Fig. 5: Caffeine downregulates COL12A1 to reduce kynurenine.
From: Caffeine enhances antitumor T-cell activity by suppressing kynurenine pathway in colorectal cancer
a Principal component analysis, b Enrichment analysis of metabolites, c Quantification of tryptophan related metabolite levels (n = 6), d Box plots of tryptophan changes from supernatant of control (A, n = 6, Min=23.3397, Max = 23.7274, Median = 23.5581, Lower Whisker = 23.3997, Upper Whisker= 23.7274, Q1 = 23.4511, Q3 = 23.6636, IQR = 0.2125) and caffeine-treated (B, n = 6, Min = 23.5735, Max = 24.0682, Median = 23.9736, Lower Whisker=23.5735, Upper Whisker=24.0682, Q1 = 23.9351, Q3 = 24.0298, IQR = 0.1026) HCT116 cells, e Heatmap of differential metabolite. f ELISA analysis kynurenine (Kyn) levels in CRC cells treated with caffeine (0, 0.1, 1, 10 mM) (Representative data from n = 3 independent experiments). g ELISA analysis Kyn levels in COL12A1-overexpressed (COL12A1-OE) CRC cells treated with caffeine (1 mM) (Representative data from n = 3 independent experiments). h Schematic depicting the treatment of caffeine or Kyn (100 mg/kg, ip) or Indoximod (100 mg/kg, ip) in subcutaneous CT26 tumor-bearing BALB/c mice. Representative images of tumors (n = 6 per group). Scale bars, 1 cm. Quantification of the tumor growth curve and weight. i Flow cytometry analysis of CD8+ T cells and GZMB, PD-1, LAG3 expression (n = 4 per group) or j Quantification of immunohistochemistry staining of CD8+ T cells and COL12A1 (n = 6) in tumor of subcutaneous CT26 tumor-bearing BALB/c mice treated with caffeine or Kyn or Indoximod or control. k Schematic depicting the treatment of caffeine or Indoximod in subcutaneous COL12A1-OE CT26 tumor-bearing BALB/c mice. Representative images of tumors (n = 5 per group). Scale bars, 1 cm. Quantification of the tumor growth curve and weight. l Flow cytometry analysis of CD8+ T cells and GZMB, PD-1, LAG3 expression (n = 4 per group) or m Quantification of immunohistochemistry staining of CD8+ T cells and COL12A1 (n = 5) in tumor of subcutaneous COL12A1-OE CT26 tumor-bearing BALB/c mice treated with caffeine or Indoximod. n Schematic of co-culture experiments with splenocytes and COL12A1-OE CT26 cells treated with caffeine (1 mM), Kyn (100 μM) or Indoximod (50 μM). Flow cytometry analysis of the GZMB, PD-1, Ki67 expression in CD8+ T cells of splenocytes (n = 4 technical replicates, Representative data from n = 3 independent experiments). Data error bars are mean ± SD. The statistical analysis of the tumor 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.
