Journal: NPJ Precision Oncology

Article Title: DDR1 is identified as an immunotherapy target for microsatellite stable colon cancer by CRISPR screening

doi: 10.1038/s41698-024-00743-2

Figure Lengend Snippet: 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 × 10 6 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).

Article Snippet: For the mouse models receiving combination therapy of the DDR1 inhibitor 7rh and RMP1-14, the treatment group was given 7rh (MCE, # HY-U00444) at a dose of 12.5 mg/kg, administered orally three times with a three-day interval in both the CT26 and MC38 mouse models.

Techniques: Knockdown, Western Blot, Transfection, shRNA, Functional Assay, Fluorescence, Expressing

Journal: NPJ Precision Oncology

Article Title: DDR1 is identified as an immunotherapy target for microsatellite stable colon cancer by CRISPR screening

doi: 10.1038/s41698-024-00743-2

Figure Lengend Snippet: a mRNA and protein expression levels of DDR1 in colon cancer cell lines compared to normal colon epithelial cells (detected by real-time PCR, fold change shown relative to GAPDH. Three independent experiments are shown). b Protein expression level of DDR1 in colorectal carcinoma tissue compared to normal adjacent tissue. c The gene expression profile of DDR1 across all tumor samples and paired normal tissues (bar plot, the highest bar represents the median expression of certain tumor types or normal tissues. The Y-axis represents transcripts per million (TPM) according to the Gene Expression Profiling Interactive Analysis (GEPIA) database ( http://gepia.cancer-pku.cn/ ). d Western blotting confirmed the construction of MSS and MSI CRC cell lines of both human and mouse origin with stable and transient knockdown of DDR1 induced by shRNA and siRNA transfection. Human MSS cell lines: HT29, SW620; human MSI cell lines: HCT116 and DLD1. Mouse MSS cell line: CT26; mouse MSI cell line: MC38. To achieve a better nockdown effect, we chose si 1 and si 2 for CT26 and MC38, si 1, si 2 and si 3 for HCT116 and SW620, sh1 and sh3 for CT26 and MC38, sh1 and sh2 for SW620 and DLD1 and sh1 and sh4 for HCT116 and HT29 for the following experiments. e Cell proliferation was assessed by CCK-8 assay in both human and mouse CRC cell lines with DDR1 knockdown vs. control siRNA (siNC) transfection. f Apoptosis was assessed based on the percentage of apoptotic cells detected by Annexin V/PI double staining by flow cytometry among the human and mouse CRC cell lines (cells with transient knockdown of DDR1 compared to control cells). g Invasion and migration capacity was assessed by Transwell assay in CRC cell lines with transient knockdown of DDR1 or control transfection. Scale bar, 100 μm. ( p < 0.05;** p < 0.01;***, p < 0.001, n.s. , not significant).

Article Snippet: For the mouse models receiving combination therapy of the DDR1 inhibitor 7rh and RMP1-14, the treatment group was given 7rh (MCE, # HY-U00444) at a dose of 12.5 mg/kg, administered orally three times with a three-day interval in both the CT26 and MC38 mouse models.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Knockdown, shRNA, Transfection, CCK-8 Assay, Control, Double Staining, Flow Cytometry, Migration, Transwell Assay

Journal: NPJ Precision Oncology

Article Title: DDR1 is identified as an immunotherapy target for microsatellite stable colon cancer by CRISPR screening

