Mechanical properties of cancer cells reveal differential responses to anti-PD-1 immunotherapy.

Cancer cell mechanobiology plays a critical role in the efficacy of anti-PD-1 immunotherapy, yet the nanomechanical evolution of tumor cells under immune attack remains poorly characterized. In this study, we used atomic force microscopy (AFM) in quantitative imaging mode to analyze the time-dependent changes in Young's modulus, adhesion, roughness, and cell morphology of four human solid tumor cell lines (A-375, HCC-4006, SGC-7901, and MDA-MB-231) during co-culture with CD8+ T cells, either alone or with the anti-PD-1 antibody. Complementary scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were employed to visualize ultrastructural damage and cytoskeletal reorganization. This AFM-based analysis revealed distinct cell-type-specific death trajectories: A-375 cells showed a gradual decline in Young's modulus and adhesion; HCC-4006 cells exhibited early synchronous softening and loss of adhesion; SGC-7901 cells displayed a delayed but severe adhesive failure; and MDA-MB-231 cells underwent a pronounced loss of mechanical integrity by the endpoint. Combined anti-PD-1 treatment significantly accelerated and amplified these mechanical deteriorations compared to CD8+ T cells alone, with the strongest effects at 24 h. We conclude that how fast and how much cancer cells lose their mechanical properties determines their differential response to immune attack. These results demonstrate that AFM offers a method to quantitatively evaluate the biomechanical heterogeneity of cancer cells during immunotherapy, which may be used for treatment monitoring and patient stratification.