ewing sarcoma ews cell line 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: Scientific Reports

Article Title: Suicide gene therapy targeting ewing sarcoma via an ewing-specific GGAA promoter

doi: 10.1038/s41598-025-14945-6

Figure Lengend Snippet: Ewing sarcoma cells are sensitive to HSV-TK/GCV-mediated toxicity. (a) A673 and MHH-ES1 Ewing sarcoma cells were infected at an MOI of 0.5 with lentivirus harboring the HSV-TK(SR39h)/HA gene under the control of the constitutive promoter EF1A (EF1A > TK) or the Ewing-specific promoter GGAAprom (GGAA > TK). Western blot confirming the expression of HSV-TK. (b) A673 and MHH-ES1 cells expressing HSV-TK under the aforementioned promoters were incubated with different concentrations of GCV for six days, and the effect on cell viability was quantified with a resazurin assay (mean ± SD of three independent experiments).

Article Snippet: The Ewing sarcoma cell line A673 (CRL-1598), the fibrosarcoma cell line HT1080 (CCL-121) and the osteosarcoma cell lines U2-OS (HTB-96) and Saos-2 (HTB-85) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Infection, Control, Western Blot, Expressing, Incubation, Resazurin Assay

Journal: Scientific Reports

Article Title: Suicide gene therapy targeting ewing sarcoma via an ewing-specific GGAA promoter

doi: 10.1038/s41598-025-14945-6

Figure Lengend Snippet: HSV-TK/GCV-mediated bystander effect in Ewing sarcoma cells. (a) A673 and MHH-ES1 cells stably transduced with GGAA > HSV-TK(SR39h) lentivirus were cocultured with cells stably transduced with the luciferase gene (LUC cells). The ratios of GGAA > TK cells to LUC cells are indicated. The cells were incubated with 10 µM GCV, and luciferase activity was quantified after 72 h. Each point represents the mean of one experiment performed in triplicate (3–4 independent experiments). (mean ± SD). (b) MHH-ES1 cells constitutively expressing EGFP and HSV-TK(SR39h) under the control of the GGAA promoter were cocultured with cells constitutively expressing mCherry (50:50 ratio) for 93 h in the absence (control) or presence of GCV (10 µM). Representative fluorescence images showing that HSV-TK-negative cells (mCherry-positive cells) were killed in the presence of HSV-TK-positive cells (EGFP-positive cells). A representative video is also included in the supplementary material.

Article Snippet: The Ewing sarcoma cell line A673 (CRL-1598), the fibrosarcoma cell line HT1080 (CCL-121) and the osteosarcoma cell lines U2-OS (HTB-96) and Saos-2 (HTB-85) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Stable Transfection, Transduction, Luciferase, Incubation, Activity Assay, Expressing, Control, Fluorescence

Journal: Scientific Reports

Article Title: Suicide gene therapy targeting ewing sarcoma via an ewing-specific GGAA promoter

doi: 10.1038/s41598-025-14945-6

Figure Lengend Snippet: HSV-TK/GCV therapy reduces tumor growth and induces immune cell infiltration in vivo. (a) Schematic representation of the experimental design. (b) Nude mice were inoculated s.c. with A673/GGAA > HSV-TK (SR39h) cells and split into two groups when the tumors reached a mean volume of approximately 200 mm 3 (dashed line). One group was treated with valganciclovir (VGCV) ( n = 8) dissolved in drinking water, and the other group was treated with vehicle ( n = 6). The tumor volume (mean ± s.e.m) and tumor volume normalized to day 14 (start of treatment) for each animal are shown. (c) Micrograph of tumors excised at the end of the experiment (day 22). Two animals in the treated group presented no residual tumors and thus were missing in the image. (d) Tumor sections were stained with an anti-mouse CD45 antibody to assess immune cell infiltration. Representative images of tumors from the VGCV and control groups demonstrating the massive death of Ewing sarcoma cells and immune cell infiltration (CD45 + cells) in the VGCV group. Images from the control and VGCV groups are shown at the same magnification.

Article Snippet: The Ewing sarcoma cell line A673 (CRL-1598), the fibrosarcoma cell line HT1080 (CCL-121) and the osteosarcoma cell lines U2-OS (HTB-96) and Saos-2 (HTB-85) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: In Vivo, Staining, Control

Journal: Scientific Reports

Article Title: Suicide gene therapy targeting ewing sarcoma via an ewing-specific GGAA promoter

doi: 10.1038/s41598-025-14945-6

Figure Lengend Snippet: The combination of adenovirus GGAA > TK (Ad-GGAA > TK) and VGCV reduces tumor growth and induces immune cell infiltration in vivo. (a) Schematic representation of the experimental design. Nude mice were inoculated s.c. with A673 Ewing sarcoma cells and split into three groups when the tumors reached a mean volume of approximately 200 mm 3 (dashed line). One group received intratumoral injections of PBS (control PBS) ( n = 7), other group received intratumoral injections of control adenovirus (Ad-GGAA > ORF, 2 × 10 10 VP/mL dose) ( n = 6) and the third group received intratumoral administration of the therapeutic adenovirus Ad-GGAA > TK (2 × 10 10 VP/mL dose) ( n = 7). PBS and adenoviruses were administered at 2–3-day intervals (four doses). All groups received VGCV dissolved in drinking water. (b) The tumor volume normalized with respect to the start of treatment for each group and each animal is shown (mean ± s.d.). Statistical significance ( p-values ) between groups is shown (2-way ANOVA, ns = not significant). (c) Micrograph of tumors excised at the end of the experiment for each experimental group. (d) Tumor sections were stained with an anti-mouse CD45 antibody to assess immune infiltration. Representative images of a tumor treated with Ad-GGAA > TK and tumors treated with PBS or the adenovirus control Ad-GGAA > ORF are shown. Control tumors (PBS and Ad-GGAA > ORF) were negative for CD45 + cells except at the periphery of the tumor, where some CD45 + cells accumulated. In contrast, Ad-GGAA > TK treatment induced massive death of Ewing sarcoma cells and immune cell infiltration (CD45 + cells). Images are shown at the same magnification.

Article Snippet: The Ewing sarcoma cell line A673 (CRL-1598), the fibrosarcoma cell line HT1080 (CCL-121) and the osteosarcoma cell lines U2-OS (HTB-96) and Saos-2 (HTB-85) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: In Vivo, Control, Staining