ews cell lines a673  (ATCC)

Journal: Science Advances

Article Title: Autocrine TGFβ2 enforces a transcriptionally hybrid cell state in Ewing sarcoma

doi: 10.1126/sciadv.ady0550

Figure Lengend Snippet: ( A ) EwS TMA sections stained for CD99 (tumor marker) with the low (left) and high (right) pSMAD2 S465/S467 signal. ( B ) Nuclear pSMAD2 S465/467 and cytoplasmic/membranous CD99 IF average intensity per segmented cell. Gates define positive versus negative populations (conservative threshold of pSMAD2 or CD99 true positives). Red box: CD99+ (likely tumor cell) population ( C ) % CD99+ cells that were also pSMAD2+ for each sample in the TMA ( n = 27, table S2); red line: median. ( D ) H&E and IF from EwS TMA sections stained for CD99, pSMAD2 S465/467, and TNC (ECM protein). Right: Zoomed insets showing the CD99 hi /pSMAD low /TNC low region versus CD99 hi /pSMAD2 hi /TNC hi region. ( E ) H&E of the tumor formed 3 weeks post–renal subcapsular injection of A673 cells in NSG mice. Right: IF for pSMAD2 S465/467 and ECM marker TNC (human-specific antibody).

Article Snippet: EwS cell lines A673, TC71, and CHLA10 were obtained from American Type Culture Collection (ATCC) and Children's Oncology Group (COG) ( https://childrensoncologygroup.org/ ) cell line repositories.

Techniques: Staining, Marker, Injection

Journal: Science Advances

Article Title: Autocrine TGFβ2 enforces a transcriptionally hybrid cell state in Ewing sarcoma

doi: 10.1126/sciadv.ady0550

Figure Lengend Snippet: ( A ) Real-time measurements of confluence (Incucyte) for TC71, A673, CHLA10, and PDX305 cells treated with vehicle controls, TGFβ1 (10 ng/ml), vactosertib (1 μM), or TGFβ1 + vactosertib. n = 2; error bars: SEM. P values: unpaired t tests of confluence at 48 hours relative to 0 hours within each line (nonsignificant comparisons not shown). hr, hours. ( B ) Measurement of the cell area at 48 versus 0 hours of Incucyte imaging in parental cell lines or BlastR control versus dominant-negative TGFBR2 transduced CHLA10 cells. n = 2. P values: unpaired t test within each line. ( C ) Cell viability measurements by CellTiter-Glo after 72 hours of culture with vehicle control, TGFβ1 (10 ng/ml), or vactosertib (1 μM) in parental cell lines or BlastR control versus dominant-negative TGFBR2 transduced CHLA10 cells. n = 3; P values: unpaired t test within each line. ( D ) Soft agar colony-forming assays of A673 (day 14) and CHLA10 (day 14) cells treated with vehicle control, TGFβ1 (10 ng/ml), or vactosertib (1 μM). ( E ) Number of colonies formed in soft agar for TC71 (day 14), A673 (day 14), CHLA10 (day 14), and PDX305 cells (day 35), relative to the number of colonies in the vehicle control well. n = 2; P values: unpaired t tests within each line. ( F ) Invasive morphology of spheroids cultured for 4 days in 3D rat tail collagen I–rich gels treated with vehicle controls, TGFβ1 (10 ng/ml), or vactosertib (10 μM). Stained with phalloidin to mark F-actin. Yellow arrows: invasive strands. Representative of n = 2. For all panels: n.s., P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001. hr, hours.

Article Snippet: EwS cell lines A673, TC71, and CHLA10 were obtained from American Type Culture Collection (ATCC) and Children's Oncology Group (COG) ( https://childrensoncologygroup.org/ ) cell line repositories.

