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.

抽象的该申请是为了响应特殊利益通知（NOSI）而提交 23-045。细胞活性生物力学特性的波动被研究了，但有证据表明它们在在核心生理过程和疾病中的关键作用。例如，组织相变来自弹性至流体状（或堵塞至无障碍）被认为部分是由于细胞连接处的噪声增加而产生的力学。这些力也可以导致外泌体等细胞材料脱落。这两个过程在癌症进展中起着核心作用，最著名的是入侵和转移。但是，主要挑战是确定这些力的特定亚细胞来源和负责的机械他们。例如，使用顶点建模方法对组织流化的研究定义了必要的上皮中的组织流体的足够几何变化。具体而言，细胞中的主动波动连接长度是改变连接拓扑并允许细胞像液体。在没有细胞分裂和细胞凋亡的情况下，肌动球蛋白动力学可以推动这些过渡，但是模型专注于肌动球蛋白在顶端/侧界面（细胞之间）的活性，但在很大程度上忽略了更多间接活动源，例如从基底表面产生的轨道衍生而来横向和顶端细胞连接。通过跟踪正常和转化的单个单元的动力学原发性人类乳腺上皮器官我们看到证据表明，将组织流动所必需的活动来自ECM处细胞与其基础界面之间的相互作用。在单细胞级别，我们推理该活动表现为细胞障碍中的波动，特别是在基底细胞ECM接口和数十分钟的时间尺度。在此补充建议中，我们将首先开发一个平台，允许在单元格接口处的动态细胞段的测量，我们将应用于单个正常和转化的乳腺上皮细胞。其次，我们将开发一个平行测定，用于测量在细胞电池接口。我们假设细胞ECM接口处的波动的大小将是几个折叠比横向界面高，并且最大的波动将发生在数十分钟时间尺度，与完整组织中的连接性波动相一致。成功开发该测定法将使我们能够研究机械波动在跨越组织流化的过程中的影响，癌细胞入侵和外泌体脱落。