Fig 2: The correlation of DDR1, CXCL5, and neutrophils infiltration at tissue microarray (TMA) in PDX tumors.(A) IHC staining showed DDR1, CXCL5, and Ly6G+ neutrophils infiltration at PDX tumors and identified using PE Vectra3. Scale bar: 50 µm. (B–D) Pearson’s correlation showed relationship of DDR1, CXCL5, and Ly6G by using H-score, which quantified the DBA signals by inForm software. n = 82.

Fig 3: The correlation of DDR1, CXCL5, and NET-like structure in samples of patient with PADC.(A) Upper and middle panel: IHC staining showed DDR1, CXCL5 expression in PDAC patient samples, and identified using PE Vectra3. Scale bar: 3 mm and 50 µm. Bottom panel: NET-like structures were analyzed by immunofluorescence staining using DAPI (blue), anti-CK19 (white), anti-MPO (green), and anti–citrullinated histone H3 (red) mAbs in samples of patient with PDAC. Scale bar: 20 µm. (B) Pearson’s correlation showed relationship of DDR1 and CXCL5 by using H-score, which quantified the DBA signals by inForm software, P < 0.0001.

Fig 4: CXCL5 involved in DDR1-mediated NET formation and cancer cell invasion.(A–F) Human neutrophils were cocultured with DDR1 knockdown or reexpression of MDA-PATC 148 or BxPC-3 by Matrigel transwell chamber, with or without anti-CXCL5 neutralized antibody or recombinant CXCL5 treatment, for 18 hours. (A) NET structures were analyzed by immunofluorescence staining using DAPI (blue), anti-NE (red), and anti–histone H3 (green) mAbs. Scale bar: 50 µm. (B and C) The NET quantification is displayed as NET histone area (µm2) per field, 6 fields per group. (D) Cit-histone H3 expression were analyzed by western blotting. (E and F) The number of invaded cells were analyzed by immunofluorescence staining using DAPI and calculated based on the number of cells found in 6 fields per chamber. All the data are mean ± SD. n = 5, 3 independent experiments. (B and E) P values were analyzed by 1-way ANOVA with Sidak post hoc testing. ***P < 0.001. (C and F) P values were analyzed by unpaired 2-tailed Student’s t test. *P < 0.05; ***P < 0.001.

Fig 5: 7rh treatment reduced NET formation through inhibition of the DDR1/PKC?/SYK/CXCL5 axis and reduced cancer metastasis.(A and B) MDA-PATC 148 cells were pretreated with 7rh for 30 minutes and then with collagen I for 3 hours. (A) Phospho-NF-?B P65, phospho-PKC?, and phospho-SYK were analyzed by western blotting. (B) CXCL5 levels were analyzed by ELISA. Data are mean ± SD. n = 4, 3 independent experiments; 1-way ANOVA with Sidak post hoc testing. *P < 0.05; ***P < 0.001. (C–E) Human neutrophils were cocultured with MDA-PATC 148 and BxPC-3 cells by Matrigel transwell chamber for 18 hours. (C) NET structures were analyzed by immunofluorescence staining using DAPI (blue), anti-NE (red), and anti–histone H3 (green) mAbs. Scale bar: 50 µm. (D) The NET quantification is displayed as NET histone area (µm2) per field, 6 fields per group. Data are mean ± SD. n = 6, 3 independent experiments; 1-way ANOVA with Sidak post hoc testing. ***P < 0.001. (E) Cit-histone H3 expression were analyzed by western blotting. (F and G) Mice were orthotopically injected with MDA-PATC 148 cells, with or without 3 mg/kg 7rh treatment for 9 weeks. (F) Liver metastasis was detected by immunofluorescence staining using DAPI (blue) and anti-CK19 (red) mAbs in liver section. Scale bar: 50 µm. The metastasis quantification is displayed as CK-19 positive signals/per x20 field, 6 fields per group. (G) Neutrophils infiltration was detected by immunofluorescence staining using DAPI (blue), anti-CK19 (red), and anti-Ly6G (green) mAbs in pancreas section. Scale bar: 50 µm. The Neutrophils infiltration quantification is displayed as Ly6G positive signals per x20 field, 6 fields per group. Data are mean ± SD. n = 5, unpaired 2-tailed Student’s t test. ***P < 0.001.