Time-delay deformation cytometry reveals a Piezo1-dependent viscomechanical transition driving glioblastoma invasion.

The mechanical properties of tumor cells critically influence their invasive behavior, yet how extracellular viscosity regulates cellular deformation-recovery dynamics remains poorly understood. Here, we developed time-delay deformation cytometry, a microfluidic platform integrating inertial focusing, hydrodynamic deformation, and sequential imaging to characterize single-cell mechanical responses and morphological recovery. Using glioblastoma models exposed to elevated extracellular viscosity, we identified a distinct mechanical phenotype characterized by increased deformation resistance and incomplete morphological recovery within the millisecond observation window. Complementary atomic force microscopy measurements revealed increased cellular stiffness and Young's modulus, accompanied by reduced mechanical hysteresis, supporting viscosity-associated mechanical reinforcement. Notably, this altered recovery phenotype was associated with enhanced cellular invasion. Mechanistically, viscosity priming increased PIEZO1 expression and intracellular Ca2+ signaling, accompanied by ROCK-associated actomyosin remodeling, focal adhesion elongation, and YAP nuclear localization. PIEZO1 knockdown attenuated Ca2+ elevation, ROCK-associated phosphorylation, invasive behavior, and incomplete morphological recovery, whereas TRPV4 knockdown showed comparatively limited effects. Pharmacological ROCK inhibition similarly attenuated the altered recovery phenotype, supporting the involvement of PIEZO1-Ca2+-ROCK signaling. Furthermore, viscosity-primed GL261 cells exhibited comparable mechanical changes and increased infiltration following orthotopic transplantation. Together, these findings establish time-delay deformation cytometry as a platform for resolving viscosity-associated mechanical responses and suggest that time-dependent morphological recovery may serve as a potential biophysical indicator of glioblastoma invasive behavior.