Method Profile
Compare target-specific CAR-T activation and killing in paired tumor and healthy lung epithelial cultures.
Preclinical functional selection of CAR targets and effector ratios with a matched healthy-tissue comparison.
Lung tumor and matched healthy EpCAM epithelial organoids; healthy-donor, nonautologous CAR T cells directed against HER2 or PD-L1. Stromal/immune cells are not retained in the epithelial culture before T cells are added.
Activation/cytokines after 12–16 hours at E:T 1:1; separate live killing assay at E:T 5:1, 1:1 and 1:5, with images every three hours through 30 hours.
Target and control systems
Matched tumor/healthy organoids, HER2- and PD-L1-directed effectors, CD19/EGFRvIII control CARs. Endogenous T-cell-receptor expression was removed in the donor T cells.
Readouts
Intact living-organoid area, T-cell activation, cytokine secretion, antigen staining and separate molecular characterization. Tissue-versus-culture antigen checks identified cMET mismatch and high MUC1 in healthy cultures.
Compare tumor and healthy killing at each ratio and against control effectors. Interpret activation and antigen abundance separately from selective functional killing.
Six long-term tumor cultures from twelve fresh/short-frozen samples; molecular analysis included eight lines from six patients. Later cases tested feasibility within about three months, without a clinical treatment trial.
Matched healthy organoids, different effector ratios and control CARs.
High ratios killed both tumor and healthy cultures. Some pairs showed tumor selectivity; two HER2 pairs with similar healthy/tumor killing were excluded from further selectivity analysis. HER2 abundance–maximum killing correlation was nonsignificant (rho 0.771, P = 0.103).
Primary study comparisons of the stated models, treatments and measurements.
Lung tumor and matched healthy epithelial organoids exposed target- and effector-ratio-dependent CAR-T responses. Some conditions injured both tissues, while antigen abundance and T-cell activation alone did not reliably establish selective tumor killing.
