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A single cell assay for tissue activity

组织活性的单细胞测定

基本信息

批准号：
10831316
负责人：
ANDREI GOGA
金额：
$ 16.13万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10831316
关键词：
Actomyosin Apical Apoptosis Area Basal Cell Basic Science Biological Assay Biomechanics Breast Epithelial Cells Cadherins Cell division Cell membrane Cells Cellular Assay Clinical Collagen Type IV Development Diffuse Disease Elasticity Environment Epithelial Cells Epithelium Extracellular Matrix Fibronectins Frequencies Grant Homeostasis Human In Situ Intercellular Junctions Invaded Laminin Lateral Length Liquid substance Mammary Neoplasms Measurement Measures Mechanics Membrane Modeling Myoepithelial cell Neoplasm Metastasis Noise Oncogenes Organoids Output P-Cadherin Patients Phase Transition Physiological Processes Play Process Production Property Role Solid Source Surface System Tissues Traction Traction Force Microscopy assay development cancer cell cancer invasiveness cell behavior cell transformation exosome interest mammary epithelium neoplastic cell polyacrylamide gels professor programs response tumor microenvironment tumor progression wound healing

项目摘要

Abstract This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA- 23-045. Fluctuations in the active biomechanical properties of cells are understudied, but evidence suggest they play a critical role in both core physiological processes and in disease. For example, tissue phase transitions, from elastic- to fluid-like (or jammed to unjammed) are thought to arise in part from increased noise in cell junctional mechanics. These forces can also result in shedding of cellular material like exosomes. Both of these processes play a central role in cancer progression, most notably in invasion and metastasis. However, a major challenge is identifying the specific subcellular origin of these forces and the machinery responsible for them. For example, studies of tissue fluidization using vertex modeling approaches have defined the necessary and sufficient geometric changes for tissue fluidization in epithelia. Specifically, active fluctuations in cell junction length are required for the T1 transitions that change junctional topology and allow cells to diffuse like a fluid. Actomyosin dynamics can drive these transition in the absence of cell division and apoptosis, but these models focus on actomyosin activity at apical/lateral interfaces (between cells), but largely ignore more indirect sources of activity, for example deriving from tractions generated at the basal surface which acts to sheer lateral and apical cell junctions. By tracking the dynamics of single cells in both normal and transformed primary human mammary epithelial organoids we see evidence that the activity necessary to fluidize a tissue derives from interactions between cells and their basal interface at the ECM. At the single cell level, we reason that this activity manifests as fluctuations in cell tractions, specifically at basal cell-ECM interfaces, and at the tens of minutes timescale. In this supplemental proposal, we will first develop a platform allowing the measurement of dynamic cell tractions at the cell-ECM interface which we will apply to single normal and transformed mammary epithelial cells. Second, we will develop a parallel assay for measuring cell tractions at the cell-cell interface. We hypothesize that the magnitude of fluctuations at the cell-ECM interface will be several fold higher than at lateral interfaces, and that the largest fluctuations will occur at the tens of minutes timescale, consistent with that of junctional fluctuations in intact tissues. Successful development of this assay will allow us to investigate the impact of mechanical fluctuations in processes spanning tissue fluidization, cancer cell invasions, and exosome shedding.

摘要本申请是为了响应被标识为NOT-CA的特别利益通知（NOSI）而提交的- 23-045. 细胞活跃的生物力学特性的波动还没有得到充分的研究，但有证据表明，它们在细胞的生长过程中起着重要的作用。在核心生理过程和疾病中起着关键作用。例如，组织相变，从弹性到流体状（或堵塞到未堵塞）被认为部分是由于细胞连接处的噪音增加而引起的。力学这些力也可以导致细胞物质如外来体的脱落。这两过程在癌症进展中起核心作用，最显著的是在侵袭和转移中。但主要的挑战是确定这些力量的特定亚细胞起源和负责这些力量的机制。他们例如，使用顶点建模方法的组织流化研究已经定义了必要的以及足够的几何变化以用于上皮中的组织流化。具体来说，细胞中的活跃波动 T1转变需要结长，T1转变改变结拓扑并允许细胞扩散，流体。肌动球蛋白动力学可以在没有细胞分裂和凋亡的情况下驱动这些转变，但这些转变可能是由肌动球蛋白动力学引起的。模型集中在顶端/侧面界面（细胞之间）的肌动球蛋白活性，但在很大程度上忽略了更间接的活动源，例如源自基底表面处产生的牵引力，侧和顶细胞连接。通过跟踪正常和转化细胞中单细胞的动态，我们看到了证据表明，人类乳腺上皮类器官的活性，来源于细胞与其ECM基底界面之间的相互作用。在单细胞水平上，我们推理这种活性表现为细胞牵引力的波动，特别是在基底细胞-ECM界面，以及在细胞膜上。几十分钟的时间尺度。在这项补充建议中，我们会首先发展一个平台，测量细胞-ECM界面处的动态细胞牵引力，我们将其应用于单个正常和转化的乳腺上皮细胞。其次，我们将开发一种平行测定法，用于测量细胞牵引力，细胞-细胞界面。我们假设细胞-ECM界面的波动幅度将是几倍高于横向接口，最大的波动将发生在几十分钟，时间尺度，与完整组织中的连接波动一致。本试验的成功开发将使我们能够研究机械波动在组织流化过程中的影响，癌细胞入侵和外泌体脱落。