Journal: Oncotarget

Article Title: EWS-FLI1 low Ewing sarcoma cells demonstrate decreased susceptibility to T-cell-mediated tumor cell apoptosis

doi: 10.18632/oncotarget.26939

Figure Lengend Snippet: ( A ) Ewing sarcoma cell lines A673 and CHLA-10 were labeled using an APC-conjugated ICAM-1 antibody and near-IR live/dead stain. Gates were generated to analyze live cell singlets with ICAM-1 staining. % denotes the frequency of ICAM-1+ cells upon analysis of a minimum of 10,000 total events. ( B ) A673 and CHLA10 cells were treated with control or EWS-FLI1 siRNA for 72 hours. Generated cDNA was analyzed for EWS-FLI1 and RPLP0 (control) expression using RT-PCR. Relative expression normalized to control siRNA is graphed. Cells treated with control or EWS-FLI1 siRNA were also either ( C ) lysed and subjected to western blot analysis for FLI1 and a vinculin loading control or ( D ) stained and analyzed by flow cytometry for ICAM-1 surface expression ( n = 3 per cell line). Error bars reflect SD. Circles on bar graphs in B and D indicate values for individual replicates. Control versus EWS-FLI1 siRNA treated cells were compared using an unpaired t -test. * p < 0.05, *** p < 0.001.

Article Snippet: Human patient-derived Ewing sarcoma cell lines were obtained from ATCC (A673, SK-N-MC), and the Children’s Oncology Group Childhood Cancer Repository (CHLA10). shA673-1c cells (with shRNA mediated tet-repressible EWS-FLI1 expression) are a kind gift from Dr. Olivier Delattre [ , ].

Techniques: Labeling, Staining, Generated, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Flow Cytometry

Journal: Oncotarget

Article Title: EWS-FLI1 low Ewing sarcoma cells demonstrate decreased susceptibility to T-cell-mediated tumor cell apoptosis

doi: 10.18632/oncotarget.26939

Figure Lengend Snippet: ( A ) A673 ( n = 4) and CHLA-10 ( n = 4) cells were treated with control or EWS-FLI1 siRNA for 48 hours, followed by treatment with 500 U/mL IFN-γ for a subsequent 24 hours. Ewing cells were analyzed by RT-PCR analysis for ICAM-1 and RPLP0 (control) expression. Graphs represent relative increase in ICAM1 mRNA expression (values normalized to untreated control). Bars represent SD. Differences in IFN-γ treatment response between ctsi and EWFsi groups for each cell line was compared using an unpaired t -test. *** p < 0.001. ( B ) Cells were treated with IFN-γ as in (A) followed by analysis of ICAM-1 surface expression by flow cytometry. MFI (median fluorescence intensity) values for both control (ct) and EWS-FLI1 (EWF) siRNA treatment conditions in both the A673 and CHLA10 cell lines are listed. ( C ) shA673-1c cells (harboring shRNA mediated tet-repressible EWS-FLI1 vector) were treated with DMSO vehicle control or 1 microgram/mL doxycycline for 48 hours ( n = 3). RT-PCR analysis of EWS-FLI1 and RPLP0 (control) expression was performed. *** p < 0.001. ( D ) An ICAM1 LightSwitch promoter reporter assay was used to examine differences in promoter activity between shA673-1c cells + DOX (doxycycline) or DMSO control cells in the absence or presence of IFN-γ. In this system, +Dox= EWS-FLI1 ‘low’ expression. Differences in promoter activity between conditions were determined using an unpaired t -test. * p < 0.05.

Article Snippet: Human patient-derived Ewing sarcoma cell lines were obtained from ATCC (A673, SK-N-MC), and the Children’s Oncology Group Childhood Cancer Repository (CHLA10). shA673-1c cells (with shRNA mediated tet-repressible EWS-FLI1 expression) are a kind gift from Dr. Olivier Delattre [ , ].

Techniques: Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Flow Cytometry, Fluorescence, shRNA, Plasmid Preparation, Reporter Assay, Activity Assay

Journal: Oncotarget

Article Title: EWS-FLI1 low Ewing sarcoma cells demonstrate decreased susceptibility to T-cell-mediated tumor cell apoptosis

doi: 10.18632/oncotarget.26939

Figure Lengend Snippet: ( A ) A673 and CHLA-10 Ewing sarcoma cells were grown alone or in co-culture with activated T-cells for 48 hours and monitored in real time using an IncuCyte system apoptosis assay. Representative images of cells at 36 hours upon real-time monitoring for apoptosis (activated caspase 3/7 activity, red) in tumor cells in culture alone (left panels) or co-cultured with activated T-cells (right panels). The black bar denotes a length of 400 micrometers. ( B–C ) Graphs represent fold increase in apoptosis in A673 and CHLA10 upon exposure to activated T-cells ( n = 3 per cell line). Differences in apoptosis ± T-cell exposure were determined using an unpaired t -test. * p < 0.05. ( D ) A673 Ewing sarcoma cells were left untreated or treated with IgG control or ICAM-1 blocking antibody, co-cultured with activated T-cells and then monitored for caspase 3/7 activity using an IncuCyte apoptosis assay. No T-cell controls were also included . Graphs represent the max % change in tumor cell apoptosis with IgG or ICAM-1 antibody treatment compared to no antibody controls ( n = 3). * p < 0.05. ( E ) A673 ( n = 4) and ( F ) CHLA-10 ( n = 3) Ewing sarcoma cells were treated with control (ctsi) or EWS-FLI1 siRNA and placed in the in the absence (black lines) or presence (red lines) of activated T-cells at a ratio of 1:10 (T-cell : tumor cell). Caspase activity was monitored in real time using an IncuCyte apoptosis assay. Error bars represent SD.

Article Snippet: Human patient-derived Ewing sarcoma cell lines were obtained from ATCC (A673, SK-N-MC), and the Children’s Oncology Group Childhood Cancer Repository (CHLA10). shA673-1c cells (with shRNA mediated tet-repressible EWS-FLI1 expression) are a kind gift from Dr. Olivier Delattre [ , ].

Techniques: Co-Culture Assay, Apoptosis Assay, Activity Assay, Cell Culture, Control, Blocking Assay