Real-time multimodal phenotyping reveals distinct tumour cell dynamics and immune escape mechanisms in T cell therapies

Adoptive T cell transfer therapy remains limited by the inability to monitor live tumour cell dynamics during treatment. Here we introduce a real-time, label-free phenotyping system that integrates electrical impedance spectroscopy, Raman spectroscopy and microscopy to analyse live tumour cells undergoing therapy. This system enables simultaneous tracking of metabolic activity, membrane integrity and cytoplasmic properties at single-cell resolution. First, analysis of glycolysis reveals that tumour-infiltrating lymphocytes suppress lactate production early, reducing tumour aggressiveness, while chimaeric antigen receptor T cells trigger tumour silent escape early and delay metabolic inhibition until later stages, culminating in cell death. Second, membrane profiling shows early phospholipid and cholesterol depletion under tumour-infiltrating lymphocyte treatment, with partial recovery, whereas chimaeric antigen receptor T cells cause progressive and irreversible membrane damage. Third, cytoplasmic analysis identifies early protein structural disruption and ionic imbalance under tumour-infiltrating lymphocyte therapy, while chimaeric antigen receptor T cells trigger delayed metabolic collapse and cytoplasmic contraction. These findings uncover distinct immune killing mechanisms and escape phases, offering mechanistic insights into tumour–immune interactions and informing the design of personalized therapeutic strategies.