Fig. 5

Pretreatment of MIF inhibitor or therapeutic targeting of MIF-CD44 axis inhibits metastatic tumor burden in orthotopic and metastatic models. Mice were fed with a CDAHFD for 4 weeks before the establishment of the liver metastasis model. The compound IPG1576 (30 mg/kg/d) was administered twice daily via oral gavage and commenced 7 days intrasplenic injection of 10⁶ KPC cells and continued for a duration of 14 days. An equivalent quantity of solution, 5%DMSO and 95% (20% [2-Hydroxypropyl]-β-cyclodextrin), was used as vehicle for IPG1576 (n = 6–7). a Representative macroscopic appearance of the liver metastases and the liver to body weight ratio in two groups. b HE staining of liver metastases in the MASLD groups with quantification of maximum tumor size and the total area of metastatic foci. c Tumor microenvironment of liver metastasis was examined by mIHC staining of 3-color mIHC staining including CD4, CD8 and GmzB on metastatic liver tissues from vehicle and IPG1576 group, respectively. Representative images including whole slide, merged channels and separate channels were shown. The ratio of CD4⁺ T cells and GmzB⁺CD8⁺ T cells were quantified by Halo software using HighPlex FL v4.2.14 module (n = 5). d, e The infiltrated cell number of CD4 and CD8 per mm² tissue across the tumor border was quantified (n = 5). f Mice were fed with a CDAHFD for 4 weeks followed by the establishment of the liver metastasis model. Five days after intrasplenic injection of KPC cells (5×10⁵), IPG1576 (30 mg/kg/d) was administered twice daily for a duration of 14 days (n = 8). Representative macroscopic appearance of the liver metastases and the liver to body weight ratio were shown. g HE staining of liver metastases in the MASLD groups with quantification of the total area of metastatic foci (n = 8). h Mice were fed with a CDAHFD for 4 weeks followed by the establishment of the liver metastasis model using KPC cells (10⁶) transfected with siCtrl or siCd44 (n = 6–7). Representative macroscopic appearance of the liver metastases in the siCtrl and siCd44 groups were shown and liver weight was measured. i HE staining of liver metastases in the siCtrl and siCd44 groups with quantification of the total area of metastatic foci (n = 6–7). Scale bar was indicated in the individual figures. Representative pictures are shown. Data are shown as mean ± SD per group. Unpaired parametric Student’s t test was performed to identify differences between the two groups. For proximity analyses, two-way ANOVA followed by Šídák’s multiple comparison test was performed to identify differences between two groups at different distances. A p value less than 0.05 was considered statistically significant. *p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; “n.s.” indicates not significant. GmzB, granzyme B