Long-term survival and melanoma lung tumor analysis. A) Survival of mice with established pulmonary metastases of B16F10 melanoma cells, intravenously injected with genetically modified ADSCs. An additional group of mice with established lung metastases derived from melanoma cells received only PBS (control group). Survival was measured from the day of melanoma cell injection until the day of death. Survival curves were drawn by the Kaplan-Meier method (n =6 in each treatment group). B) Representative microscopic images of immunohistochemistry (IHC) staining for cell proliferation marker Ki67 (Inlays show negative control and positive controls), TUNEL staining for analysis of tumor cell apoptosis, IHC staining for PD-L1 (Scale bars =50 μm), and IHC satining for neovasculature marker CD31 (Scale bar =100 μm). Microscopic images clearly shows injected IFNγ-expressing ADSCs significantly upregulate PD-L1 expression in melanoma lung tumor cells (compared to untreated control and EGFP-ADSC injected groups). C) Bar graphs showing statistically significant effect of IFNγ-ADSCs on growth of metastatic melanoma tumors. (Left) Quantification of proliferation and apoptosis was performed by averaging percentage of positively stained cells to total cells within 10 randomly selected areas at x200 magnification. IFNγ-producing ADSCs significantly decreased the melanoma cell proliferation and increased tumor cell apoptosis in groups treated with IFNγ-producing ADSCs in a statistically meaningful manner (ANOVA; p < .001). (Right) Quantification of angiogenesis was performed by counting the number of vessels in 10 randomly selected areas of CD31 stained sections at x200 magnification. Results indicate statistically significant inhibition of angiogenesis after injection of IFNγ-ADSCs. Each group consisted of four mice. ***P < .001 (ANOVA). The statistical tests were two-sided. PDL1 = programmed cell death 1 ligand 1.