Figure S4 from SPP1 Drives Colorectal Cancer Liver Metastasis and Immunotherapy Resistance by Stimulating CXCL12 Production in Cancer-Associated Fibroblasts

Figure S4 CAF enhances SPP1-induced suppression of T-cell cytotoxicity. A Schematic representation of the tumor cell and T cell coculture system with or without CAFs. B, C After 24 hours of coculture with or without CAFs, propidium iodide staining was used to identify LoVo (B) or HCT116 (C) cells killed by T cells. Fluorescence microscopy assessed the effects of SPP1 overexpression on T-cell cytotoxicity, n = 3 biologically independent experiments. Scale bar, 100 μm. D Schematic representation of the tumor cell and T cell coculture system, with or without CAFs and SPP1 protein (1 µg/mL) treatment. E, F After 24 hours of coculture with or without CAFs, propidium iodide staining was used to identify LoVo (E) or HCT116 (F) cells killed by T cells. Fluorescence microscopy assessed the effects of SPP1 protein (1 µg/mL) treatment on T-cell cytotoxicity, n = 3 biologically independent experiments. Scale bar, 100 μm. G Schematic representation of the tumor cell and T cell coculture system, with or without CXCL12 protein (100 ng/mL) or CXCL12-neutralizing antibody (100 ng/mL) treatment. H, I After 24 hours of coculture, propidium iodide labeling identified LoVo and HCT116 cells killed by T cells. Fluorescence microscopy assessed the effects of CXCL12 protein (100 ng/mL) or CXCL12-neutralizing antibody (100 ng/mL) treatment on T-cell cytotoxicity, n = 3 biologically independent experiments. Scale bar, 100 μm. All results are presented as mean ± SEM. P values were determined by one-way ANOVA. ns, no significant difference, *P < 0.05, ***P < 0.001. PI, propidium iodide.