Journal: Theranostics

Article Title: GD2-mediated impairment of macrophage phagocytosis drives pulmonary metastasis in osteosarcoma

doi: 10.7150/thno.113887

Figure Lengend Snippet: GD2 is associated with poor prognosis of osteosarcoma. ( A-C ) Analysis of GD2 expression in patient samples. Shown are presentative images ( A ), quantitation of GD2 immunostaining of osteosarcoma patient samples with or without lung metastasis ( B ) and overall survival analyses of the patients according to different GD2 expression status ( C ). ( D ) Correlation between B4GALNT1 expression and GD2 expression in patient samples. ( E-G ) Analysis of B4GALNT1 expression in GDC TARGET-OS dataset. Shown are progress-free survival ( E ), overall survival ( F ) analyses of the patients according to different B4GALNT1 expression status and lung metastasis rate between patients with low B4GALNT1 expression and high B4GALNT1 expression ( G ). ( H ) B4GALNT1 expression in osteosarcoma cells with different metastasis potential in GSE18947 dataset. Scale bars: 100 μm. P values were obtained by log-rank test ( C , E and F ), chi-squared test ( G ), Pearson's correlation analysis ( D ) and 2-tailed unpaired t test ( B, H ). Box plots display values of minimum, first quartile, median, third quartile, and maximum. Data are represented as mean ± SD.

Article Snippet: The RNA of BMDMs treated with 200 μM GD2 (HY-165740, MedChemExpress) or PBS (as control) were used for transcriptome sequencing.

Techniques: Expressing, Quantitation Assay, Immunostaining

Journal: Theranostics

Article Title: GD2-mediated impairment of macrophage phagocytosis drives pulmonary metastasis in osteosarcoma

doi: 10.7150/thno.113887

Figure Lengend Snippet: GD2 promotes lung metastasis of osteosarcoma. ( A-D ) Intravenous injection of U2OS with or without B4GALNT1 knockdown into nude mice for lung metastasis analysis. Weekly BLI quantitation of tumor burden of the mice and representative images ( A , n = 6 mice per group), ex vivo BLI analysis of lungs and representative images ( B ), number of lung nudes and representative images ( C ) and animal survival ( D , n = 6 mice per group). ( E-G ) Intratibial injection of 143B with or without B4GALNT1 knockdown into nude mice for lung metastasis analysis. Ex vivo BLI analysis of lungs ( E ) and hindlimbs ( F ) at week 6 and representative images ( G ). Scale bars: 3 mm. P values were obtained by Mann-Whitney U test ( A , B , E and F ), 2-tailed unpaired t test ( C ) and log-rank test ( D ). Box plots display values of minimum, first quartile, median, third quartile, and maximum. Data are represented as mean ± SD.

Article Snippet: The RNA of BMDMs treated with 200 μM GD2 (HY-165740, MedChemExpress) or PBS (as control) were used for transcriptome sequencing.

Techniques: Injection, Knockdown, Quantitation Assay, Ex Vivo, MANN-WHITNEY

Journal: Theranostics

Article Title: GD2-mediated impairment of macrophage phagocytosis drives pulmonary metastasis in osteosarcoma

doi: 10.7150/thno.113887

Figure Lengend Snippet: GD2 suppresses phagocytosis of osteosarcoma cells. ( A - C ) Immunofluorescence analyses of lung metastasis niches at day 28 after intravenous injection of U2OS cells (with or without B4GALNT1 knockdown). Shown are representative images ( A ), quantitation of tumor infiltrated macrophages ( B ) and percentage of GFP + macrophages ( C ). ( D , E ) GO analyses of enriched genes ( D ) in the RNA-seq profiles of GD2-treated mouse bone marrow derived macrophages vs control mouse bone marrow derived macrophages and GSEA analyses of Phagosome gene sets ( E ). ( F , G ) Representative flow cytometry plots depicting the phagocytosis of GFP-labeled U2OS cells (with or without B4GALNT1 knockdown) co-cultured with mouse bone marrow derived macrophages ( F ) and flow-cytometry-based quantification of phagocytosis of U2OS in the presence of mouse bone marrow derived macrophages ( G ). Scale bars: 50 μm. P values were obtained by 2-tailed unpaired t test ( B , C and G ). Data are represented as mean ± SD.

