Journal: PLoS ONE

Article Title: High Expression of CD109 Antigen Regulates the Phenotype of Cancer Stem-Like Cells/Cancer-Initiating Cells in the Novel Epithelioid Sarcoma Cell Line ESX and Is Related to Poor Prognosis of Soft Tissue Sarcoma

doi: 10.1371/journal.pone.0084187

Figure Lengend Snippet: A. Expression of CD109 mRNA in ES cell lines. Bars represent mean±SEM. ※※※ p <0.001, determined by the Mann-Whitney test. N.S.: not significant. B. Expression of CD109 mRNA in human sarcoma cell lines. Cell lines of epithelioid sarcoma (FU-EPS-1 and VA-ES-BJ), osteosarcoma (OS2000, KIKU, NY, U2OS, Saos-2, HuO9 and HOS), Ewing sarcoma (SKES, WES and RDES), synovial sarcoma (Fuji and YaFuSS) and malignant fibrous histiocytoma (MFH2003 and MFH2004) were used. C. Expression of CD109 mRNA in human fetal tissues (upper panel) and human adult tissues (lower panel). ESX was used as a positive control. D. Immunohistochemistry of CD109 in normal adult tissues.

Article Snippet: Human osteosarcoma cell lines (NY, U2OS and HOS), human Ewing sarcoma cell lines (SKES, WES, and RDES), the human synovial sarcoma cell line FUJI, and the human ES cell line VA-ES-BJ were purchased from the Japanese Collection of Research Bioresources Cell Bank (Tokyo, Japan) and American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, MANN-WHITNEY, Positive Control, Immunohistochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Pre-clinical and clinical significance of heparanase in Ewing’s sarcoma

doi: 10.1111/j.1582-4934.2010.01190.x

Figure Lengend Snippet: Antitumor activity of SST0001 against TC71 Ewing’s sarcoma

Article Snippet: The Ewing’s sarcoma cell line TC71 was maintained in Iscove’s modified Dulbecco’s Medium (Lonza, Verviers, Belgium) supplemented with 10% foetal bovine serum at 37°C in 5% CO 2 atmosphere.

Techniques: Activity Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Pre-clinical and clinical significance of heparanase in Ewing’s sarcoma

doi: 10.1111/j.1582-4934.2010.01190.x

Figure Lengend Snippet: Heparanase staining in Ewing’s sarcoma patients

Article Snippet: The Ewing’s sarcoma cell line TC71 was maintained in Iscove’s modified Dulbecco’s Medium (Lonza, Verviers, Belgium) supplemented with 10% foetal bovine serum at 37°C in 5% CO 2 atmosphere.

Techniques: Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Pre-clinical and clinical significance of heparanase in Ewing’s sarcoma

doi: 10.1111/j.1582-4934.2010.01190.x

Figure Lengend Snippet: Immunohistochemical staining of heparanase in Ewing’s sarcoma specimens. Formalin-fixed, paraffin-embedded 5 μm sections were subjected to immunostaining, applying anti-heparanase polyclonal antibody (Ab 733), as described under ‘Materials and methods’. Shown are representative photomicrographs of heparanase-positive specimens categorized as weak (+1, A, B) or strong (+2, C, D) intensity. Nuclear localization of heparanase is shown in (E). Specimens that were similarly stained with pre-immune serum, or applying the above procedure but lacking the primary antibody, yielded no detectable staining (F). Original magnification: (A, C) 100×; (B, D–F) 400×.

Article Snippet: The Ewing’s sarcoma cell line TC71 was maintained in Iscove’s modified Dulbecco’s Medium (Lonza, Verviers, Belgium) supplemented with 10% foetal bovine serum at 37°C in 5% CO 2 atmosphere.

Techniques: Immunohistochemical staining, Staining, Formalin-fixed Paraffin-Embedded, Immunostaining

Journal: Cancer Research Communications

Article Title: BRCA1-Associated RING Domain-1 (BARD1) Loss and GBP1 Expression Enhance Sensitivity to DNA Damage in Ewing Sarcoma

doi: 10.1158/2767-9764.CRC-21-0047

Figure Lengend Snippet: The landscape of germline BARD1 variants in a cohort of pediatric oncology patients.

Article Snippet: Ewing sarcoma cell lines were queried in the Broad Institute Cancer Cell Line Encyclopedia (CCLE) database ( https://portals.broadinstitute.org/ccle ).

