Cellular Traction and Morphological Adaptation to Cell-Scale Stiffness Gradients on Length-Encoded Micropillar Arrays

Abstract

In tumour microenvironments, cancer cells routinely encounter abrupt and spatially heterogeneous stiffness variations at the scale of individual cells. How cells sense and respond to such steep, local stiffness gradients is not well understood. Here, we developed length-encoded micropillar arrays in which stiffness is set by the effective pillar length, while the pillar tops remain at the same height and spacing. Pillars with diameters of 2, 2.5 and 3 {micro}m gave stiffness values of 4.0-82.6 nN/{micro}m (effective modulus 2.9-39.4 kPa), varying periodically over distances comparable to the size of individual cancer cells. This platform allows simultaneous measurement of cell morphology, alignment, spreading, pillar displacement, and traction forces. In MDA-MB-231 breast cancer cells, pillar displacement showed a biphasic response. It decreased with increasing stiffness up to about 10 nN/{micro}m and then remained close to 0.5 {micro}m. In contrast, traction forces increased across the full stiffness range. Cells contacted soft pillars more than stiff pillars, with about 23% greater coverage of soft regions. Cells also avoided large stiffness differences along their length: among cells whose two ends sat on pillars of equal stiffness, 48-50% lay along a single stiffness row, compared with about 14% expected by chance. Cell alignment was enriched along the two principal array directions (27-31% of cells within 10{degrees}) and increased slightly with pillar diameter. Length-encoded micropillar arrays therefore provide a platform for studying how cells respond to cell-scale mechanical heterogeneity, with relevance to cancer invasion.