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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.

摘要本申请是为了回应被确认为非CA-CA的特殊利益通知(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，它们在在核心生理过程和疾病中都起着关键作用。例如，组织相变，来自弹性-流体样(或被堵塞到不堵塞)被认为部分是由于细胞交界处增加的噪音引起的机械师。这些力量也可能导致细胞物质的脱落，如外切体。这两个都是过程在癌症的进展中起着核心作用，尤其是在侵袭和转移方面。然而，a 主要的挑战是确定这些部队的具体亚细胞来源以及负责他们。例如，使用顶点建模方法对组织流态化的研究已经定义了必要的和足够的几何变化，使组织在上皮内流态化。具体地说，细胞中的活跃波动 T1转换需要连接长度，这会改变连接拓扑并允许细胞像这样扩散一种液体。肌动球蛋白的动力学可以在没有细胞分裂和凋亡的情况下推动这些转变，但这些模型主要关注顶端/外侧界面(细胞之间)的肌动球蛋白活性，但在很大程度上忽略了更多的间接活动的来源，例如来自在作用于赤道的基面产生的牵引力侧向和顶端细胞连接。通过跟踪正常和转化的单个细胞的动力学我们看到证据表明，使组织流态化所必需的活动源于细胞及其在细胞外基质上的基础界面之间的相互作用。在单细胞水平上，我们推断这种活动表现为细胞牵引力的波动，特别是在基础细胞-细胞外基质界面和几十分钟的时间刻度。在这项补充建议中，我们将首先开发一个平台，允许测量细胞-细胞外基质界面上的动态细胞牵引力，我们将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检测方法，用于测量细胞牵引力细胞-细胞界面。我们假设细胞-细胞外基质界面的波动幅度为比侧界面高几倍，最大的波动将出现在几十分钟时间尺度，与完整组织中连接波动的时间尺度一致。该检测方法的研制成功将使我们能够调查跨越组织流态化过程中的机械波动的影响，癌细胞入侵和外体脱落。