Figure S5 from SPP1 Drives Colorectal Cancer Liver Metastasis and Immunotherapy Resistance by Stimulating CXCL12 Production in Cancer-Associated Fibroblasts

Figure S5 SPP1-CD44 interaction activates β-catenin/HIF-1α signaling in CAFs. A GSEA analysis of RNA-seq data from CAFs derived from primary tumors (PT-CAFs) and liver metastases (LM-CAFs) of CRC patients. B qRT-PCR and western blot assessed the knockdown efficiency of si-CTNNB1 in CAFs. C qRT-PCR was used to evaluate the mRNA expression of HIF1A in CAFs with CTNNB1 knockdown (left) or MSAB treatment (right), n = 3 biologically independent experiments. D Western blot analysis was performed to assess the protein expression of HIF-1α in CAFs with CTNNB1 knockdown or MSAB treatment. E qRT-PCR and western blot assessed the knockdown efficiency of si-HIF1A in CAFs. F qRT-PCR was used to measure CD44 mRNA levels in T cells and different kind of CAFs. NAF: normal-associated fibroblasts; PT: primary tumor; LM: liver metastasis, n = 3 biologically independent experiments. G qRT-PCR and western blot assessed the knockdown efficiency of si-CD44 in CAFs. H, I Western blot analysis was conducted to evaluate HIF-1α and β-catenin protein expression in control (si-NC) and CD44 knockdown CAFs, with or without conditional medium from HCT116-HM (H) or LOVO-HM (I). J CAF cells were transfected with si-NC or si-CD44 and subsequently treated with conditioned medium from SPP1-overexpressing HCT116 or LoVo cells. The expression levels of β-catenin and HIF-1α were then assessed by western blot. K Western blot analysis was conducted to evaluate HIF-1α and β-catenin protein expression in control (si-NC) and CD44 knockdown CAFs, with or without rhSPP1 protein (1 µg/mL) treatment. L Nuclear-cytoplasmic fractionation was performed to assess the nuclear translocation of HIF-1α in control (si-NC) and CD44 knockdown CAFs, with or without treatment with rhSPP1 protein (1 µg/mL). Western blot experiments in (A, C, F-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 (C) and one-way ANOVA (B, E-G). ns, no significant difference, **P < 0.01, ***P < 0.001.