering tissues that precisely mimic functional tissues in vivo remains challenging. Increased understanding of 3-dimensional (3D) tissue systems that model complex, in vivo tissue structures should advance therapeutic strategies for replacing diseased or defective tissues. Here, I propose to employ nanoplasmonic rulers, capable of long term, real time, and quantitative measurements of secreted proteinases, in 3D models for the purpose of understanding branching morphogenesis and engineering branched structures.