Fig 1: Epithelial-derived IL1α drives tumor IL6/CXCL8 production. A,In vitro lung epithelial–osteosarcoma organotypic coculture. Red, mCherry-expressing osteosarcoma cells; green, phalloidin (actin); blue, DAPI. Scale bar, 50 µm. B and C, scRNA-seq analysis of coculture with Seurat-based clustering on Uniform Manifold Approximation and Projection (UMAP) with epithelial cells annotated by expression of KRT19 and osteosarcoma cells annotated by expression of COL1A1. D, NicheNet heatmap and dot plot analysis demonstrating candidate epithelial-derived ligands (rows) and osteosarcoma-derived receptors (columns). The strength of evidence for ligand–receptor interaction is indicated by the shade of blue in the heatmap, whereas the level of expression and percentage of cells expressing ligand or receptor are noted in dot plots. E, Heatmap demonstrating the regulatory potential of epithelial-derived ligands for the top 50 osteosarcoma differentially expressed genes (coculture vs. monoculture). Red arrows, IL6, CXCL3, and CXCL8, which are strongly regulated IL1 ligands. F, Dot plot of osteosarcoma and epithelial (HBEC3-KT) ligands. Note that IL6 upregulation in coculture occurs in a minority of cells. G, Stimulation with IL1α or IL1β significantly increases secretion of IL6 and CXCL8 in human osteosarcoma cells. H, Epithelial-induced [HBEC3-KT conditioned media (CM)] osteosarcoma IL6 and CXCL8 production requires IL1 signaling (IL1Ra, IL1 receptor antagonist anakinra). ANOVA with Dunnett multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Fig 2: IL1 signaling is required for osteosarcoma metastasis progression. A, Functional validation of IL1R1 CRISPR knockout by loss of epithelial-induced IL6 secretion measured by ELISA. n = 4 biological replicates done in triplicate. B, Representative hematoxylin and eosin–stained lungs from mice inoculated with OS-17 electroporated control (NG) or OS-17 IL1R1 CRISPR knockout cell lines (B and C). Scale bar, 2 mm. C, Number of metastatic lesions/slice quantified by a blinded examiner. n = 15 mice/condition. ANOVA with Dunnett multiple comparisons test. D and E, Representative images and quantification of the number of metastatic lesions and percentage of metastasis burden (relative to whole lung area) in mice inoculated via tail vein with OS-17 cells, then treated with vehicle (PBS) or anakinra 1 day after tumor injection. Welch t test. Scale bar, 2 mm. F and G, Representative images and quantification of the number of metastatic lesions and percentage of metastasis burden (relative to whole lung area) in mice inoculated via tail vein with F420 cells then treated with vehicle (PBS) or anakinra 1 day after tumor injection. Scale bar, 2 mm. Welch t test. **, P < 0.01; ****, P < 0.0001.
Supplier Page from BioLegend for Recombinant Human IL-1α (carrier-free)