Techniques: Sequencing

Journal: Cancer Research Communications

Article Title: BRCA1-Associated RING Domain-1 (BARD1) Loss and GBP1 Expression Enhance Sensitivity to DNA Damage in Ewing Sarcoma

doi: 10.1158/2767-9764.CRC-21-0047

Figure Lengend Snippet: GSEA analysis and validation of a primary cell line from a Ewing tumor with a BARD1 pathogenic variant. A, Schematic overview of tumor samples associated with analyses in B – F . B, Gene-set enrichment analysis (GSEA) of RNA-seq data comparing the lung relapse of the Ewing tumor with a germline BARD1 pathogenic variant to the original primary/pretreatment biopsy. Genesets significantly impacted ( P < 0.05) are included. C, Phase contrast image (400×) of the PSaRC318 Ewing tumor cell line. D, Flow cytometry showing presence of surface CD99 expression in the PSaRC318 cell line. E, Schematic detailing the difference between Type 1 and Type 3 EWS-FLI fusions (top) and Western blot analysis with anti-FLI1 antibody of Ewing sarcoma cell lines with type 3 (PSaRC318) versus type 1 (A673, CHLA9, CHLA10, and TC71) EWS-FL1 fusions. F, Western blot demonstrating BARD1 protein expression in the same Ewing sarcoma cell lines as in E . PSaRC318 cells demonstrate significantly ( P < 0.05) less BARD1 expression as compared with other Ewing cell lines. Densitometry values below the blot indicate relative expression values. Experiments in D – F were completed minimally in biological triplicate.

Article Snippet: Ewing sarcoma cell lines were queried in the Broad Institute Cancer Cell Line Encyclopedia (CCLE) database ( https://portals.broadinstitute.org/ccle ).

Techniques: Biomarker Discovery, Variant Assay, RNA Sequencing, Flow Cytometry, Expressing, Western Blot

Journal: Cancer Research Communications

Article Title: BRCA1-Associated RING Domain-1 (BARD1) Loss and GBP1 Expression Enhance Sensitivity to DNA Damage in Ewing Sarcoma

doi: 10.1158/2767-9764.CRC-21-0047

Figure Lengend Snippet: Loss of BARD1 enhances Ewing sarcoma cell sensitivity to PARP inhibition. A, Western blot analysis for PARP1 expression in Ewing sarcoma cells. B, p-γH2AX and DAPI immunofluorescence staining of PSaRC318, A673, and CHLA10 cells treated with DMSO or 100 nmol/L talazoparib (Tal). Cells imaged at 630× and p-γH2AX foci were quantified (bottom graphs). C, IncuCyte assay comparing the confluence of PSaRC318 cells treated with DMSO versus 100 nmol/L talazoparib over 1 week. D, qRT-PCR showing BARD1 mRNA expression in A673 or CHLA10 cells treated with control (ctsi) or BARD1 ( BARD1 si) siRNA. E, Western blot analysis for BARD1 expression in untreated (NT) A673 or CHLA10 cells or cells treated with Ctsi or BARD1 si. F, IncuCyte monitoring of cell confluence at increasing concentrations of talazoparib versus DMSO controls in A673 and CHLA10 cells treated with Ctsi versus BARD1 si. A673 and CHLA10 cell data is graphed at the 60-hour time point. Normalized expression values from densitometry analyses are included under Western blots. Experiments were completed minimally in biological triplicate. NS, not significant; *, P < 0.01. Error bars, SD.

Article Snippet: Ewing sarcoma cell lines were queried in the Broad Institute Cancer Cell Line Encyclopedia (CCLE) database ( https://portals.broadinstitute.org/ccle ).

Techniques: Inhibition, Western Blot, Expressing, Immunofluorescence, Staining, Quantitative RT-PCR, Control

Journal: Cancer Research Communications

Article Title: BRCA1-Associated RING Domain-1 (BARD1) Loss and GBP1 Expression Enhance Sensitivity to DNA Damage in Ewing Sarcoma

doi: 10.1158/2767-9764.CRC-21-0047

Figure Lengend Snippet: BARD1 loss enhances Ewing sarcoma cell apoptosis in response to niraparib plus radiation. A, Relative apoptosis (caspase 3/7 activity) data from IncuCyte assays showing the effect of 0.5 μmol/L niraparib (Nir) versus DMSO control plus either 0 or 2 Gy radiation on PSaRC318 cells. B, Confluence data from IncuCyte assays showing the effect of 0.5 μmol/L niraparib versus DMSO control plus either 0 or 2 Gy radiation on PSaRC318 cells. C and D, A673 cells were treated with control (Ctsi) or BARD1 ( BARD1 si) siRNA, niraparib (at doses indicated) versus DMSO control, and either 0 or 2 Gy radiation and monitored via IncuCyte apoptosis assay ( C ) or confluence assay ( D ). For these experiments, cells were seeded in the presence of niraparib and radiation was performed at 12–15 hours. Relative apoptosis (caspase 3/7 dye activity) is calculated as green fluorescence in μm 2 divided by confluence. Experiments were completed minimally in technical and biological triplicates. *, P < 0.01 as determined by ANOVA analysis with Tukey multiple comparisons test. Error bars, SD.

