FIGURE 5.

The TDO2‐AHR pathway is required for the transactivation of PD‐L1 and TDO2 and for the maintenance of stemness in colon cancer spheroids. A, B, qPCR analyses of PD‐L1, TDO2, and IL‐1b expression in cancer spheroids (CRC‐29 M) treated with the indicated concentrations of an inhibitor of TDO2 (A) or AHR (B) for 96 h. C, Schematic representation of predicted AHR‐binding sites in the promoters of TDO2. D, ChIP analyses of the AHR‐bound promotors of the TDO2 gene in spheroid cells (CRC‐29 M) treated with the TDO2 inhibitor (100 μmol/L) or AHR inhibitor (50 μmol/L) for 96 h. E, Relative proliferation of spheroid cells (CRC‐29 M) measured by CellTiter‐Glo assays (n = 4). The spheroids were treated with the indicated concentrations of the TDO2 or AHR inhibitor for 7 d. F, Limiting dilution assay with FACS‐sorted spheroid cells. An average frequency of self‐renewing cells (upper lanes) and confidence interval (lower lanes) are shown. G, H, Western blot analyses of CD44 protein expression in the indicated spheroid cells treated with the inhibitor of TDO2 (G) or AHR (H) for 96 h. I, Western blot analyses in spheroid cells (CRC‐29 M) introduced with doxycycline‐inducible Cas9 and the indicated sg‐RNA in the presence or absence of 1 μmol/L doxycycline for 5 d. J, qPCR analyses of the indicated genes in the spheroids shown in (I). K, Relative proliferation of spheroid cells shown in (I). Values represent the mean ± SD; *P < .05, **P < .01, ***P < .001