371 il6  (Cell Signaling Technology Inc)

Journal: Stem Cell Research & Therapy

Article Title: The treatment of acute tendon injury with small extracellular vesicles originating from TNFAIP6 − ADSCs subpopoulation both in vitro and in vivo

doi: 10.1186/s13287-025-04789-2

Figure Lengend Snippet: Let-7c-5p inhibits inflammatory expression in tendon injury. A The expression of CCR7 + , CD163 + , IL-6 + , and IL-10 + cells were detected by immunofluorescence at week 1. B Positive ratio of inflammation-related factors ( n = 6). Bars 50 μm. Data are represented as mean ± SD. *vs Control group; # vs. ADSC-sEVs; n = 6. * P < 0.05, ** P < 0.01, # P < 0.05, ## P < 0.01, ### P < 0.001

Article Snippet: For immunofluorescence analysis, tissue sections were incubated with primary antibodies: anti-CCR7 (ab32527; Abcam), anti-CD163 (ab182422; Abcam), anti-interleukin (IL)-6 (TA500067S; Origene), and anti-IL-10 ( ab33471; Abcam), followed by incubation with secondary antibodies (SA00013; ProteinTech, Chicago, IL, USA).

Techniques: Expressing, Immunofluorescence, Control

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: Metastatic osteosarcoma cells promote osteosarcoma chemotaxis via the IL6/CXCL8 mechanism. A, Schematic for testing migration to IL6 and CXCL8 in a Transwell setup and quantification of migrated cells relative to media control (down arrow). B, Schematic for testing the role of IL6 and CXCL8 signaling in osteosarcoma-induced migration and quantification relative to vehicle-treated osteosarcoma (OS)–conditioned media (CM; arrow). ANOVA with Dunnett multiple comparisons test. n = 4. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Primary antibodies used are as follows: rabbit anti-CXCL8 (Abcam, ab289967, IF), mouse anti-human vimentin (Abnova, SRL33, IF, AR = TE), rabbit anti-p21 [Cell Signaling Technology, 2947, IF, AR = CB], rabbit anti–NF-κB (Cell Signaling Technology, 8242, IF), hamster anti-podoplanin (Developmental Studies Hybridoma Bank, 8.1.1, AR = TE), rat anti–Ki-67 (Invitrogen, 14-5698-82, IF, AR = CB), mouse anti-human IL6 (R&D Systems, MAB2061, IF), mouse anti-human IL1α membrane form 488 (R&D Systems, FAB2001G, IF), goat anti-mouse IL1α (R&D Systems, AF-400-NA, IF, AR = TE).

Techniques: Chemotaxis Assay, Migration, Control

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: 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.

Article Snippet: Primary antibodies used are as follows: rabbit anti-CXCL8 (Abcam, ab289967, IF), mouse anti-human vimentin (Abnova, SRL33, IF, AR = TE), rabbit anti-p21 [Cell Signaling Technology, 2947, IF, AR = CB], rabbit anti–NF-κB (Cell Signaling Technology, 8242, IF), hamster anti-podoplanin (Developmental Studies Hybridoma Bank, 8.1.1, AR = TE), rat anti–Ki-67 (Invitrogen, 14-5698-82, IF, AR = CB), mouse anti-human IL6 (R&D Systems, MAB2061, IF), mouse anti-human IL1α membrane form 488 (R&D Systems, FAB2001G, IF), goat anti-mouse IL1α (R&D Systems, AF-400-NA, IF, AR = TE).

Techniques: Derivative Assay, In Vitro, Expressing

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: Primary antibodies used are as follows: rabbit anti-CXCL8 (Abcam, ab289967, IF), mouse anti-human vimentin (Abnova, SRL33, IF, AR = TE), rabbit anti-p21 [Cell Signaling Technology, 2947, IF, AR = CB], rabbit anti–NF-κB (Cell Signaling Technology, 8242, IF), hamster anti-podoplanin (Developmental Studies Hybridoma Bank, 8.1.1, AR = TE), rat anti–Ki-67 (Invitrogen, 14-5698-82, IF, AR = CB), mouse anti-human IL6 (R&D Systems, MAB2061, IF), mouse anti-human IL1α membrane form 488 (R&D Systems, FAB2001G, IF), goat anti-mouse IL1α (R&D Systems, AF-400-NA, IF, AR = TE).

