Regulation of Adherent Cell Proliferation by Matrix Viscoelasticity

基质粘弹性对贴壁细胞增殖的调节

• 批准号: 10735701
• 负责人: Ovijit Chaudhuri
• 金额: $ 38.6万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10735701
• 关键词: 3-Dimensional; ATAC-seq; Actomyosin; Address; Adherent Culture; Adhesions; Adhesives; Alginates; Automobile Driving; Basement membrane; Binding; Biocompatible Materials; Bioinformatics; Biological Assay; Biological Process; Biophysical Process; Biophysics; Breast Epithelial Cells; CRISPR screen; CRISPR/Cas technology; Cell Adhesion; Cell Proliferation; Cell Volumes; Cells; Characteristics; Chromatin; Collagen Type I; Data; Deposition; Disease; Elasticity; Epigenetic Process; Epithelial Cells; Exhibits; Extracellular Matrix; Fibroblasts; Gene Expression; Genes; Genetic Transcription; Goals; Human; Hydrogels; Integrin Binding; Integrins; Ion Channel; Knock-out; Knowledge; Ligands; Liquid substance; MAP Kinase Gene; Mechanics; Mediating; Mesenchymal Stem Cells; Mission; Molecular; Molecular Analysis; Morphogenesis; PIK3CG gene; Pathway interactions; Process; Proliferating; Regulation; Relaxation; Research; Role; Signal Pathway; Solid; Sp1 Transcription Factor; Stress; Tissues; Transcriptional Regulation; United States National Institutes of Health; Viscosity; Work; biophysical techniques; cancer cell; cell behavior; density; disability; epigenetic regulation; epigenome; genome-wide; genome-wide analysis; in vivo; innovation; mechanotransduction; migration; novel; reconstitution; response; stem cell differentiation; three dimensional cell culture; tool; transcriptome sequencing; viscoelasticity; whole genome

Cell proliferation is a fundamental biological process that often occurs for cells in a 3D context in vivo, in which cells are surrounded by extracellular matrix (ECM) and other cells, and various applications rely on the proliferation of cells within a biomaterial. It has long been known that changes in matrix stiffness impact cell behaviors through mechanotransduction, and mechanisms of stiffness-sensing in 2D culture are now established. However, the mechanisms mediating the impact of changes in matrix stiffness on cell proliferation in 3D remain unclear. Further, living tissues and ECMs are viscoelastic, exhibiting some characteristics of elastic solids and some of viscous liquids. Matrix viscoelasticity is sensed through mechanotransduction, and we have found that changes in matrix viscoelasticity impact cell spreading, migration, proliferation, stem cell differentiation, matrix deposition, morphogenesis, and gene expression. However, the mechanisms mediating the impact of matrix viscoelasticity on these processes, particularly proliferation remain unclear. The goal of the proposed work is to determine the mechanism mediating the impact of matrix stiffness and viscoelasticity on cell proliferation in 3D matrices. Our overall hypothesis is that mechanosensitive ion channel-mediated pathways and integrin-mediated pathways interplay to sense matrix viscoelasticity and stiffness, and subsequently control proliferation through changes in chromatin accessibility, YAP-independent transcription, and a set of molecular regulators not implicated from 2D culture studies. We will address this hypothesis in 3 aims, using an approach that involves the use of alginate hydrogels with independently tunable viscoelasticity, stiffness, and RGD ligand density for 3D culture of adherent cells, including fibroblasts, epithelial cells, and mesenchymal stem cells. In aim 1, we will determine the biophysical mechanisms underlying the impact of hydrogel viscoelasticity, stiffness, and adhesivity on the proliferation of adherent cells in 3D culture. In Aim 2, we will elucidate transcriptional and epigenetic regulation of mechanotransduction and proliferation, using RNA-seq and ATAC-seq combined with advanced bioinformatics analyses. In Aim 3, we will identify novel regulators of proliferation and mechanotransduction in 3D using genome-wide CRISPR screening. Innovative aspects of this approach include the study of mechanisms mediating mechanotrasduction and proliferation in 3D matrices, the focus on viscoelasticity (beyond stiffness), the potential for discovering YAP-independent mechanisms of mechanotransduction, the identification of how the epigenome regulates mechanotransduction and proliferation in 3D, and the application of a CRISPR screen to identify novel molecular regulators of mechanotransduction. The significance of this work is that it will determine the biophysical and molecular mechanisms by which ECM or biomaterial stiffness and viscoelasticity regulate cell proliferation in 3D. Given the importance of cell proliferation, the ubiquity of matrix viscoelasticity in ECMs, and the potential relevance of discovered mechanisms of mechanotransduction to other processes, the significance is expected to be high.

细胞增殖是一个基本的生物学过程，在体内3D上下文中通常发生细胞，其中细胞发生在其中，其中 细胞被细胞外基质（ECM）和其他细胞包围，各种应用都依赖于 生物材料内细胞的增殖。早就知道矩阵刚度影响细胞的变化 通过机械转导的行为，现在是2D培养中刚度感应的机制 已确立的。但是，介导基质刚度变化对细胞增殖的影响的机制 在3D中仍不清楚。此外，活组织和ECM是粘弹性的，表现出弹性的某些特征 固体和一些粘性液体。通过机械传输来感知矩阵粘弹性，我们有 发现基质粘弹性影响细胞扩散，迁移，增殖，干细胞的变化 分化，基质沉积，形态发生和基因表达。但是，介导的机制 矩阵粘弹性对这些过程的影响，尤其是增殖尚不清楚。目标的目标 建议的工作是确定介导基质刚度和粘弹性对细胞影响的机制 3D矩阵中的增殖。我们的总体假设是机械敏感的离子通道介导的途径 和整联蛋白介导的途径相互作用，以了解矩阵粘弹性和刚度，然后控制 通过染色质可及性，非YAP独立转录和一组分子的变化增殖 监管因子不涉及2D文化研究。我们将使用一种方法来解决3个目标的这一假设 这涉及使用具有独立可调粘弹性，刚度和RGD配体的藻酸盐水凝胶 粘附细胞3D培养的密度，包括成纤维细胞，上皮细胞和间质干细胞。在 AIM 1，我们将确定水凝胶粘弹性，刚度，刚度的影响的生物物理机制 以及3D培养物中粘附细胞增殖的粘附性。在AIM 2中，我们将阐明转录和 使用RNA-SEQ和ATAC-SEQ对机械转导和增殖的表观遗传调节结合 高级生物信息学分析。在AIM 3中，我们将确定扩散和 使用全基因组CRISPR筛选在3D中的机械转导。这种方法的创新方面包括 对3D矩阵中介导的机制和增殖的机制的研究，重点是 粘弹性（超出刚度），发现独立于YAP的机制的潜力 机械转导，表观基因组如何调节机械转导和增殖的鉴定 在3D中，以及CRISPR屏幕的应用来识别机械转导的新分子调节剂。 这项工作的意义在于它将确定ECM的生物物理和分子机制 或生物材料刚度和粘弹性调节3D中的细胞增殖。鉴于细胞的重要性 ECM中基质粘弹性的扩散，无处不在的潜在以及发现的潜在相关性 机械转移到其他过程的机制，其显着性有望很高。