tc71 cell line  (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: iScience

Article Title: RNA helicase DDX3 regulates RAD51 localization and DNA damage repair in Ewing sarcoma

doi: 10.1016/j.isci.2024.108925

Figure Lengend Snippet: Inhibition of DDX3 radiosensitizes EWS (A) Immunofluorescent images of TC71 EWS cells at 1 or 24 h following 2 Gy IR that were treated with either DMSO (top) or 2 μM RK-33 (bottom). Green = γ-H2A.X detection, marking double-stranded DNA breaks (DSB); Red = DDX3; Blue = DAPI stain. Mag bar: 20 μm. (B) Quantitation of γ-H2A.X foci in stable genetically modified shDDX3 MHH-ES-1 cell lines 2D7 and 2C7 at 0 (i.e., no treatment), 6, and 24 h following 2 Gy IR. Data are representative of three independent experiments. Data are mean ± SD. ∗p < 0.05 and ∗∗∗p < 0.001 determined by two-Way ANOVA followed by Šídák’s multiple comparisons test. (C) Quantitation of γ-H2A.X foci in three independent EWS cell lines (TC71, MHH-ES-1, and TC32) where cells were irradiated with 2 Gy in the presence of 0, 0.5, 1, 2, or 4 μM RK-33. Data represent three independent experiments per cell line. Data are mean ± SD. ∗p < 0.05 and ∗∗∗p < 0.001 determined by two-Way ANOVA followed by Šídák’s multiple comparisons test. (D and E) TC71, MHH-ES-1, and A4573 EWS cells were treated with either DMSO, 2 Gy, 2 μM RK-33, or 2 μM RK-33 + 2 Gy and plated at densities of 200 or 400 cells/well 6 h post-IR. Cells were then grown in conditioned media for five days and stained with crystal violet for (D) visualization and (E) quantification of clonogenic survival fractions (n = 6 technical replicates per cohort per cell line). Results represent one experiment of three independent experiments per cell line. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 determined by one-way ANOVA followed by Šídák’s multiple comparisons test.

Article Snippet: Established EWS cell lines TC71 (RRID:CVCL_2213) and TC32 (RRID:CVCL_7151) were acquired from Children’s Cancer Respository ( https://cccells.org ), A4573 (RRID:CVCL_6245) was a kind gift from the laboratory of Katia Scotlandi, and MHH-ES-1 (ACC-167; RRID:CVCL_1411) was purchased from German Collection of Microorganisms and Cell Cultures (dsmz.de, Braunschweig, Germany).

Techniques: Inhibition, Staining, Quantitation Assay, Genetically Modified, Irradiation

Journal: iScience

Article Title: RNA helicase DDX3 regulates RAD51 localization and DNA damage repair in Ewing sarcoma

doi: 10.1016/j.isci.2024.108925

Figure Lengend Snippet: DDX3 interacts with components of the homologous DDR pathway (A) Western blots of DR-U2OS and EJ5-U2OS cell lysates following siRNA knockdown of DDX3. (B) DR-U2OS, EJ5-U2OS, and SSA-U2OS cell lines were treated with either scramble or siDDX3 and then transfected with the I Sce I-pCAGGS vector or an empty vector to induce DNA damage. Effective DDR was visualized by induction of GFP expression and quantified using flow cytometry. Results represent three independent experiments per cell line. Data represent frequency of DNA recombination events ±SEM. ∗∗∗p < 0.001 determined by multiple unpaired t tests followed by Šídák’s multiple comparisons test. I, induction of DNA damage with I Sce I-pCAGGS; UI, non-induction with empty pCAGGS vector; HR, homologous recombination; NHEJ, non-homologous end-joining; SSA, single-strand annealing. (C) Immunoprecipitation (IP) of TC71 EWS cell lysates using anti-DDX3 antibodies conjugated to magnetic beads. Western blots demonstrate co-immunoprecipitation of various DDR proteins with endogenous DDX3. Iso IgG, immunoprecipitation of TC71 cell lysates using control isotype antibodies. Results are representative of three independent experiments. See also Figure S2 . (D) Subcellular fractionations of TC71 cells demonstrate the presence of DDX3, RAD51, RECQL1, RPA32, and XRCC2 in both cytoplasmic and nuclear compartments. Results are representative of three independent experiments. See also Figure S2 . CL, whole-cell lysate. (E) Immunofluorescent images of TC71 EWS cells 6 h after treatment with 5 μM RK-33 and 2 Gy IR. Green = γ-H2A.X staining of double-stranded DNA breaks (DSB); Red = DDX3; Blue = DAPI stain. Mag bar: 20 μm.

