title: Collective cell behaviour in long-range mechanosensing of extracellular matrix dimensions
Creators: name: Muresan (Tusan), Camelia Gratiela
Publisher: name: University of Southampton
Year: 2019

description: Tissue healing and regeneration is strongly influenced by the mechanical properties of the extracellular matrix (ECM). Most mammalian cells attach to ECM and, by applying force, are able to mechanosense its stiffness as a result of its resistance to deformation. An increase in stiffness is known to affect cell proliferation, differentiation and migration. However, the stiffness that cells detect is determined not only by the matrix elastic modulus but also by the material thickness. Single cells are known to sense rigid boundaries through a soft hydrogel when the depth is less than 10 Î¼m. However, most of the tissues are composed of cohesive layers of cells, which behave very differently from single cells. Therefore, this study tested the hypothesis that colonies exert more force by acting collectively and produce more deformation than individual cells, allowing them to mechanosense more deeply into the underlying substrate than individual cells.The effect of substrate elasticity and thickness on cells and colonies was determined by culturing cells on customised extracellular-matrix-coated polyacrylamide (PA) hydrogels of varying elasticity (0.5 â 40 kPa) and thickness (1 â 1000 Î¼m), attached basally to glass substrates. Cell spreading, attachment and density were measured by epifluorescence and confocal microscopy. Cell-induced surface deformations were quantified by imaging the fluorescent fiducial-marker-labelled hydrogels during a time-lapse experiment and by computing cumulative deformations using MATLAB code. Immunofluorescent of E-cadherin and involucrin was used to test phenotypical changes in primary keratinocytes.MG63 (human osteosarcoma) single cells area decreased as a function of substrate thickness; the data was fitted to an exponential model, characterised by a half-maximum response at 3.2 Î¼m thickness. MG63 colonies were found to mechanosense the stiffness of the underlying support at greater thicknesses than individual cells. The half-maximal response for MG63 colonies (54 Âµm) was 17 times greater than that for individual cells. The abilityof cells to mechanosense matrix depth was dependent on Rho-associated protein kinase mediated cellular contractility. Colony-induced surface deformations were significantly greater on thick hydrogels than on thin hydrogels (3.9 Â± 0.8 versus 1.9 Â± 1.2 Î¼m; p &lt; 0.05). In addition, deformations extended greater distances from the colony border on thick hydrogels compared to thin hydrogels. A fibrous ECM protein (Col) was found to amplify the depth at which both single cells and colonies sense the underlying rigid substrate. The expression of E-cadherin in keratinocytes colonies was identified only on thin hydrogels. Overall, the novel findings presented in this thesis suggest that by acting collectively, groups of cells mechanosense stiff boundaries at greater thicknesses than single cells. This integrative phenomenon may empower colonies or sheets of cells to interrogate their environment mechanically in a range of biological contexts such as tissue regeneration, embryogenesis, cancer metastasis or biomaterials design.