Mechanical memory of cells to fluid shear stress and substrate stiffness revealed by atomic force microscopy

Mechanical forces present in the primary microenvironment can imprint cancer cell behavior to promote distant metastasis, but current understanding of how cancer cells perceive these mechanical signals and form a memory of them remains limited. In particular, atomic force microscopy (AFM) has become an important and standard technique for delineating the mechanical properties of single living cells under physiological conditions. Here, based on the integration of AFM, reversible bonding microfluidics, and hydrogels, the mechanical memory of cells in response to fluid shear stress and substrate stiffness was investigated. A hydrogel with tunable stiffness was used to mimic the extracellular matrix (ECM), which was combined with a microfluidic chip employing reversible bonding, thus constructing an in vitro tumor microenvironment model. The model allows precise control of fluid shear stress and ECM stiffness, and is compatible with AFM, enabling in situ mechanical measurements of individual cancer cells under multiple tumor microenvironmental stimuli. The experimental results distinctly show that an increase in fluid shear stress/ECM stiffness leads to changes in cell mechanics (stiffening), and cells retain this change as mechanical memory in the succedent growth environment. Moreover, the synergistic effects of fluid shear stress and substrate stiffness in imprinting cellular mechanics were revealed. The study provides an experimental method based on AFM for exploring the mechanical memory behavior of cancer cells, which will benefit the advancement of cancer mechanobiology.