Article Snippet: Established EWS cell lines TC71 (RRID:CVCL_2213) and TC32 (RRID:CVCL_7151) were acquired from Children’s Cancer Respository ( https://cccells.org ), A4573 (RRID:CVCL_6245) was a kind gift from the laboratory of Katia Scotlandi, and MHH-ES-1 (ACC-167; RRID:CVCL_1411) was purchased from German Collection of Microorganisms and Cell Cultures (dsmz.de, Braunschweig, Germany).

Techniques: Western Blot, Knockdown, Transfection, Plasmid Preparation, Expressing, Flow Cytometry, Homologous Recombination, Non-Homologous End Joining, Immunoprecipitation, Magnetic Beads, Control, Staining

Journal: iScience

Article Title: RNA helicase DDX3 regulates RAD51 localization and DNA damage repair in Ewing sarcoma

doi: 10.1016/j.isci.2024.108925

Figure Lengend Snippet: DDX3 interacts with and modulates cytoplasmic oligonucleotide substrates in EWS (A–C) DAPI staining of dsDNA in three independent EWS cell lines (A), three independent xenografts (B), and three independent patient samples (C). White arrows show examples of extra-nuclear dsDNA substrates. Mag bars: 10 μm. (D) Immunofluorescent images of TC71 EWS cells. Data are representative of two independent experiments. Green = single-stranded DNA (ssDNA); Red = RNA:DNA hybrid structures; Blue = DAPI stain. Mag bar: 24 μm. (E) Representative images of S9.6 staining of umbilical-cord-derived human mesenchymal stem cells (MSC), TC71, and A4573 EWS cell lines. Mag bar: 10 μm. (F and G) Three independent EWS cell lines, TC71, A4573, and MHH-ES-1, were transduced with lentivirus overexpressing either RNaseH1 WT or enzymatically dead RNaseH1 D210N for 48 h. Cells were then stained, and immunofluorescent (IF) confocal images were obtained (F) and basal RNA:DNA hybrid abundance quantified (G). Results are representative of four to five high power field (hpf) per condition from two independent experiments per cell line. Data represent mean fluorescent intensity per cell per hpf +/− SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 determined by unpaired t tests. Red = HA-tag; Green = RNA:DNA hybrids (S9.6 staining); Blue = DAPI stain. Mag bar: 10 μm. (H) TC71 and A4573 EWS cell lines were transduced with either RNaseH1 WT or enzymatically dead RNaseH1 D210N for 48 h. Cells were then stained with J2 to visualize dsRNA. Representative IF confocal images are shown. Mag bar: 10 μm. (I and J) Representative IF images of TC71 cells at 1 and 3 h post-2 Gy IR (I). Colocalization of DDX3 and RNA:DNA hybrid fluorescence was analyzed and quantified using Pearson’s coefficient. Data are representative of five high power fields (hpf) per cohort from one of three independent experiments (J). Data are mean ± SEM. ∗∗∗∗p < 0.0001 determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. Green = DDX3; Red = RNA:DNA hybrid structures; Blue = DAPI stain. Mag bar: 24 μm. (K and L) TC71 and A4573 cells were treated with either vehicle control (DMSO) or 2 μM RK-33. Immunofluorescent z stack confocal images were obtained (K). TC71 images were measured for volume of RNA:DNA hybrid structures per cell (L). Results are representative of five hpf per condition from two independent experiments. Data represent mean fluorescent intensity per cell +/− SEM. ∗∗∗p < 0.001 determined by unpaired t test. Mag bar: 10 μm. (M) Representative double-stained IF images analyzing RNA:DNA hybrid and double-stranded RNA (dsRNA) distribution with a rabbit-S9.6 and J2 antibodies, respectively, in TC71 and A4573 EWS cell lines treated with either DMSO or 2 μM RK-33. Mag bar: 10 μm.

Article Snippet: Established EWS cell lines TC71 (RRID:CVCL_2213) and TC32 (RRID:CVCL_7151) were acquired from Children’s Cancer Respository ( https://cccells.org ), A4573 (RRID:CVCL_6245) was a kind gift from the laboratory of Katia Scotlandi, and MHH-ES-1 (ACC-167; RRID:CVCL_1411) was purchased from German Collection of Microorganisms and Cell Cultures (dsmz.de, Braunschweig, Germany).

