Fig. 3: Pre-activated T cells also kill CCA organoids in a patient-specific manner.

Bright field images (a) of CCA organoids cultured with and without T cells for 7 days demonstrate that CCA1 organoids are not inhibited by T cells while CCA2 and CCA3 organoids are smaller and disintegrated, indicating cell death. Flow cytometric cell count for EpCAM-positive, CD45-negative, and DAPI-negative cells (b) shows that CCA2 and CCA3 organoids sustain significant cell death after 7 days of co-culture with anti-CD3/CD28 bead pre-activated T cells, while CCA1 organoids are not affected (n ≥ 3 biological replicates for each organoid line; n = 2 different PBMC batches). Flow cytometric cell count for live (DAPI-negative) CD8⁺ and CD8^- T cells (c). Relative difference in CYFRA concentration in supernatant after 7 days of CCA organoid and T cell co-culture (d) demonstrates a significant increase in CCA2 (n ≥ 3 biological replicates for each organoid line). Representative confocal images of Cell Trace Far Red-stained T cells (red), Hoechst 33342-stained CCA2 organoids (blue), and co-cultures in the presence of a Caspase 3/7-probe (green) at timepoints 0, 90, and 180 h (e). Quantification of dead cell surface (Caspase 3/7 probe-positivity) in organoid area (Hoechst 33342 labelled) (f) and number of T cells (g) in nine fields of view of confocal time-lapse imaging every 6 h for 180 h. Organoid death is higher in co-cultures with T cells compared to CCA2 organoids alone. The number of T cells (Cell Trace Far Red labelled) is comparable between solo culture and co-culture. Scale bar: 200 µm (left and middle panel of a), 50 µm (right panel of a), 100 µm (e). All values with error bars represent mean with SEM. *p < 0.05, **p < 0.01, ***p < 0.001.