Identifying and reprogramming softness-driven cancer stem-like cells overcomes CAR-T cell resistance in solid tumours

Solid tumours show substantial mechanical heterogeneity, yet how such cues influence the susceptibility of cancer cells to T cell-based therapies remains unclear. Here we discover that cancer cells grown on soft matrices are less sensitive to chimeric antigen receptor T cell cytotoxicity and exhibit elevated extracellular adenosine triphosphate and sustained calcium activity. To understand the mechanisms underlying this reduced killing, we sought to identify the cells that respond to mechanical softness. We engineered a doxycycline-gated calcium-activated transcriptional mechano-recorder that integrates softness-induced calcium activity over a defined recording window and converts this prior signalling history into a stable fluorescent output that persists for days. Unlike real-time calcium indicators, which report instantaneous calcium activity only at the moment of imaging, this recorder preserves a sortable transcriptional mark, enabling selective labelling and profiling of cells according to their past mechanosensing activity. Transcriptomic analyses showed that recorder-positive cells adopt a stem-like programme, including epithelial-mesenchymal transition, hypoxia responses, oncogenic signalling and elevated stemness markers, across cancer cell lines and patient-derived samples. To render these resistant cells targetable, we rewired the mechano-recorder into a mechano-reprogrammer by replacing the fluorescent output with the clinically validated antigen CD19, enabling softness-responsive cells to be recognized by CD19-directed T cells. This rewired system improved elimination of stem-like cancer cells in culture and animal models, converting mechanobiological resistance into therapeutic vulnerability.