Fig. 1: CAF-derived factors induce NETosis in vitro and in vivo.

A Confocal microscopy of myeloperoxidase (MPO) and podoplanin (PDPN) expressed on neutrophils and CAFs respectively in pancreatic, skin tumors. B Confocal microscopy of NETs in murine pancreatic, skin tumors showing expression of myeloperoxidase (MPO) and Citrullinated histone H3 (CitH3) by NETting neutrophils and podoplanin (PDPN) by CAFs. C Quantification of the area of the field covered by SYTOX green positive neutrophil-derived extracellular DNA relative to the number of neutrophils in each field after treatment with pancreatic, lung or skin FBs CMed, CAF CMed or PMA for 3 h. D The percentage of dead neutrophils after treatment with pancreatic, lung or skin CAF CMed or PMA based on the number of SYTOX green positive neutrophils. E Confocal microscopy of bone marrow neutrophils stained with MPO, CitH3 and live/dead cell viability dye after induction of NETosis by treatment with CAF CMed for 3 h. Quantification of the relative NET coverage of neutrophils treated with CAF CMed with or without pre-treatment with F N-acetyl-cysteine (NAC) or G anti-granulocyte colony-stimulating factor (α-GCSF). Data are mean ± SEM; *p < 0.05, **p < 0.01 and ***p < 0.001 using (C, F and G) one-way ANOVA with a Tukey post hoc test (with the exception of 1C, untreated vs PMA, which was performed with a paired t-test) and (D) paired t-test. Assays were performed on (A–B) Representative images of n = 6 tumors, (C) n = 7, 4 and 7 (Pancreatic for unstimulated, FB CMed and CAF CMed treated, respectively), n = 11, 3 and 10 (Lung for unstimulated, FB CMed and CAF CMed treated, respectively), n = 4, 4 and 6 (Skin for unstimulated, FB CMed and CAF CMed treated, respectively) and n = 9 (PMA), (D) n = 4, (E) n = 2, F n = 4 (Pancreatic and Skin) n = 5 (Lung) and (G) n = 4 independent experiments. Scale bars are 50 µm.