Fig. 6

CYP19A1 inhibition potentiates CD8⁺ T cell-mediated antitumor immunity in vitro coculture model. A The HT29 or HCT116 cells were treated with the letrozole or vehicle for 24 h. Aromatase activity was evaluated by culturing cells with testosterone and measuring estradiol levels in the culture medium with enzyme-linked immunosorbent assay. B The viabilities of HT29 and HCT116 cells were determined by MTT assay. C The level of CYP19A1 protein expression was measured in HT29 and HCT116 cells transfected with CYP19A1 siRNA (si-CYP19A1) or si-Scram. Human PBMCs were cocultured with the letrozole (5 μM) or si-CYP19A1-treated HT29 cells or HCT116 cells for 24 h. D Tumor cells pre-incubated with anti-EpCAM antibody were stained by 7-AAD, and then the proportion of EpCAM⁺ 7-AAD⁺ cells were analyzed by flow cytometry. E Carboxyfluorescein succinimidyl ester (CFSE) dilution was used to measure the proliferation of CD8⁺ T cells. F and G The percentage of IFN-γ⁺ or CD107a⁺ CD8⁺ cells were determined by flow cytometry. H Transwell migration analysis of human brain vascular pericytes (HBVP) and human umbilical vein endothelial cells (HUVEC) treated with the conditioned medium (CM) from the above treated-HT29 cells and HCT116 cells. The number of the migrated cells was counted. The values are presented as the mean ± standard error of the mean, n = 5. ^*, P < 0.05; ^**, P < 0.01 vs. control. ^##, P < 0.01 vs. si-Scram