Article Snippet: The RNA of BMDMs treated with 200 μM GD2 (HY-165740, MedChemExpress) or PBS (as control) were used for transcriptome sequencing.

Techniques: Immunofluorescence, Injection, Knockdown, Quantitation Assay, RNA Sequencing, Derivative Assay, Control, Flow Cytometry, Labeling, Cell Culture

Journal: Theranostics

Article Title: GD2-mediated impairment of macrophage phagocytosis drives pulmonary metastasis in osteosarcoma

doi: 10.7150/thno.113887

Figure Lengend Snippet: GD2 functions by interacting with SIGLECs. ( A ) Siglece, Siglecf, Siglecg and Siglech expression in mouse bone marrow derived macrophages in GSE190235 and GSE271727 datasets. ( B ) Flow cytometric histograms of U2OS stained with recombinant mouse SIGLECs. ( C ) Flow cytometric histograms of U2OS (with or without B4GALNT1 knockdown) stained with recombinant mouse SIGLECE. ( D ) Quantitation of SIGLECE mean fluorescence intensity (MFI) in panel ( C ). ( E ) Flow cytometric analysis of SIGLECE expression in mouse bone marrow derived macrophages (with or without Siglece knockdown). ( F ) Quantitation of SIGLECE mean fluorescence intensity (MFI) in panel ( E ). ( G , H ) Representative flow cytometry plots depicting the phagocytosis of GFP-labeled U2OS cells co-cultured with mouse bone marrow derived macrophages (with or without Siglece knockdown) ( G ) and flow-cytometry-based quantification of phagocytosis of U2OS in the presence of mouse bone marrow derived macrophages ( H ). P values were obtained by 2-tailed unpaired t test ( C , E and G ). Data are represented as mean ± SD.

Article Snippet: The RNA of BMDMs treated with 200 μM GD2 (HY-165740, MedChemExpress) or PBS (as control) were used for transcriptome sequencing.

Techniques: Expressing, Derivative Assay, Staining, Recombinant, Knockdown, Quantitation Assay, Fluorescence, Flow Cytometry, Labeling, Cell Culture

Journal: Theranostics

Article Title: GD2-mediated impairment of macrophage phagocytosis drives pulmonary metastasis in osteosarcoma

doi: 10.7150/thno.113887

Figure Lengend Snippet: GD2 activates SH2-containing protein tyrosine phosphatase 2. ( A ) Western blot analysis of phosphorylated SHP2 protein level in mouse bone marrow derived macrophages after treated with GD2. ( B , C ) Representative images ( B ) and quantitation ( C ) of phosphorylated SHP2 immunostaining of mouse bone marrow derived macrophages after treated with GD2. ( D ) Western blot analysis of phosphorylated SHP2 protein level in mouse bone marrow derived macrophages after treated with GD2 and/or SHP099. ( E , F ) Representative flow cytometry plots depicting the phagocytosis of GFP-labeled U2OS cells co-cultured with mouse bone marrow derived macrophages (with or without SHP099 treatment) ( E ) and flow-cytometry-based quantification of phagocytosis of U2OS in the presence of mouse bone marrow derived macrophages ( F ). Scale bars: 100 μm. P values were obtained by 2-tailed unpaired t test ( C and F ). Box plots display values of minimum, first quartile, median, third quartile, and maximum. Data are represented as mean ± SD.

Article Snippet: The RNA of BMDMs treated with 200 μM GD2 (HY-165740, MedChemExpress) or PBS (as control) were used for transcriptome sequencing.

Techniques: Western Blot, Derivative Assay, Quantitation Assay, Immunostaining, Flow Cytometry, Labeling, Cell Culture