Regulation of Adherent Cell Proliferation by Matrix Viscoelasticity

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.