Fig. 4: Caffeine activates CD8⁺ T cells by KLF4/COL12A1 axis in CRC cells.

a Western blot and b qPCR analysis of COL12A1 expression in CRC cells treated with caffeine (0, 1, 10 mM) (Representative data from n = 3 independent experiments). The samples derive from the same experiment but different gels for COL12A1, GAPDH were processed in parallel. The quantification provided under the blots is for the representative blot from n = 3 independent experiments. c Western blot and d qPCR analysis COL12A1 expression in COL12A1-overexpressed (COL12A1-OE) and control cells treated with caffeine (1 mM) (Representative data from n = 3 independent experiments). The samples derive from the same experiment but different gels for COL12A1, GAPDH were processed in parallel. The quantification provided under the blots is for the representative blot from n = 3 independent experiments. e Schematic depicting the treatment of caffeine in subcutaneous COL12A1-OE MC38 tumor-bearing C57BL/6 mice. Representative images of tumor from caffeine-treated or COL12A1-OE groups (n = 5 per group). Scale bars, 1 cm. Quantification of the tumor growth curve and tumor weight. f Flow cytometry analysis of CD8⁺ T cells and GZMB, PD-1, LAG3 expression (n = 4 per group) or g Quantification of immunohistochemistry staining of CD8⁺ T cells and COL12A1 (n = 5) in tumor of subcutaneous MC38 with COL12A1-OE or control (COL12A1-NC) tumor-bearing C57BL/6 mice treated with caffeine or control. h Representative images of tumors from the treatment of anti-CD8 monoclonal antibody (CD8 mAb) (200 μg, ip. twice weekly) in subcutaneous MC38 with COL12A1 knockdown (sh-COL12A1) or control (sh-NC) tumor-bearing C57BL/6 mice (n = 5 per group). Scale bars, 1 cm. Quantification of the tumor growth curve and tumor weight. i Flow cytometry analysis (n = 4 per group) and j Quantification of immunohistochemistry staining (n = 5) of CD8⁺ T-cell infiltration in subcutaneous MC38 with sh-COL12A1 tumor-bearing C57BL/6 mice treated with CD8 mAb. k Schematic of co-culture experiments with splenocytes and COL12A1-OE MC38 cells treated with caffeine (1 mM). Flow cytometry analysis the GZMB, PD-1 and Ki67 expression in CD8⁺ T cells of splenocytes (n = 4 per group). Data error bars are mean ± SD. The statistical analysis of the growth curve is Two-way ANOVA, the others are Student’s two-tailed unpaired t-test. The images of mice and co-culture elements were drawn by Figdraw. Source data is provided as a Source Data file.