Fig. 1: FGL1 facilitates the progression of colorectal cancer through immunosuppression in the liver microenvironment.

A Schematic of establishment of an orthotopic liver metastasis mouse model of CRC by injection of MC38-GFP cells into the cecum termini and the FACS sorting strategy. FACS, fluorescence-activated cell sorting. B Immunoblotting (IB) detection of indicated immunosuppressive molecule expression in MC38 cells sorted from primary and liver metastatic tumor tissue by flow cytometry. C Quantitative estimates of the expression of indicated immunosuppressive molecules using ImageJ based on IB analysis. D Representative luciferase images and H&E staining of metastatic livers from C57BL/6 J mice intraportally injected with the control (shNC) and FGL1 knockdown MC38 cells. Scale bar for bright-field images, 1 cm; scale bar for H&E images, 2.5 mm. E Experimental strategies. i.p.v., intraportal vein injection; i.v., intravenous injection. F Representative luciferase images and H&E staining of metastatic livers from C57BL/6 J mice intraportally implanted with MC38 cells and subjected to AAV-shNC or AAV-shFGL1 (#1, #2) treatment. Scale bar for bright-field images, 1 cm; scale bar for H&E images, 2.5 mm. AAV, adeno-associated virus. G Bioluminescent quantification and liver weight of mice with knockdown of FGL1 in MC38 cells or hepatocytes (n = 5 mice per group). H, I Quantification of liver metastases (H) and ELISA detection of plasma FGL1 levels (I) in mice with FGL1 knockdown in MC38 cells and hepatocytes (n = 5 mice per group). J Flow cytometric analysis of the number of IFN-γ⁺ CD8⁺ and IFN-γ⁺ CD4⁺ T cells in liver metastases from mice with knockdown of FGL1 in MC38 cells or hepatocytes (n = 5 mice per group). K Survival curve analysis of mice in indicated groups (Mean survival times of shNC, shFGL1 #1, shFGL1 #2, AAV-shNC, AAV-shFGL1 #1 and AAV-shFGL1 #2 mice were 27.5, 41.6, 40.8, 24.5, 35 and 35.8 days, respectively) (n = 8 mice per group). IB experiments in B were repeated three times, the data are representative of three biologically independent experiments. The data in (C, G–J) are presented as the mean ± SD (n = 5). The data in K were determined by Kaplan‒Meier analysis with the log-rank test. P values were determined by one-way ANOVA (C, G–J).