Multiscale ECM Stiffness Characterization and Quantitative Single-Cell Analysis Reveal ITGA3-Mediated...

Extracellular matrix (ECM) stiffening is a defining feature of papillary thyroid carcinoma (PTC) and influences the emergence of cancer cell subpopulations with distinct behaviors. While tissue-scale measurements provide important insights, they do not capture the local stiffness at the cellular scale, which defines the microenvironment that guides subpopulation organization through integrin-dependent mechanotransduction. This study aimed to quantitatively assess ECM stiffness across tissue and cellular scales and investigate how it shapes PTC subpopulation composition, focusing on integrin α3 (ITGA3) as a key mediator of stiffness-responsive cellular remodeling.

Human PTC tissues were examined to assess ECM stiffness at multiple scales and to evaluate ITGA3 expression. A stiffness-mimicking hydrogel system was used to investigate how modulation of ITGA3 influences cellular responses to matrix rigidity. Single-cell imaging and computational analyses were applied to resolve stiffness-responsive cell-state patterns, and additional experiments were performed to further examine adhesion-associated functional effects.

ECM stiffness was elevated at both tissue and cellular scales in human PTC. Experimental models reproducing these stiffness conditions revealed that ITGA3 is required for cells to properly respond to a stiff mechanical environment. Single-cell analyses identified distinct stiffness-responsive subpopulations, and loss of ITGA3 disrupted their redistribution across different mechanical conditions. Additional perturbation experiments further supported a central role for ITGA3 in stiffness-responsive remodeling of cell states.

This study integrates multi-scale stiffness measurements with single-cell analysis to reveal how ECM mechanics influence phenotypic heterogeneity in PTC. By identifying ITGA3 as a key mediator of stiffness-responsive remodeling, our findings link matrix stiffness with the organization of distinct cellular subpopulations. Together, these results provide conceptual insight into how the mechanical microenvironment shapes cell behavior in PTC.

The online version contains supplementary material available at 10.1007/s12195-026-00895-0.