Journal: Cancer Research

Article Title: Metastasis-Initiating Osteosarcoma Subpopulations Establish Paracrine Interactions with Lung and Tumor Cells to Create a Metastatic Niche

doi: 10.1158/0008-5472.CAN-24-3360

Figure Lengend Snippet: Persistent IL6 and CXCL8 production is limited to a small subpopulation of cells. A, Uniform Manifold Approximation and Projection (UMAP) of tumor cells subsetted from coculture demonstrating that IL6 and CXCL8 expression is limited to a single cluster of cells. B and C, IF of osteosarcoma cells stimulated with IL1α (72 hours) and stained for IL6 (green) and DAPI (blue). Scale bar, 50 µm. The percentage of IL6+ cells was quantified in n = 3 experiments, 100 cells/experiment. D, scRNA-seq analysis of OS-17 tibial xenograft tumors, OS-17 experimental lung metastasis, human primary tumors, and human lung metastasis samples demonstrating the heterogeneity of IL1R1 expression. E, Flow cytometry analysis quantifying surface IL1R1 expression in human osteosarcoma cell lines.

Article Snippet: After the blocking incubation, excess blocking IgG was not removed, and cells were stained for IL1R1 (IL1R1 PE-conjugated, R&D Systems, FAB269P) or control antibody (Goat IgG Control PE-conjugated, R&D Systems, IC108P) at 10 μL/10 6 cells for 30 minutes at room temperature in the dark.

Techniques: Expressing, Staining, Flow Cytometry

Journal: Cancer Research

Article Title: Metastasis-Initiating Osteosarcoma Subpopulations Establish Paracrine Interactions with Lung and Tumor Cells to Create a Metastatic Niche

doi: 10.1158/0008-5472.CAN-24-3360

Figure Lengend Snippet: IL1α-induced IL6 and CXCL8 production requires NF-κB signaling in a subpopulation of cells in the G 1 cell-cycle phase. A, OS-17 cells were stimulated with IL1α and then fixed and stained for NF-κB (green) or IL1R1 (red) at the indicated time points. Note the nuclear translocation of NF-κB. Scale bar, 50 µm. B, IKK inhibitors IKK-16 and TPCA-1 abrogate IL1α-induced IL6 and CXCL8 secretion as measured by ELISA. n = 2 biological replicates done in technical triplicates. ANOVA with Dunnett multiple comparisons test. **, P < 0.01; ****, P < 0.0001. C, scRNA-seq analysis of OS-17 tibial xenograft tumors, OS-17 experimental lung metastasis, human primary tumors, and human lung metastasis samples demonstrating restriction of NF-κB activity (see Materials and Methods) in a small subpopulation of G 1 cells.

Article Snippet: After the blocking incubation, excess blocking IgG was not removed, and cells were stained for IL1R1 (IL1R1 PE-conjugated, R&D Systems, FAB269P) or control antibody (Goat IgG Control PE-conjugated, R&D Systems, IC108P) at 10 μL/10 6 cells for 30 minutes at room temperature in the dark.

Techniques: Staining, Translocation Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Cancer Research

Article Title: Metastasis-Initiating Osteosarcoma Subpopulations Establish Paracrine Interactions with Lung and Tumor Cells to Create a Metastatic Niche

doi: 10.1158/0008-5472.CAN-24-3360

Figure Lengend Snippet: 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.

Article Snippet: After the blocking incubation, excess blocking IgG was not removed, and cells were stained for IL1R1 (IL1R1 PE-conjugated, R&D Systems, FAB269P) or control antibody (Goat IgG Control PE-conjugated, R&D Systems, IC108P) at 10 μL/10 6 cells for 30 minutes at room temperature in the dark.

Techniques: Functional Assay, Biomarker Discovery, CRISPR, Knock-Out, Enzyme-linked Immunosorbent Assay, Staining, Control, Injection