tc 71 cvcl 2213 cell lines  (DSMZ)

Journal: International Journal of Biological Sciences

Article Title: AVN944 Elicits Apoptotic Responses and Impedes Tumorigenic Potential in Ewing's Sarcoma Cells

doi: 10.7150/ijbs.116651

Figure Lengend Snippet: Kaplan-Meier survival analysis and differential expression of IMPDH2 in sarcoma. (A) Overall survival curve based on IMPDH2 expression in patients with sarcoma from the TCGA cohort (n = 262). Patients were stratified into low IMPDH2 expression (blue line) and high IMPDH2 expression (red line) groups. Kaplan-Meier curves show that higher expression of IMPDH2 is associated with a trend towards reduced survival, although this did not reach statistical significance (log-rank p = 0.061). The hazard ratio for high expression was 1.5, indicating a 50% increase in the risk of death for patients with high expression of IMPDH2 ( p = 0.063). (B) Differential expression of IMPDH2 between sarcoma (Tumor, n = 262) and normal tissues (Normal, n = 2) from the TCGA dataset. IMPDH2 expression, measured in transcripts per million (TPM), is significantly higher in tumor samples than in normal tissues. (C) Western blot analysis to detect expression of IMPDH2 in Ewing's sarcoma cell lines (RD-ES, SK-ES-1, and TC71). HeLa (human cervical carcinoma cell line) and MCF-7 (human breast cancer cell line) cells were used as positive controls. α/β-tubulin served as a loading control.

Article Snippet: By contrast, the TC71 cell line was acquired from the Leibniz Institute DSMZ (Germany).

Techniques: Quantitative Proteomics, Expressing, Western Blot, Control

Journal: International Journal of Biological Sciences

Article Title: AVN944 Elicits Apoptotic Responses and Impedes Tumorigenic Potential in Ewing's Sarcoma Cells

doi: 10.7150/ijbs.116651

Figure Lengend Snippet: Effects of AVN944 on growth and colony formation by TC71 Ewing's sarcoma cells. (A) Representative images of TC71 cells treated with 1 μM AVN944 or control (vehicle) over 5 days. Morphological changes were observed, with AVN944-treated cells showing lower cell density than control cells. Scale bar, 100 μm. (B) Growth curve for TC71 cells treated with 1 μM AVN944 (orange) or control (blue) for 5 days. AVN944 inhibited cell proliferation significantly when compared with the control. Data represent the mean ± SD of three independent experiments (n = 3, ** p < 0.01). (C) Representative images from a BrdU incorporation assay in which TC71 cells were treated with AVN944 for 3 h on Day 1. BrdU-positive cells (green) are actively proliferating. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. (D) Quantification of BrdU-positive TC71 cells treated (or not) with AVN944. AVN944 reduced BrdU incorporation significantly, indicating a reduction in DNA synthesis (n = 3, ** p <0.01). (E) MTT assay of TC71 cells treated with AVN944. AVN944 treatment resulted in a significant reduction in cell viability compared with the control (n = 8, ** p <0.01). (F) Colony formation assay in which TC71 cells were treated with increasing concentrations of AVN944 (0-1 μM). Colonies were stained with crystal violet, which revealed dose-dependent inhibition of colony formation. (G) Quantification of colony formation in F. The number of colonies was counted using ImageJ software, and presented as a percentage of the control value. AVN944 reduced colony formation significantly and in a dose-dependent manner (* p < 0.05, ** p < 0.01 vs. control). Data represent the mean ± SD of three independent experiments (n = 3). (H) Measurement of intracellular ATP levels in TC71 cells treated with AVN944 for 24 h. AVN944 reduced cellular ATP levels significantly compared with the control, indicating a decline in cellular metabolic activity (** p < 0.01 vs. control). Data represent the mean ± SD of eight independent experiments (n = 8).

Article Snippet: By contrast, the TC71 cell line was acquired from the Leibniz Institute DSMZ (Germany).

Techniques: Control, BrdU Incorporation Assay, Staining, DNA Synthesis, MTT Assay, Colony Assay, Inhibition, Software, Activity Assay

Journal: International Journal of Biological Sciences

Article Title: AVN944 Elicits Apoptotic Responses and Impedes Tumorigenic Potential in Ewing's Sarcoma Cells

doi: 10.7150/ijbs.116651

Figure Lengend Snippet: Dose-dependent inhibition of TC71 cell proliferation by AVN944, and determination of the IC 50 . (A) Representative phase-contrast images of TC71 cells treated with varying concentrations of AVN944 (0-1 μM) for 3 days. Cells were imaged on Days 1, 2, and 3 post-treatment. AVN944 caused a concentration-dependent reduction in cell density, as well as changes in cell morphology, particularly at higher concentrations. Scale bar, 100 μm. (B) Dose-response curve for TC71 cells treated with AVN944 for 3 days. Cell viability was measured, and the half-maximal inhibitory concentration (IC 50 ) was calculated as 0.0535 μM, with an R² value of 0.998, indicating a solid fit of the data to the model. Data are presented as the mean ± SD of three independent experiments (n = 3).

