Fig. 3: Knockdown of DDR1 or treatment with its kinase inhibitor 7rh sensitizes CRC tumors to ICIs and increases intratumoral functional CD8⁺ T cell infiltration.

a The knockdown effect of DDR1 was confirmed by western blotting, and sh1 and sh3 were chosen for the following animal experiments in CT26 and MC38 models, respectively. b Flow chart of the animal experiments for CT26 and MC38. RMP1-14 (mouse PD-1 blockade, 10 mg/kg) was given 7 days after subcutaneous inoculation of each group with tumor cells (1 × 10⁶ cells for each mouse, right flank), and 4 doses were given in total. Mice were sacrificed 3 days after the last dose of RMP1-14, and tumors were harvested and dissociated into single cells for FACS. c Effect of Ddr1 knockdown and the combination of PD-1 blockade on CT26- and MC38-bearing mice shown by the tumor volume curve of each group. Mice bearing CT26 and MC38 tumors were transfected with DDR1 shRNA, treated with PD-1 blockade and their combination. The image on the right side shows the sizes of the tumors in each group (n = 5 in each group).Data are the mean ± SEM. Information on sample sizes, experimental number, times, biological replicates, statistical tests, and p values is available in Methods. d Flow cytometric determination of the percentage of each type of immune cell infiltrated in the tumor with the gating strategy in (sup Fig. 3B) in the Ddr1 knockdown CT26 animal experiment, which showed increased infiltration of CD8⁺ T cells, especially GZMB+ and IFNγ + CD8 + T cells, in the shDdr1+PD-1 blockade group compared to the other groups. Error bars show the mean ± SEM ; p values were calculated by one-way ANOVA. e Treatment schedule, tumor growth curve and representative tumor images for treating CT26 and MC38 tumors with DDR1 inhibitor 7rh and PD-1 blockade (n = 5 tumors per group. Error bars show the mean ± SEM. p values were determined by two-way ANOVA). f Flow cytometric determination of the percentage of each type of immune cell infiltrated in the tumor in the 7rh and PD-1 blockade combination in CT26 mouse models. The results showed increased CD8⁺ Tcell infiltration, especially functional infiltration, such as GZMB⁺CD8⁺ T cells, in the 7rh + PD-1 blockade group and shDdr1+7rh+PD-1 blockade group. n = 5 tumors per group. Error bars show the mean ± SEM. ; p values were calculated by one-way ANOVA. g Representative fluorescence confocal images of Ddr1 (red), collagen I (yellow) and CD8 (green) expression in the TME showing increased CD8⁺ T cell infiltration in areas with low Ddr1 and collagen I expression. Scale bar, 50 μm. h Representative fluorescence confocal images of dMMR CRC patient samples. Intratumoral CD8⁺ T cells were more enriched in the tumor area with low expression of DDR1 and collagen I than in that with high expression of these factors (scale bar: 50 μm).