Figure 7: IL-33-recruited macrophages mediate cancer metastasis in a pancreatic tumour model.

(a) ELISA detection of IL-33 protein expression levels in various xenograft tumour tissues and in cultured tumour cell lines (n=6 samples per group). PDGF-BB-stimulated and non-stimulated pericytes served as positive controls. (b) Immunostaining of PDGFRβ⁺ signals and Iba1⁺ macrophages in T241, LLC and Panc02 tumours. Upper two panels were counter-stained with H&E, or PDGFRβ and haematoxylin. Lower middle panels were double stained with DAPI (blue). Black and white arrowheads point to PDGFRβ⁺ signals. Red arrowheads indicate Iba1⁺ macrophages. Quantification of PDGFRβ⁺ signals and Iba1⁺ macrophages (n=8 random fields per group). NS, not significant. (c) Immunoblotting detection of total and phosphorylated PDGFRβ in Panc02 tumour tissues. Vector- and PDGF-BB-T241 or -LLC tumour tissues were used as controls. (d) Pulmonary and hepatic metastasis in wt T241-, LLC- and Panc02-tumour-bearing mice. Metastases were detected by gross examination of lung surface and liver surface, and histological staining with H&E. Arrowheads indicate lung and liver surface metastases in Panc02 tumour-bearing mice. Dashed lines encircle the borders between tumour nodules and surrounding tissues. T, tumour. Quantification of lung and liver surface metastases in tumour-bearing mice (n=8–10 mice per group). (e) Detection of Iba1⁺ macrophages (red) and tumour cells (green) in clodronate- and vehicle-treated Panc02-GFP⁺ tumours implanted in wt mice. Iba1⁺ macrophages (red) were also detected in Panc02-GFP⁺ tumours implanted in Il33^−/− mice. Wild type of mice serves as a control. Arrowheads indicate Iba1⁺ macrophages. Quantification of Iba1⁺ macrophages (n=8 random fields per group). (f) Pulmonary and hepatic metastasis in clodronate- and vehicle-treated Panc02-GFP⁺ tumour-bearing mice, and wt or Il33^−/− mice with Panc02-GFP⁺ implantation. Metastases were detected by gross examination of lung surface and liver surface, and histological staining with H&E. Arrowheads indicate lung and liver surface metastases in tumour-bearing mice. Dashed lines encircle the borders between tumour nodules and surrounding tissues. T, tumour. Quantification of lung and liver surface metastases in tumour-bearing mice (n=8–10 mice per group; mean±s.e.m., NS, not significant, Student’s t-test). Full-gel images for c are shown in Supplementary Fig. 9.