Dynamic Resilience Assessment of Single Cells under Cyclic Mechanical Loading Reveals Chemotherapy-Induced Cytoskeletal Microdamage

Cancer cells depend on mechanical resilience to survive within confined microenvironments and the deformability to traverse vascular barriers. However, current microfluidic single-cell mechanophenotyping lacks comprehensive characterization of post-deformation recovery dynamics and standardized quantification frameworks for mechanical resilience─limitations that hinder the development of mechanical biomarkers critical for understanding tumor progression. Here, we introduce dynamic resilience as a novel biomechanical metric to characterize the cellular capacity for maintaining structural homeostasis through sequential deformation-recovery cycles. To operationalize this concept, we utilize cyclical mechanical loading microchannels (CMLMs) to apply controlled mechanical stimuli to individual cells, including static uniform compression, low-frequency, mid-frequency, and high-frequency localized loading. Within this framework, we quantified the mechanical deformation process using the aspect ratio (AR) for anisotropy and circularity deformation (CD) for contour irregularity. Next, we introduced a resilience metric d_res by calculating the dynamic time warping (DTW) distance between pre-load vs post-load deformation trajectories. Under a controlled flow rate (20-300 μ L/h), AR proved to be a robust resilience indicator, effectively capturing recovery behavior. Single-cell analysis indicates that mechanical resilience depends more on cytoskeletal organization and cross-linking density than on cell size. To validate the performance of the dynamic resilience characteristics, we applied this approach to characterize two distinct cell lines (HeLa and BxPC-3) and conducted chemotherapeutic testing with daunorubicin to assess the drug susceptibility. Daunorubicin treatment established concentration-dependent modulation of cellular resilience, with mechanosensitivity specifically manifesting under large-strain deformations. This mechanoresponsive behavior demonstrates that cyclic-loading-characterized mechanical resilience quantifies drug-induced cytoskeletal microdamage in a dose-dependent manner. These findings offer an evaluation framework for cellular resilience and provide new insights into chemotherapy susceptibility under microenvironmental stresses.