doi: 10.1038/s41698-024-00743-2

Figure Lengend Snippet: a Volcano and Venn plots showing significantly elevated CXCL10 RNA expression by chemokine profile analysis in the shDdr1 mouse MSS CRC cell line CT26 and the human MSS CRC cell lines HT29 and SW620 after stimulation with collagen I and IFNγ (20 ng/ml) compared to control RNA-transfected cells. |Log2fold change | >1.2, | p adj | >0.05). b , c GO and KEGG enrichment analysis of the DEGs (DEGs, differentially expressed genes; GO,gene ontology). Dot plot and bar plot showed DEGs enriched in histone modification in the BP (biological process) category, chromosome region in the CC (cellular component) category and proteoglycans in cancer in shDDR1 CRC cell lines compared to shNC cell lines after collagen I and IFNγ (20 ng/ml) by KEGG and GO analysis. d Chord diagram showing the top 100 ranked upregulated genes by GO analysis in SW620-shDDR1 cells compared to SW620-shNC cells stimulated with collagen I and IFNγ (20 ng/ml). e Western blotting of DDR1 knockdown CRC cell lines showed decreased levels of DDR1 and phosphorylated DDR1 kinase domain (Tyr792) but increased supernatant CXCL10 levels in shDDR1 cell lines stimulated with collagen I and a small dose of IFNγ (20 ng/ml (WB protein levels were normalized to β-actin level). f ELISA verified elevated supernatant CXCL10 levels and increased RNA expression of CXCL10 in the shDDR1 human MSS CRC cell lines HT29 and SW620 compared to the control group stimulated with collagen I and a small dose of IFNγ (20 ng/ml) (ELISA was quantified with the same volume of supernatant of 1 × 10 7 cells). g , h CXCL10 RNA levels were quantified by RT-PCR and normalized to GAPDH levels (detected by real-time PCR, fold change shown relative to GAPDH). Three independent experiments are shown with stimulation by IFNγ (20 ng/ml) and stimulation by both collagen I and IFNγ (20 mg/ml).

Article Snippet: For the mouse models receiving combination therapy of the DDR1 inhibitor 7rh and RMP1-14, the treatment group was given 7rh (MCE, # HY-U00444) at a dose of 12.5 mg/kg, administered orally three times with a three-day interval in both the CT26 and MC38 mouse models.

Techniques: RNA Expression, Control, Transfection, Modification, Western Blot, Knockdown, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: NPJ Precision Oncology

Article Title: DDR1 is identified as an immunotherapy target for microsatellite stable colon cancer by CRISPR screening

doi: 10.1038/s41698-024-00743-2

Figure Lengend Snippet: a EZH2 is highly expressed in human CRC cell lines (relative to NAPDH in both MSS and MSI CRC cell lines) compared to normal epithelial cells (CCD841con). b Downregulation of EZH2 has been observed in shDDR1 both human and mouse MSS CRC cell lines either by stimulation byIFN-γ (20 ng/ml, same as below) or by the combination of IFNγ and collagen I which were quantified and normalized to GAPDH level by qt-PCR. c With stimulation using collagen I and IFNγ, we found decreased levels of EZH2 and H3K27me3 in shDDR1 MSS CRC cell lines compared with the cell with only stimulation of IFNγ. d ChIP‒qPCR results of shDDR1 SW620 and HT29 cell lines showed increasing H3K27me3 binding at the CXCL10 promoter with stimulation by both collagen I and IFNγ. e IFNγR2was not changed in shDDR1 CRC cell lines stimulated by both collagen I and IFNγ. f Protein expression of DDR1, EZH2 and H3K27me3 in the third and fourth round of CT26 mouse models’ tumor specimens. ×400, scale bar 50 μm). g , h Protein expression of DDR1 and collagen I in tumor tissue of CRC patients.The level of DDR1 expression was classified as negative,weak,moderate and strong (×40 and ×200, scale bar 500 μm and 100 μm respectively, same as below). i Expression of DDR1 and disposition of collagen I in dMMR compared to the one in pMMR patients (×200, scale bar 100 μm). j , k Immunohistochemical score of DDR1 and collagen I in 13 dMMR and 13 pMMR CRC patients which indicated both DDR1 and collagen I expression showed higher level than the ones in pMMR group. DDR1 showed no correlation with collagen I in dMMR group but positively correlated with collagen I in pMMR group. l Schematic diagram of collagen I and DDR1 function in CXCL10 transcription in MSS CRC cells.

Article Snippet: For the mouse models receiving combination therapy of the DDR1 inhibitor 7rh and RMP1-14, the treatment group was given 7rh (MCE, # HY-U00444) at a dose of 12.5 mg/kg, administered orally three times with a three-day interval in both the CT26 and MC38 mouse models.

Techniques: Binding Assay, Expressing, Immunohistochemical staining