Figure 4: CAR.PDZ NK cells have enhanced cytolytic activity and invasive properties.

(a) Cytotoxicity assay scheme with A549 lung adenocarcinoma cell viability determined by a chromogenic MTS assay after 24-hour co-culture with NK cells.

(b) CAR vs CAR.PDZ NK cells: significant differences at all effector to target (E:T) ratios except for 5:1. All other comparisons are significantly different. Two-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR. Statistical difference delineated by q<0.05 *, q<0.01 **, n=3 donors, mean±SEM shown.

(c) CAR vs CAR.PDZ vs CAR.PDZmut NK cells: significant differences at all effector to target (E:T) ratios 2.5:1. Two-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR. n=3 donors, mean±SEM shown.

(d) UN, CAR Δ, CAR, CAR.PDZ NK cells with 10 micromolar PDZ blocking or control (CTRL) peptides. Technical triplicates with mean±SEM shown.

(e) Area between the curve analysis (shaded regions in d) for two independent experiments with unique donors normalized to untransduced.

(f) Representative images from a tumoroid droplet cytotoxicity assay 143b osteosarcoma cells in mCherry. White bars indicate 1mm.

(g) Quantification of the resulting tumor reduction as determined by tumor area normalized to the 1-hour (hr) mark. CAR.PDZ vs CAR Δ NK cells: significant differences starting at 40hrs of culture; CAR.PDZ vs CAR NK cells: significant differences starting at 58hrs of culture. Two-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR. Dashed lines indicate comparison groups and what hour they become statistically different at least q<0.05 *. n=3 donors, mean±SEM shown.

(h) Representative images from a tumoroid droplet invasion assay showing NK cells in green. Co-cultures were imaged hourly for 2 days. White bars indicate 1mm.

(i) NK cell invasion ratio represents the counted NK cells per mm² and normalized to the 1-hour mark.