Fig. 7: Benzethonium chloride inhibits tumor cell progression in the liver microenvironment by reducing the secretion of FGL1.

A Graph of the z scores (red line = −3) showing the effect of 1430 FDA-approved drugs (blue spheres) on the secretion of FGL1 in HT29 cells. B Structure of benzethonium chloride. C ELISA detection of FGL1 secreted by indicated cells treated with benzethonium chloride, n = 3 biologically independent experiments. B.C., benzethonium chloride. D IB detection of FGL1 expression in indicated cells. E Representative H&E staining of metastatic livers from C57BL/6 J mice intraportally injected with MC38 cells followed by treatment with PBS or benzethonium chloride as indicated. Scale bar for bright-field images, 1 cm; scale bar for H&E images, 2.5 mm. F Quantification of liver weight and the number of liver metastases of the indicated groups of mice (n = 5 mice per group). G Representative H&E staining of metastatic livers from C57BL/6 J mice intraportally injected with MC38 cells followed by treatment as indicated. Scale bar for bright-field images, 1 cm; scale bar for H&E images, 2.5 mm. H Quantification of liver weight and the number of liver metastases of the indicated groups of mice (n = 5 mice per group). I–K Flow cytometric analysis of the number of IFN-γ⁺ CD8⁺ (I), IFN-γ⁺ CD4⁺ (K), Ki67⁺ CD8⁺ and Ki67⁺ CD4⁺ T cells (J) in liver metastases in indicated groups of mice (n = 5 mice per group). L Survival curve analysis of mice in the indicated groups (Mean survival times of IgG+PBS, αPD-1 + PBS, IgG+B.C. and αPD-1 + B.C. mice were 26.5, 27, 33.9 and 43.5 days, respectively) (n = 8 mice per group). M Graphical abstract describing how TAMs upregulate FGL1 levels in tumor cells and mediate immune evasion as well as how benzethonium chloride reduces FGL1 in the liver microenvironment to promote antitumor immunity. The data are presented as the mean ± SD. z’ in A was calculated as previously described⁴⁰. IB experiments in (D) were repeated three times, the data are representative of three biologically independent experiments. The data in (L) were determined by Kaplan‒Meier analysis with the log-rank test. P values were determined by one-way ANOVA (C, F, H–K).