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