Fig 1: 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 2: PDGF-BB-PDGFRß-signalling induces IL-33 expression.(a) Heatmap of top 10 most upregulated and downregulated genes by genome-wide expression profiling of PDGF-BB-stimulated lung pericytes cultured in vitro. (b) Heatmap of top 10 most upregulated and downregulated inflammation-related signalling molecules by genome-wide expression profiling of PDGF-BB-stimulated lung pericytes cultured in vitro. (c) qPCR quantification of Il33 mRNA expression levels in PDGF-AA- or PDGF-BB-stimulated lung pericytes cultured in vitro. (PC; n=6 samples per group). NS, not significant. (d) qPCR quantification of Il33 mRNA expression levels in PDGF-BB-stimulated bone marrow stromal fibroblasts cultured in vitro (SF; n=6 samples per group). (e) Quantification of mouse IL-33 protein levels of vector- and PDGF-BB-T241 tumours, and scrambled and Pdgfb shRNA-transfected A431 tumours (n=6 samples per group). (f) qPCR quantification of Il33 mRNA of Adv-Gfp- and Adv-Pdgfb-infected liver tissues (n=6 samples per group). Adv-Gfp-infected hepatocytes were visualized by a fluorescent microscope. (g) qPCR quantification of Il33 mRNA expression levels in CD31+, PDGFRß+ and NG2+ cell populations isolated from vector and PDGF-BB T241 tumours (n=6 samples per group). Vector and PDGF-BB T241 tumour cells served as controls. (h) qPCR analysis of Il33 mRNA of vehicle-, anti-PDGFRa-, anti-PDGFRß- or imatinib-treated PDGF-BB-stimulated or non-stimulated lung pericytes cultured in vitro. (PC; n=6 samples per group; mean±s.e.m., NS, not significant, Student's t-test).
Fig 3: IL-33 mediates PDGF-BB-stimulated cancer metastasis through a TAM-dependent mechanism.(a) Clodronate effectively inhibited Iba1+ macrophage infiltration (green) in IL-33-T241 tumours. Tumour cells were labelled with RFP (red). Arrowheads indicate TAMs. Quantification of Iba1+ macrophage in clodronate-treated and non-treated vector- and IL-33-T241 tumours (n=8 random fields per group). (b) Lung metastasis in clodronate-treated and non-treated vector- and IL-33-T241 tumour-bearing mice. Arrowheads indicate lung surface metastatic nodules. Dashed line marks the border between the RFP+ metastatic nodule and surrounding lung tissues. T, tumour. Quantification of percentage of animals with pulmonary metastasis on the surface of lungs (n=10–16 mice per group). (c) Detection of Iba1+ macrophages (red) and tumour cells (green) in vehicle- and soluble ST2-treated PDGF-BB-T241 tumours implanted in wt mice. Iba1+ macrophages (red) were also detected in PDGF-BB-T241 tumours implanted in St2-/- mice. Vector-T241 tumour serves as a control. The detection of Iba1+ macrophages (red) and tumour cells (green) in PDGF-BB-LLC tumours implanted in wt and Il33-/- mice. Vector-LLC tumour serves as a control. Arrowheads indicate Iba1+ macrophages. Quantification of Iba1+ macrophages (n=8 random fields per group). (d) Pulmonary metastasis in vehicle- and soluble ST2-treated PDGF-BB-T241 tumour-bearing mice. Pulmonary metastasis in PDGF-BB-LLC tumour-bearing wt and Il33-/- mice. Metastases were detected by gross examination of lung surface, fluorescent detection for GFP+ signals 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 surface metastases in tumour-bearing mice (n=8–10 mice per group; mean±s.e.m., NS, not significant, Student's t-test).
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).
Fig 5: 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 PDGFRß+ signals and Iba1+ macrophages in T241, LLC and Panc02 tumours. Upper two panels were counter-stained with H&E, or PDGFRß and haematoxylin. Lower middle panels were double stained with DAPI (blue). Black and white arrowheads point to PDGFRß+ signals. Red arrowheads indicate Iba1+ macrophages. Quantification of PDGFRß+ signals and Iba1+ macrophages (n=8 random fields per group). NS, not significant. (c) Immunoblotting detection of total and phosphorylated PDGFRß 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.
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