Nanoplasmonics-enabled Quantitative Spatiotemporal Activity Mapping in Engineered

描述(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，类似于体内分支乳腺的结构。设计显示适当的细胞空间组织和组织结构的功能特性的分枝组织结构对于替换有缺陷的组织可能是潜在有用的。了解细胞分泌的信号是如何引导分支形成的，对于设计替代组织结构以及修复有缺陷的组织结构非常重要。基质金属蛋白酶(MMPs)以前被认为是分支形态发生的关键介质，可能是通过降解脂肪垫中的细胞外基质(ECM)来实现的。然而，随着时间的推移，这些蛋白酶的信号如何向分支组织提供空间依赖的图案信息仍不清楚。我假设，分枝模式至少在一定程度上是局部微环境中蛋白水解酶的时空活性分布的结果。MMPs还被证明通过提供肿瘤细胞侵袭ECM所需的蛋白分解活性，在肿瘤进展中发挥重要作用。有理由怀疑，正常组织和恶性组织中分支的图案化信息不同，这些差异将反映在蛋白酶信号的时空活性谱中。为了回答这个问题，需要长期、实时和定量地测量局部微环境中分泌的蛋白酶的方法，但目前还缺乏。尺子由多肽连接的贵金属纳米颗粒组成，可以通过光学监测其随时间的光散射光谱来定量测量具有单分子灵敏度的蛋白酶活性。纳米等离子尺子不会出现光漂白或眨眼(即因此，纳米等离子尺子能够对分泌的蛋白酶进行长期和连续的光学测量。我建议使用纳米等离子尺子，能够长期，实时和定量地测量分泌的蛋白水解酶，目的是了解分支过程和工程分支结构。以下具体目标支持这一目标的实现：(1)利用先前表征的纳米等离子尺子作为性能的积极模型，验证纳米等离子尺子适用于长期、实时和定量的蛋白酶活性测量。(2)与恶性器官培养物相比，鉴定非恶性器官培养物分支形成过程中分泌的蛋白水解酶的时空活性。(3)确定蛋白酶的调制是否改变了时空活性分布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.