Article Snippet: By contrast, the TC71 cell line was acquired from the Leibniz Institute DSMZ (Germany).

Techniques: Inhibition, Concentration Assay

Journal: International Journal of Biological Sciences

Article Title: AVN944 Elicits Apoptotic Responses and Impedes Tumorigenic Potential in Ewing's Sarcoma Cells

doi: 10.7150/ijbs.116651

Figure Lengend Snippet: Time-dependent induction of apoptosis of TC71 Ewing's Sarcoma cells by. AVN944. (A) Flow cytometry analysis of DNA content in PI-stained TC71 cells treated with 5 μM AVN944 for 0, 48, 72, and 96 h. The sub-G1 (M1), G1 (M2), S phase (M3), and G2/M phase (M4) populations are indicated, along with a time-dependent increase in the sub-G1 population, indicative of apoptotic cells. (B) Measurement of the sub-G1 population at 48, 72, and 96 h post-treatment with AVN944. Data are presented as the mean ± SD from three independent experiments (n = 3, ** p < 0.01). (C) Flow cytometry analysis of apoptosis (using Annexin V and PI staining) in TC71 cells treated with 5 μM AVN944 for the indicated times. Annexin V-positive cells exhibit early-stage apoptosis, while PI-positive/Annexin V-negative cells are necrotic. An apparent time-dependent increase in the percentage of Annexin V-positive cells indicates progression from early apoptosis at 48 h to advanced apoptosis by 72 and 96 h. (D) Quantification of Annexin V-positive apoptotic cells over time. A time-dependent increase in Annexin V-positive cells was observed, with significant elevations at 48, 72, and 96 h, indicating progression of apoptosis. By 96 h, more than 78% of TC71 cells were Annexin V-positive, confirming marked induction of apoptosis (** p < 0.01). Data are presented as the mean ± SD from three independent experiments (n=3). (E) Percentage of viable cells over time post-treatment with AVN944. Viable cell populations decreased significantly at 48, 72, and 96 h (** p < 0.01). Data are presented as the mean ± SD from three independent experiments (n=3).

Article Snippet: By contrast, the TC71 cell line was acquired from the Leibniz Institute DSMZ (Germany).

Techniques: Flow Cytometry, Staining

Journal: International Journal of Biological Sciences

Article Title: AVN944 Elicits Apoptotic Responses and Impedes Tumorigenic Potential in Ewing's Sarcoma Cells

doi: 10.7150/ijbs.116651

Figure Lengend Snippet: AVN944 modulates expression of cell cycle regulators and apoptosis-associated proteins in TC71 cells. (A) Western blot analysis of p53, Cyclin D1, Cyclin E, Bax, Bcl-2, and PARP1 (full-length and cleaved forms) expression in TC71 cells treated with 5 μM AVN944 for 0, 24, and 48 h. α/β-Tubulin was used as a loading control. AVN944 treatment led to a time-dependent increase in expression of p53, Bax, and cleaved PARP1, while there was a marked reduction in expression of Cyclin D1, Cyclin E, Bcl-2, and full-length PARP1. (B-H) Quantification of protein levels, normalized to α/β-Tubulin, using ImageJ software. Protein levels were measured using ImageJ software, normalized to α/β-tubulin, and further normalized to the 0-h time point for each condition. The resulting -fold changes relative to 0-h were annotated directly on the graphs to facilitate quantitative interpretation. The graphs depict relative levels of (B) p53, (C) Cyclin D1, (D) Cyclin E, (E) Bax, (F) Bcl-2, (G) full-length PARP1, and (H) cleaved PARP1. Data are presented as the mean ± SD of three independent experiments (n=3). Statistical significance is indicated (* p < 0.05 and ** p < 0.01).

Article Snippet: By contrast, the TC71 cell line was acquired from the Leibniz Institute DSMZ (Germany).