Article Snippet: Ewing sarcoma cell lines were queried in the Broad Institute Cancer Cell Line Encyclopedia (CCLE) database ( https://portals.broadinstitute.org/ccle ).

Techniques: Activity Assay, Control, Apoptosis Assay, Fluorescence

Journal: Cancer Research Communications

Article Title: BRCA1-Associated RING Domain-1 (BARD1) Loss and GBP1 Expression Enhance Sensitivity to DNA Damage in Ewing Sarcoma

doi: 10.1158/2767-9764.CRC-21-0047

Figure Lengend Snippet: Impact of BARD1 loss on the Ewing sarcoma cell transcriptome. A, Volcano plot of genes up-/downregulated upon loss of BARD1 as compared with A673 Ewing sarcoma cells transfected with Ctsi in three biological replicates. Horizontal dashed lines denote P = 0.05 (5 on −log 10 scale). Vertical dashed lines denote a value of 1 on the log 2 scale. BARD1 is circled in blue and highlighted as to verify that it is significantly downregulated upon RNA-seq analysis. In addition, the inset image demonstrates RT-PCR analysis of BARD1 expression as a second means by which to validate reduction of BARD1 expression in these RNA samples (×3 biological replicates) as compared with Ctsi-treated cells, *, P < 0.05; error bars, SD. B, List of most significantly ( P adj ) upregulated and downregulated genes with a log 2 fold change of 2 or greater when comparing cells treated with BARD1 siRNA versus Ctsi. C, Pathway analysis (C2 and Hallmark genesets) of BARD1 siRNA-treated cells as compared with Ctsi-treated cells. NES, normalized enrichment score; P adj , adjusted P value.

Article Snippet: Ewing sarcoma cell lines were queried in the Broad Institute Cancer Cell Line Encyclopedia (CCLE) database ( https://portals.broadinstitute.org/ccle ).

Techniques: Transfection, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Cancer Research Communications

Article Title: BRCA1-Associated RING Domain-1 (BARD1) Loss and GBP1 Expression Enhance Sensitivity to DNA Damage in Ewing Sarcoma

doi: 10.1158/2767-9764.CRC-21-0047

Figure Lengend Snippet: Guanylate-binding protein 1 (GBP1) contributes to Ewing cell sensitivity to DNA damage noted upon loss of BARD1. A, IHC analysis of GPB1 expression in the PSaRC318 patient tumor and in two additional independent Ewing tumors. Images provided at both low (200×) and high (1,000×) power. B, Western blot for GBP1 in Ewing sarcoma cell lines. Numbers under the blot indicate normalized expression as determined by densitometry analysis. C, Normalized GPB1 mRNA expression of PSaRC318 cells treated with control (ct) or GBP1 siRNA (si). *, P < 0.0001. D, PSaRC318 cells were transfected with control or GBP1 siRNA for 72 hours. Cells were then seeded into 96-well plates in quadruplicate, allowed to adhere and then radiated (dose = 1 Gy). Live-cell IncuCyte monitoring of cell confluence and apoptosis (caspase 3/7 activity) was monitored. The graph displays the relative apoptosis (apoptosis/confluence) in control versus GBP1 siRNA-treated cells over time. *, P < 0.05. E, PSaRC318 cells treated with siRNA and seeded as in D and then treated with DMSO, 0.5 μmol/L niraparib (Nir), or 0.75 μmol/L niraparib. The graph displays the relative apoptosis (apoptosis/confluence) over time. Representative IncuCyte images at 48 hours are included (right). NS, not significant; *, P < 0.05. Experiments completed minimally in biological triplicate. Error bars, SD.

Article Snippet: Ewing sarcoma cell lines were queried in the Broad Institute Cancer Cell Line Encyclopedia (CCLE) database ( https://portals.broadinstitute.org/ccle ).

Techniques: Binding Assay, Expressing, Western Blot, Control, Transfection, Activity Assay