Techniques: Imaging, Control, Dominant Negative Mutation, Cell Culture, Staining

Journal: Science Advances

Article Title: Autocrine TGFβ2 enforces a transcriptionally hybrid cell state in Ewing sarcoma

doi: 10.1126/sciadv.ady0550

Figure Lengend Snippet: ( A ) % GFP+ after transduction with SBE-GFP SMAD reporter −/+ TGFβ1 (48 hours, n = 2). ( B ) TGFB1 and TGFB2 expression in CAF-like (top 30% expression CAF-like gene signature) versus non-CAF-like cells by scSeq (nine EwS lines). ( C ) ELISAs of TGFβ1 in conditioned media (3 days of culture). n = 3. Dashed line indicates the TGFβ1 level detected in media-only controls. ( D ) ELISAs of secreted total TGFβ2 in conditioned media (3 days of culture). n = 3. TGFβ2 not detected in media-only controls. ( E ) Genes significantly ( P adj < 0.05) up-regulated by TGFβ1 or TGFβ2 in A673, CHLA10, and PDX305 (bulk RNA-seq, n = 3). ( F ) Crystal violet–stained cells 24 hours after seeding on Matrigel-coated transwells to measure invasion. Each cell marked with an asterisk. ( G ) Quantification of Matrigel-coated transwell invasion 24 hours postseeding ( n = 3 to 4; each dot is a biological replicate). P values: unpaired t tests within each line. ( H and I ) ELISAs of secreted total (active and latent) (H) TGFβ2 and (I) TGFβ1 after 3 days of culture of CHLA10 dominant-negative TGFBR2 or empty vector transduced cells. n = 3. P values: unpaired t tests. ( J ) Expression of TNC , TGFB1 , and TGFB2 in PDX305 cells treated with TGFβ1 (10 ng/ml) −/+ vactosertib, TGFβ2 (10 ng/ml) −/+ vactosertib, or vactosertib alone (1 μM). n = 2; P values: unpaired t tests of treatment versus vehicle (nonsignificant comparisons not shown). ( K ) Expression of TGFβ-induced genes in CHLA10 cells treated with TGFβ ligand–blocking antibodies (10 μg/ml, 4 days). n = 2; P values: unpaired t tests. ( L ) IF of TNC in CHLA10 cells treated with TGβ ligand–blocking antibodies versus IgG control (10 μg/ml, 4 days). P values: unpaired t tests. For all panels: n.s., P > 0.05; * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: EwS cell lines A673, TC71, and CHLA10 were obtained from American Type Culture Collection (ATCC) and Children's Oncology Group (COG) ( https://childrensoncologygroup.org/ ) cell line repositories.

Techniques: Transduction, Expressing, RNA Sequencing, Staining, Dominant Negative Mutation, Plasmid Preparation, Blocking Assay, Control

Journal: Cancers

Article Title: In Vitro and In Vivo Efficacy of Romidepsin Alone and in Addition to Standard of Care for Treatment of Ewing Sarcoma

doi: 10.3390/cancers17244018

Figure Lengend Snippet: The addition of romidepsin to IE decreases tumor burden in a xenograft model. 2 × 10 6 A673 cells were injected subcutaneously into mice. Once tumors were palpable, the mice were divided into groups and treated with two 21-day cycles of vehicle control (DMSO), romidepsin (2 mg/kg; twice per week), IE (90 mg/kg ifosfamide, 120 mg/kg etoposide; days 1–3 per cycle), or a combination of romidepsin and IE. Vehicle n = 19, romidepsin n = 20, IE n = 18, IE + romidepsin n = 18. ( A ) Average tumor volume over time. Two-way ANOVA indicates significance ( p < 0.05 *) between the IE group and the IE + romidepsin group at day 11. ( B ) Average tumor volume on day 5 of treatment. One-way ANOVA with Tukey’s multiple comparisons test indicates significance ( p < 0.05 *). ( C ) Survival curve, log-rank was test used to test for statistical differences, ns: not significant, p < 0.05 *, p < 0.0001 ****. ( D ) Average mouse weight for each group throughout the first cycle of treatment.

Article Snippet: A673 cells were obtained from ATCC and cultured in high glucose DMEM media (ThermoFisher Scientific, High Point, NC, USA) supplemented with 10% FBS, 100 U/mL Penicillin, and 100 μg/mL Streptomycin.

Techniques: Injection, Control