Techniques: Expressing, Western Blot, Control, Software

Journal: International Journal of Biological Sciences

Article Title: AVN944 Elicits Apoptotic Responses and Impedes Tumorigenic Potential in Ewing's Sarcoma Cells

doi: 10.7150/ijbs.116651

Figure Lengend Snippet: In vivo anti-tumor activity of AVN944 in a TC71 xenograft model . (A) The body weight of mice treated with AVN944 (50 mg/kg) or control was monitored over 10 days. There was no significant difference in body weight changes between the AVN944-treated and the control groups, suggestive of no major systemic toxicity. Data are presented as the mean ± SD (control: n = 6; AVN944: n = 9). (B) Volume of tumors in TC71 xenograft-bearing mice treated with AVN944 (50 mg/kg, n = 9) compared with that of tumors in mice treated with the control (n = 6). Tumor growth in the AVN944-treated group was inhibited significantly, and tumor volume remained significantly smaller throughout the experiment (** p < 0.01). (C) Representative images of tumors excised from control (n = 6) and AVN944-treated mice (n = 9) at the end of the experiment. Tumors from AVN944-treated mice were markedly smaller than those from control mice, providing visual confirmation of the significant inhibition of tumor growth observed in Figure B. Each tumor was measured and aligned for comparison, demonstrating a clear reduction in tumor mass after AVN944 treatment . (D) AVN944-induced reductions in tumor weight in TC71 xenografts. Tumor weight was measured post-excision; AVN944 treatment led to a significant reduction in tumor mass when compared with the control (** p < 0.01). Data are presented as the mean ± SD (control: n = 6; AVN944: n = 9).

Article Snippet: By contrast, the TC71 cell line was acquired from the Leibniz Institute DSMZ (Germany).

Techniques: In Vivo, Activity Assay, Control, Inhibition, Comparison

Journal: Cancer immunology research

Article Title: Engagement of CD99 Activates Distinct Programs in Ewing Sarcoma and Macrophages.

doi: 10.1158/2326-6066.CIR-23-0440

Figure Lengend Snippet: Figure 1. The human anti-CD99 dAbd C7 inhibits tumor growth and increases macrophages infiltration. A, Tumor volume after treatment with the anti-CD99 dAbd C7 in xenografts derived from injection of 6647 cells in nude mice. Control, n ¼ 10; dAbd C7, n ¼ 6 (left) or PDX-EW#2 tumors developed in NSG mice; control, n ¼ 4; dAbd C7, n ¼ 5 (right). Dots represent single tumor volumes, mean SD are displayed. Mann–Whitney U test, , P < 0.05. B, Representative H&E images of 6647 (left) or PDX-EW#2 (right) xenografts treated or not with dAbd C7 (scale bar, 50 mm). The tumor regions with nuclear condensation and the total area of the samples were identified by manually marking with Image J software. Histogram represents the percentage of tumor area with nuclear condensation (vs. total tumor area). Data are expressed as the median and range (minimum–maximum). Mann–Whitney U test, , P < 0.05. C, Representative images of double staining IHC for F4/80 murine macrophages (brown) and CD99 EWS cells (red) in 6647 (left) or PDX-EW#2 (right) xenografts after treatment with dAbd C7 (scale bar, 50 mm). The enlarged box shows a mouse macrophage with engulfed EWS cells; the histograms represent the percentage of F4/80-positive cells calculated after evaluation of at least ten fields. Data are expressed as the median and range (minimum–maximum. Mann–Whitney U test; , P < 0.001.

Article Snippet: In vitro cell cultures We used the following human patient-derived EWS cell lines: TC-71 (DSMZ, Catalog no. ACC-516, RRID: CVCL_2213) and 6647 (RRID: CVCL_H722) both kindly provided by T. J. Triche (Children’s Hospital, Los Angeles, CA); H-825 kindly provided by Prof. A. Llombart-Bosch (University of Valencia, Spain); and A673 cells (RRID: CVCL_0080) kindly provided by H. Kovar (St. Anna Kinderkrebsforschung).

Techniques: Derivative Assay, Injection, Control, MANN-WHITNEY, Software, Double Staining

Journal: Cancer immunology research

Article Title: Engagement of CD99 Activates Distinct Programs in Ewing Sarcoma and Macrophages.

doi: 10.1158/2326-6066.CIR-23-0440

Figure Lengend Snippet: Figure 2. Macrophage-mediated phagocytosis and cytotoxicity of EWS cells after anti-CD99 antibodies treatment. A, Phagocytic index of M0-like macrophages cocultured for 6 hours with TC-EGFP cells being exposed to anti-CD99 antibodies (12E7 mAb; 0662 mAb; dAbd C7 or to irrelevant MOPC21 antibody used as isotype control) for 30 minutes. Phagocytic index indicated the number of EWS cells phagocytosed per 100 macrophages. Dots represent single fields and data are expressed as the median and range (minimum–maximum) of at least three independent experiments. One-way ANOVA: , P < 0.05; , P < 0.01; , P < 0.001. B, Representative images of M0-like macrophages phagocytosing EWS cells after treatment with anti-CD99 antibodies. Arrows point to phagocytosed tumor cells (scale bar, 50 mm). C, EWS cell survival in the presence or not of M0-like macrophages (Mj) detected by Trypan blue vital counting. Data are expressed as percentage compared with untreated TC-71 cells. Bars represent the mean SD of at least three independent experiments. Mann–Whitney U test: , P < 0.05; , P < 0.01.

