Mechanical preconditioning by shear stress enhances memory formation and anti-tumor function of CAR-T cells

Chimeric antigen receptor T-cell (CAR-T) therapy has achieved remarkable success in the treatment of hematological malignancies, and its application to solid tumors is currently under active investigation. Upon infusion into the bloodstream, CAR-T cells are exposed to fluid shear stress (SS) generated by blood flow. However, the impacts of SS on CAR-T cells function are not well understood. In this study, we exposed anti-mesothelin (MSLN) CAR-T cells to a shear stress of 5 dynes/cm2, and observed reduced cell viability, enhanced activation, and decreased expression of exhaustion markers. Notably, we found that a one-hour SS stimulation applied seven days after T cell isolation and activation significantly increased the proportion of stem cell memory T cells (Tscm), mitigated exhaustion, and enhanced the anti-tumor efficacy of anti-MSLN CAR-T cells. Furthermore, SS-preconditioned CAR-T cells exhibited resistance to SS-induced cell death and demonstrated increased migratory capacity. In vivo experiments, SS-preconditioned CAR-T cells exhibited greater tumor infiltration, improved persistence, superior tumor control, and a favorable safety profile in tumor-bearing mice. Transcriptomic analysis revealed upregulation of genes associated with activation, infiltration, and microtubule dynamics, alongside downregulation of genes linked to exhaustion, apoptosis, and immunosuppression. Collectively, our findings demonstrate for the first time that mechanical preconditioning with fluid shear stress can reprogram CAR-T cells toward a more functional and persistent phenotype, offering a novel strategy to optimize CAR-T therapy for solid tumors.