Fig 2: IL-33 stimulates Raw cell migration through activation of the ST2-intracellular signalling pathways.(a) Left panels: FACS analysis of F4/80+ST2+ mouse Raw macrophage cell line (n=6 samples per group). Non-immune IgG served as a negative control. Right panel: St2 mRNA expression in macrophages and stromal fibroblasts. (b) Dose-dependent stimulation of Raw cell migration by IL-33 (n=6 samples per group). Vehicle-treated macrophages served as controls. (c) Inhibition of IL-33-induced Raw cell migration by a siRNA specifically targeting St2 (n=6 samples per group). Scrambled siRNA serves as a control. (d) Inhibition of IL-33-induced morphological changes of Raw cells by a siRNA specifically targeting St2. Scrambled siRNA serves as a control. Arrowheads indicate filopodia sprouts of the IL-33-activated macrophages. Scale bar, 50 µm. (e) Cell morphologies of Raw cells co-cultured 48 h with PDGF-BB- or buffer-stimulated lung pericytes. F4/80 were shown in green (yellow overlapped with phalloidin red). Soluble ST2 was added to block IL-33 function (200 ng ml-1). PC, pericyte. Scale bar, 50 µm. Arrowheads indicate filopodia sprouts of the IL-33-activated macrophages. Recombinant IL-33-stimulated mouse Raw cells served as a positive control. (f) Migration of Raw cells co-cultured with PDGF-BB- or buffer-stimulated lung pericytes in the presence of a soluble ST2 or vehicle (n=6 samples per group). (g) IL-33 induces phosphorylation of MAPK and p38 at 10 min, and I?Ba at 5 min in mouse Raw cells. Beta-actin indicates the loading level in each lane. (h) SiRNA specifically targeting St2 inhibited IL-33-induced phosphorylation of MAPK, p38 and I?Ba in Raw cells. Beta-actin indicates the loading level in each lane. (i) Inhibition of IL-33-induced mouse Raw cell migration by MAPK, p38 and I?Ba specific inhibitors (n=6 samples per group). Vehicle-treated cells served as controls. (j) Inhibition of IL-33-induced Raw cell shape changes by MAPK, p38 and I?Ba specific inhibitors (n=6 samples per group). Vehicle-treated cells served as controls. Arrowheads indicate filopodia sprouts of the IL-33-activated Raw cells. Scale bar, 50 µm (mean±s.e.m., Student's t-test). Full-gel images for g,h are shown in Supplementary Fig. 9.

Fig 3: Genome-wide profiling of wt and St2-/- TAMs and survival correlation of IL-33 expression in grafted tumours.(a) Heatmap of top 10 metastasis-related genes by genome-wide expression profiling of F4/80+ cells isolated from Panc02 tumours implanted in wt and St2-/- mice (n=3 samples per group). (b) Heatmap of top 10 up- and top 10 down-most cytokines, chemokines and their receptor genes by genome-wide expression profiling of F4/80+ cells isolated from Panc02 tumours implanted in wt and St2-/- mice (n=3 samples per group). (c) Schematic diagram shows in pericytes and stromal fibroblasts the PDGF-BB-PDGFRß-IL-33-ST2 axis-recruited macrophages in switching a noninvasive tumour to a highly invasive tumour.

Fig 4: IL-33 induces infiltration of M2-like TAMs and metastasis.(a) IL-33 expression levels in vector- and IL-33-T241 tumour xenografts (n=6 samples per group). (b) FACS analysis of the total F4/80+ macrophages in vector- and IL-33-T241 tumour tissues (n=5 samples per group). (c) Heatmap of M1 and M2 related genes by genome-wide expression profiling of F4/80+ cells isolated from Panc02 tumour grafts implanted in wt and St2-/- mice (n=3 samples per group). (d) In vitro matrigel invasion of GFP+ LLC tumours in the presence of IL-33 or vehicle-stimulated macrophages (n=6 samples per group). Arrowheads point to spread GFP+ tumour cells. Scale bar, 100 µm. (e) FACS analysis and quantification of RFP+ circulating tumour cells in the peripheral blood of vector- and IL-33-T241 tumour-bearing mice (n=5 samples per group) at the time point of the average tumour size of 1.5 cm3. (f) Bioluminescent imaging of tumour-bearing mice implanted in livers with luciferase+ vector- and IL-33-T241 tumours. Arrowheads point to luciferase+ tumours. Quantifications of bioluminescence signals and liver weights (n=5 samples per group). NS, not significant. (g) Bioluminescent imaging of lungs of luciferase+ vector- and IL-33-T241 tumour-bearing mice. Arrowheads point to luciferase+ metastatic nodules. Quantifications of luciferase+ pulmonary metastases (n=8 animals per group; mean±s.e.m., NS, not significant, Student's t-test).