Exploring Mechano-metabolomic Pathways in CAR T cells

Mechanical force generation at the immunological synapse is a hallmark of CAR T cell cytotoxicity and a key indicator of functional potency, yet the underlying mechanisms are not fully understood. Here, we performed real-time single-cell analysis to quantify the spatiotemporal dynamics of synaptic force and metabolic activity in CAR T cells upon CD19 antigen engagement. We identified a Piezo1–Ca²?–Wnt signaling axis that integrates mechanical sensing with cytoskeletal remodeling and metabolic reprogramming. This mechano-metabolic coupling drives CAR T cell activation and varies across different CAR constructs, patient-derived products, and manufacturing conditions. Notably, functional heterogeneity among CAR T cells correlates with differences in their mechanical force profiles and energetic output. Our findings reveal a reciprocal relationship between mechanotransduction and metabolic switching, providing new insight into CAR T cell biology. This framework may inform the development of screening tools and engineering strategies to enhance CAR T cell mechanical fitness, energetic resilience, and therapeutic efficacy. Overall design: CAR T cells were acitvated by anti-FMC63 Antibody µBeads for 24 hours.