Fig. 4: Effect of metformin and anti-PD-L1 combination on lymphocytes antitumor activity towards NSCLC cells.

A Cell viability assay of LKB1wt (H1299) and LKB1mut (H460) NSCLC cells exposed to NSCLC patient-derived PBMCs pre-treated with metformin (MET) and anti-PD-L1 for 48 h. Data are expressed as mean ± SD. At least three independent experiments were performed. B Western blotting of H460 and H1299 NSCLC cell lines after 48 h of co-culture with NSCLC patient-derived PBMCs. β-actin was used to ensure equal loading. C H460 and H1299 NSCLC cell lines were seeded in a 12-well plate (3000 cells/well). The cells were preincubated for 48 h with NSCLC patient-derived PBMCs pre-treated with drugs for 5 days. Afterward, the cells were fixed and stained with 0.5% crystal violet solution. Images of representative wells were scanned and are shown. Bar plots of colony numbers for H1299 NSCLC cells are shown in blue, while H460 NSCLC cells are shown in red. Results are expressed as the mean of three technical replicates ± SD. D Real-time PCR analysis of in vitro STING, c-GAS, CXCL10 and CCL5 mRNA expression in patient-derived monocytes (n = 11). Changes in mRNA levels were normalized to the expression of housekeeping genes (18S). Data are expressed as means ± SEM derived from n = 2 technically calculated by the comparative method 2 − ∆∆Ct. One-tailed unpaired Student’s t-test with CI = 95%. E Cell viability assay of NSCLC cell lines cultured with NSCLC patient-isolated monocytes treated with MET and/or anti-PD-L1 for 48 h conditioned media. Each experiment was performed in triplicate. Data are expressed as mean ± SD. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.