Figure S2 from SPP1 Drives Colorectal Cancer Liver Metastasis and Immunotherapy Resistance by Stimulating CXCL12 Production in Cancer-Associated Fibroblasts

Figure S2 SPP1 promotes the occurrence of CRC metastasis and immunotherapy resistance. A The effect of overexpression of SPP1 on the migration and invasion of HCT116 cells was assessed using transwell assays, n = 3 biologically independent experiments. Scale bar, 200 μm. B The effect of SPP1 knockdown on the migration and invasion of HCT116-HM cells was assessed using transwell, n = 3 biologically independent experiments. Scale bar, 200 μm. C Wound healing assays were used to evaluate the effect of SPP1 overexpression (left) or knockdown (right) on the migration of HCT116 and HCT116-HM cells, n = 3 biologically independent experiments. D Western blot analysis was used to evaluate the effect of SPP1 overexpression or knockdown on the expression of EMT markers in LoVo and HCT116 cells. E A flowchart illustrating the establishment of an MC38-R string with acquired resistance to anti-PD-1 antibody. F, G Representative tumor gross morphology with tumor volume and tumor weight data (n = 5/group). H H&E analysis in tumor tissues from each group, with expression levels of α-SMA and CD8 evaluated by mIHC analysis. Scale bar, 100 μm. IOD: Integrated Optical Density. I The expression levels of SPP1 in MC38-WT and MC38-R were evaluated using western blot analysis. J qRT-PCR and western blot were used to assess the knockdown efficiency of si-Spp1 in MC38 cells. K C57BL/6 mice were subjected to subcutaneous co-injection of CAFs and MC38-R tumors. Mice were randomized to receive either si-NC or si-SPP1 via intratumoral injection, followed by intraperitoneal administration of anti-PD-1. L-M Representative tumor gross morphology with tumor weight and tumor volume data (n = 5/group). N, O ELISA was performed to assess the expression levels of granzyme B and IFN-γ in tumor tissues from each group. The expression of the control group was taken as 1, and the expression in the remaining groups was expressed as a multiple of the control group. P Western blot analysis of SPP1 protein levels in tumor tissues from each group. Q H&E analysis in tumor tissues from each group. Scale bar, 100 μm. R, S Collagen content assessed by Masson’s trichrome staining, and expression levels of α-SMA and CD8 evaluated by IHC analysis. Scale bar, 100 μm. T TUNEL and Ki-67 staining in MC38 allografts to assess cell apoptosis and proliferation. Scale bar, 100 μm. Western blot experiments in (D, I, J) were repeated three times, the data are representative of three biologically independent experiments. All results are presented as mean ± SEM. P values were determined by two-tailed unpaired Student’s t test (A, C, F-H, L-P, R-T) and one-way ANOVA (B, C, J). *P < 0.05, **P < 0.01, ***P < 0.001.