Fig. 7: Activation of the autophagic pathway confers a superior anti-tumor function to PD-L1 CAR-NK cells.

A Scheme depicting the structure of PD-L1.CAR NK-92 used in the study. B Flow cytometry analysis of PD-L1.CAR expression on PD-L1.CAR NK-92 and parental NK-92 cells, by human recombinant PD-L1-Fc protein combined with anti-Fc secondary antibody. Filled gray areas indicate negative controls stained with secondary antibody only. Representative data from at least 3 independent experiments are shown. C Flow cytometry analysis of PD-L1 expression in PC3 cell line. Left plot represents Fluorescence Minus One (FMO) control, right plot represents cells stained with PD-L1 antibody. D Cytotoxicity of PD-L1.CAR NK-92 cells, treated or not with tumor-conditioned medium, with or without Metformin, against PC3 target cells (n = 4 biological replicates, independently collected batches of tumor-conditioned medium). E, F Tumor growth curves (E) and tumor volume at the end of the experiment (F) referred to mice treated as described in 4G (n = 7 control, n = 7 CAR NK-92 cells, n = 7 Metformin-treated CAR NK-92 cells). G Graph showing the absolute number of tumor-infiltrating NK-92 cells. n = 7 control, n = 7 CAR NK-92 cells, n = 7 Metformin-treated CAR NK-92 cells. H, I Graph showing the frequency of LC3B⁺ cells (H) and effector function expression (I) in tumor-infiltrating CAR NK-92 cells. n = 7 control, n = 7 CAR NK-92 cells, n = 7 Metformin-treated CAR NK-92 cells. Data in (D) and (F) are presented as Min to Max box-and-whisker plot, the box extends from the 25th to 75th percentiles and the whiskers reach the sample maximum and minimum values, the median is indicated at center line and the mean value is indicated as “+”; one-way ANOVA test with Holm-Šídák’s multiple-comparisons test. Symbols in (E) represent mean and error bars indicate SEM; one-way ANOVA test with Holm-Šídák’s multiple-comparisons test, comparing area under the curve (AUC). Data in (G), (H), and (I) are presented as Min to Max box-and-whisker plot, the box extends from the 25th to 75th percentiles and the whiskers reach the sample maximum and minimum values, the median is indicated at center line and the mean value is indicated as “+”; two-tailed unpaired t test.