Microfluidic Cytotongue system enables early, label-free detection of CAR-T-induced mechanical softening in tumor spheroids.

Quantitative assessment of chimeric antigen receptor T (CAR-T) activity in solid tumors remains challenging, as immune engagement can induce early mechanical softening of tumor spheroids before overt cell death becomes detectable. To address this limitation, we developed Cytotongue, a three-dimensional (3D)-printed microfluidic aspiration system for real-time, treatment-integrated aspiration-response phenotyping of live tumor spheroids. Using human epidermal growth factor receptor 2 (HER2)-positive BT-474 breast cancer spheroids exposed to CAR-T cells, the Cytotongue system quantified aspiration-induced elongation dynamics and assay-specific deformation indices derived from an empirical biphasic deformation framework. This approach enabled detection of CAR-T-induced mechanical softening at low effector-to-target ratios (1:1-2:1) within 24 h, conditions under which conventional propidium iodide (PI) staining showed minimal response. System-level validation demonstrated robust performance, with high Z' factors (0.74-0.93), large effect sizes, and low coefficients of variation. Moreover, Cytotongue distinguished CAR-T-associated mechanical softening accompanied by apoptosis from doxorubicin-induced deformation responses despite comparable PI readouts. Collectively, this work introduces a treatment-integrated microfluidic aspiration system that enables real-time mechanical phenotyping of tumor spheroids, providing a new physical dimension for evaluating immunotherapeutic and drug responses.