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: Primary antibodies used are as follows: rabbit anti-CXCL8 (Abcam, ab289967, IF), mouse anti-human vimentin (Abnova, SRL33, IF, AR = TE), rabbit anti-p21 [Cell Signaling Technology, 2947, IF, AR = CB], rabbit anti–NF-κB (Cell Signaling Technology, 8242, IF), hamster anti-podoplanin (Developmental Studies Hybridoma Bank, 8.1.1, AR = TE), rat anti–Ki-67 (Invitrogen, 14-5698-82, IF, AR = CB), mouse anti-human IL6 (R&D Systems, MAB2061, IF), mouse anti-human IL1α membrane form 488 (R&D Systems, FAB2001G, IF), goat anti-mouse IL1α (R&D Systems, AF-400-NA, IF, AR = TE).

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: Sustained IL6 production is maintained by a small subset of hypoproliferative cells that anchor osteosarcoma cells to the lung niche. A, IF expression of IL6 (red) and CXCL8 (green) over time (8–72 hours) following stimulation with IL1α. White, proliferating cells (Ki-67); blue, DAPI. Scale bar, 50 µm. B and C, ELISA and IF quantitation demonstrating that IL6 and CXCL8 production remains constant over time, whereas IL6 and CXCL8+ expression by IF becomes progressively restricted to Ki-67− cells. ELISA, two biological replicates done in technical triplicate. Percentage of IL6, CXCL8, and Ki-67 quantified in n = 3 experiments, 100 cells/experiment. ANOVA with Dunnett multiple comparisons test. ***, P < 0.001; ****, P < 0.0001. D, IL6, CXCL8, and Ki-67 expression in NCH-OS-4 cells following stimulation with IL1α (72 hours). Scale bar, 50 µm. E, F420 murine cells stimulated with murine IL1α (72 hours) and stained with Ki-67 (red), IL6 (green), and DAPI (blue). Scale bar, 50 µm. F, Quantitation of IL6 and Ki-67 staining in NCH-OS-4 and F420 cells.

Article Snippet: Primary antibodies used are as follows: rabbit anti-CXCL8 (Abcam, ab289967, IF), mouse anti-human vimentin (Abnova, SRL33, IF, AR = TE), rabbit anti-p21 [Cell Signaling Technology, 2947, IF, AR = CB], rabbit anti–NF-κB (Cell Signaling Technology, 8242, IF), hamster anti-podoplanin (Developmental Studies Hybridoma Bank, 8.1.1, AR = TE), rat anti–Ki-67 (Invitrogen, 14-5698-82, IF, AR = CB), mouse anti-human IL6 (R&D Systems, MAB2061, IF), mouse anti-human IL1α membrane form 488 (R&D Systems, FAB2001G, IF), goat anti-mouse IL1α (R&D Systems, AF-400-NA, IF, AR = TE).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Staining

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: Primary antibodies used are as follows: rabbit anti-CXCL8 (Abcam, ab289967, IF), mouse anti-human vimentin (Abnova, SRL33, IF, AR = TE), rabbit anti-p21 [Cell Signaling Technology, 2947, IF, AR = CB], rabbit anti–NF-κB (Cell Signaling Technology, 8242, IF), hamster anti-podoplanin (Developmental Studies Hybridoma Bank, 8.1.1, AR = TE), rat anti–Ki-67 (Invitrogen, 14-5698-82, IF, AR = CB), mouse anti-human IL6 (R&D Systems, MAB2061, IF), mouse anti-human IL1α membrane form 488 (R&D Systems, FAB2001G, IF), goat anti-mouse IL1α (R&D Systems, AF-400-NA, IF, AR = TE).

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