Article Snippet: In vitro cell cultures We used the following human patient-derived EWS cell lines: TC-71 (DSMZ, Catalog no. ACC-516, RRID: CVCL_2213) and 6647 (RRID: CVCL_H722) both kindly provided by T. J. Triche (Children’s Hospital, Los Angeles, CA); H-825 kindly provided by Prof. A. Llombart-Bosch (University of Valencia, Spain); and A673 cells (RRID: CVCL_0080) kindly provided by H. Kovar (St. Anna Kinderkrebsforschung).

Techniques: Control, MANN-WHITNEY

Journal: Cancer immunology research

Article Title: Engagement of CD99 Activates Distinct Programs in Ewing Sarcoma and Macrophages.

doi: 10.1158/2326-6066.CIR-23-0440

Figure Lengend Snippet: Figure 3. Modulation of ‘eat-me’ and ‘don’t eat-me’ molecules on EWS cell surface after CD99 engagement. A, Cytofluorimetric profile of PS and CALR expression in EWS cells treated with anti-CD99 antibodies (expressed as percentage of positive cells). B, MFI of CD47 expression in EWS cells treated with anti-CD99 antibodies. All cells are 100% positive to CD47 even after treatments. Bars indicated the mean SD of at least three independent experiments. One-way ANOVA: , P < 0.05; , P < 0.01; , P < 0.001. Gating strategy and representative profiles of each type of experiment (A–B), are reported in Supplementary Figs. S4 and S5. C, Confocal microscopy images of CD47 in TC-71 cells treated with 0662 mAb (10 mg/mL), indicating intracellular localization of CD47 (arrows). Enlarged views of the boxed regions are also displayed (scale bar, 50 mm, representative images of two independent experiments are shown). D, Western blot analysis of CD47 on total cell lysates from 6647 and TC-71 cells treated with anti-CD99 antibodies (0662 mAb; dAbd C7) in the presence or absence of the proteasome inhibitor MG132. Data are representative of at least three independent experiments. Beta-tubulin was included as loading control. E, Representative images of IHC staining of CD47 in 6647 (left) or PDX-EW#2 (right) xenografts after treatment with dAbd C7 (scale bar, 50 mm). Histograms represent the percentage of CD47 positive cells calculated after evaluation of at least ten fields. Data are presented as the median and range (minimum–maximum). Mann–Whitney U test: , P < 0.05; , P < 0.001.

Article Snippet: In vitro cell cultures We used the following human patient-derived EWS cell lines: TC-71 (DSMZ, Catalog no. ACC-516, RRID: CVCL_2213) and 6647 (RRID: CVCL_H722) both kindly provided by T. J. Triche (Children’s Hospital, Los Angeles, CA); H-825 kindly provided by Prof. A. Llombart-Bosch (University of Valencia, Spain); and A673 cells (RRID: CVCL_0080) kindly provided by H. Kovar (St. Anna Kinderkrebsforschung).

Techniques: Expressing, Confocal Microscopy, Western Blot, Control, Immunohistochemistry, MANN-WHITNEY

Journal: Cancer immunology research

Article Title: Engagement of CD99 Activates Distinct Programs in Ewing Sarcoma and Macrophages.

doi: 10.1158/2326-6066.CIR-23-0440

Figure Lengend Snippet: Figure 4. The presence of CD99 on EWS cell surface influences CD47 status. A,ExpressionofCD47inCD99-silenced cells. Bars indicate MFI. Mean SD of at least three independent experiments, Kruskal–Wallis test: , P < 0.05. B, Cytofluorimetric profile of CD47 expression in TC-CD99-shRNA#2 EWS cells treated with anti-CD99 antibodies. Bars indicate the mean SD of at least three independent experiments. Gating strategy and representative profiles of each type of experiment (A and B), are reported in Supplementary Fig. S7. C, Immunoprecipitation of CD99 with CD47 in 6647, TC-71, and TC-CD99- shRNA#2 EWS cells. Blots are repre- sentative of at least three independent experiments.

Article Snippet: In vitro cell cultures We used the following human patient-derived EWS cell lines: TC-71 (DSMZ, Catalog no. ACC-516, RRID: CVCL_2213) and 6647 (RRID: CVCL_H722) both kindly provided by T. J. Triche (Children’s Hospital, Los Angeles, CA); H-825 kindly provided by Prof. A. Llombart-Bosch (University of Valencia, Spain); and A673 cells (RRID: CVCL_0080) kindly provided by H. Kovar (St. Anna Kinderkrebsforschung).

Techniques: Expressing, shRNA, Immunoprecipitation