Fig. 5

Cytotoxic activity of CD70-CAR NK cells and CD70-CAR-IL-15 NK cells against CD70⁺ CAFs. A-C CD70 expression on RLT-PSC, hPSC21, and CT5.3hTERT CAF cell lines. A Representative flow cytometry histograms. B-C Quantification of percentage CD70⁺ cells and intensity of CD70 expression (ΔMFI). D Representative graphs of longitudinal survival follow-up (i.e., Cell Index; CI) with the xCELLigence RTCA system and corresponding quantification after 24 h co-culture (indicated with black arrow; normalized to untreated) of RLT-PSC (n = 6), hPSC21 (n = 3), and CT5.3hTERT (n = 4) CAF cell lines with CD70-CAR NK cells, CD70-CAR-IL-15 NK cells, or MOCK NK cells. Monocultures of CAF cell lines treated with culture medium were used as control. To ensure proper adhesion, CAF cell lines were grown over 24 h after which treatment with (CAR) NK cells started (indicated by a red arrow) and the CI was followed up 48-h post-treatment. E Schematic representation of the experimental setup of PDAC patient-derived microtumors, containing tumor organoids with fluorescent red labelled RLT-PSC cells, in co-culture with (CAR) NK cells for two days. Monocultures of microtumors treated with medium were included as control. Follow up using live cell imaging was done using the Spark Cyto multimode reader. F–H Co-cultures of PDAC patient 087 microtumors (P87) with different treatment conditions: untreated, MOCK control NK cells, CD70-CAR NK cells, and CD70-CAR-IL-15 NK cells. F Representative brightfield images overlayed with the red fluorescent signal. G Growth rate, normalized against T0h, over time for the different treatment conditions. H Quantification of three different timepoints (12-h, 24-h, and 36-h post-treatment), comparing the treatment conditions normalized to the untreated control at that timepoint (n = 3). Images were cropped with ImageJ. Error bars represent mean values ± standard error of mean. Linear mixed models with Tukey’s correction for multiple comparison were applied to compare means of cell survival. * p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001