Fig. 6: Gefitinib, a small molecule inhibitor of EGFR inhibits adhesion formation in vivo.

a Schematic illustration of signaling pathways downstream of the epidermal growth factor receptor (EGFR). Gefitinib inhibits the kinase domain of EGFR, Ly294002 inhibits phosphoinositide 3-kinase (PI3K), and Selumetinib inhibits mitogen-activated protein kinase kinase (MEK). b Western blot stained for phospho-EGFR and the respective downstream molecules illustrated in (a). Primary mesothelial cells were isolated and cultured for two passages before they were treated with heparin-binding epidermal growth factor (HB-EGF) and inhibitors for 20 min. c Collagen deposited by primary mesothelial within 3 days of culturing. Data represent n = 3 technical replicates examined over 2 independent experiments. Data are presented as mean ± standard deviation. d–f Primary mesothelial cell cultures were treated with Gefitinib vs. dimethylsulfoxide (DMSO) control and scratch healing was assessed using real-time microscopy (d) and automated image analysis (e, f). Data represent n = 8 technical replicates examined over 2 independent experiments. Data are presented as individual values and boxplots (median, first and third quartile). P-values by t-test (two-tailed) with Holm Bonferroni correction for multiple testing. Scale bar of (d): 1 mm. g Adhesion index of mice 7 days after sterile injury (PB) in combination with bacterial contamination (CLP). Gefitinib 100 mg/kg once daily i.p., Selumetinib 50 mg/kg once daily p.o., Ly294002 25 mg/kg once daily i.p., or DMSO 20% once daily i.p. Data represent n = 20 for DMSO, 10 for Gefitinib, 4 for Selumetinib, and 6 for Ly294002 independent animals examined and pooled over 3 independent experiments. Data are presented as individual values and boxplots (median, first and third quartile). Wilcoxon test (two-sided) with Holm–Bonferroni correction for multiple-testing. DMSO vs. Gefitinib: p = 0.017, DMSO vs. Selumetinib: p = 0.0058, DMSO vs. Ly294002: p = 0.16. h Whole-mount immunohistochemistry of cleared adhesion biopsies 7 days after surgery in Wt1^CreERT2 Rosa26^tdTomato mice. TdTomato and alpha smooth muscle actin (α-SMA) positive cells are indicated by red and green respectively. Scale bar 50 µm. i Cell count of tdTomato+ cells in adhesion biopsies such as represented in (h). Data represent n = 4 independent animals per group (averaged over 2 biopsies per mouse, 2–4 fields of view each) examined over one independent experiment. Data are presented as individual values and boxplots (median, first and third quartile). t-test (two-tailed), p = 0.028. Statistical difference by. *P < 0.05, **P < 0.01, ****P < 0.0001, n.s. P ≥ 0.05. i.p. intraperitoneal p.o. per os. Source data are provided as a Source Data file.