Techniques: Staining, Derivative Assay, Transduction, Fluorescence, Control

Journal: iScience

Article Title: RNA helicase DDX3 regulates RAD51 localization and DNA damage repair in Ewing sarcoma

doi: 10.1016/j.isci.2024.108925

Figure Lengend Snippet: Inhibition of DDX3 helicase activity sequesters RAD51 in the cytoplasm in an RNA:DNA-hybrid-dependent manner following IR (A) Representative immunofluorescent images of two EWS cell lines, TC71 and A4573, demonstrating cytoplasmic colocalization of endogenous RAD51 (green) and DDX3 (red) at basal levels. Data are representative of three independent experiments. Blue = DAPI stain. Mag bar: 10 μm. (B) Representative immunofluorescent images of TC71 cells demonstrating colocalization of RAD51 (green) with DSBs, evidenced by γ-H2A.X (purple) staining, 3 h following 2 Gy IR. Data are representative of two independent experiments. Red = DDX3; Blue = DAPI stain. Mag bar: 10 μm. (C) Immunofluorescent images of TC71 cells at 3 h following treatment with either DMSO (top), 2 μm RK-33 (second row), DMSO +2 Gy IR (third row), or 2 μM RK-33 + 2 Gy (bottom). Data are representative of three independent experiments. Green = DDX3; Red = RAD51; Cyan = RNA:DNA hybrids; Blue = DAPI stain. Mag bar: 20 μm. (D) Three independent EWS cell lines, TC71, A4573, and MHH-ES-1, were transduced with lentivirus overexpressing either RNaseH1 WT or enzymatically dead RNaseH1 D210N for 48 h prior to performing radiosensitization assays. Cells were stained and immunofluorescent, z-stacked confocal images were obtained. RAD51 foci were quantified for each experimental cohort. Results represent nuclear RAD51 foci as a percentage of total RAD51 foci from all Z-planes of 4–5 representative hpf from each experimental cohort of each cell line. Data represent one of three independent experiments. Data are mean ± SEM. ∗p < 0.05 and ∗∗∗p < 0.001 determined by two-way ANOVA followed by Šídák’s multiple comparisons test.

Article Snippet: Established EWS cell lines TC71 (RRID:CVCL_2213) and TC32 (RRID:CVCL_7151) were acquired from Children’s Cancer Respository ( https://cccells.org ), A4573 (RRID:CVCL_6245) was a kind gift from the laboratory of Katia Scotlandi, and MHH-ES-1 (ACC-167; RRID:CVCL_1411) was purchased from German Collection of Microorganisms and Cell Cultures (dsmz.de, Braunschweig, Germany).

Techniques: Inhibition, Activity Assay, Staining, Transduction

Journal: iScience

Article Title: RNA helicase DDX3 regulates RAD51 localization and DNA damage repair in Ewing sarcoma

doi: 10.1016/j.isci.2024.108925

Figure Lengend Snippet:

Article Snippet: Established EWS cell lines TC71 (RRID:CVCL_2213) and TC32 (RRID:CVCL_7151) were acquired from Children’s Cancer Respository ( https://cccells.org ), A4573 (RRID:CVCL_6245) was a kind gift from the laboratory of Katia Scotlandi, and MHH-ES-1 (ACC-167; RRID:CVCL_1411) was purchased from German Collection of Microorganisms and Cell Cultures (dsmz.de, Braunschweig, Germany).

Techniques: Control, Plasmid Preparation, Microarray, Recombinant, Transfection, Magnetic Beads, DC Protein Assay, Extraction, Polymer, Software

Journal: Frontiers in Cell and Developmental Biology

Article Title: A 3D Platform to Investigate Dynamic Cell-to-Cell Interactions Between Tumor Cells and Mesenchymal Progenitors

doi: 10.3389/fcell.2021.767253

Figure Lengend Snippet: ddPCR assays are able to simultaneously detect tumor cells and gene-modified MSCs. (A) , A 3-plex ddPCR assay was developed to quantitively evaluate tumor cells and MSCs in the VITVO50. Custom primers and probes were designed on Luc and GFP target genes to identify tumor cells and MSCs, respectively. hRPP30 was used as reference gene to estimate the total number of human cells. Due to the DNA distribution within the droplets, in the 3-plex ddPCR assay droplets were sorted out in eight different colored clusters. (B) and (C) , 4-plex ddPCR assays were set up to simultaneously investigate MSC biodistribution and antitumor effect in the ES metastatic model. gDNA was obtained from lungs and liver of mice. Because of the random distribution of DNA into the droplets, in 4-plex ddPCR assays droplets are clustered into sixteen different colored groups. The control 4-plex ddPCR assay (B) was developed combining Luc and GFP target assays with hRPP30 and mTfrc reference assays to detect human cells, TC71 Luc cells and control MSCs, localized in mice organs. The sTRAIL 4-plex ddPCR assay (C) was set up matching Luc and sTRAIL target assays with the mTfrc and hRPP30 reference assays to distinguish between TC71 Luc tumor cells and effector MSCs in mice organs.

Article Snippet: The human ES cell line TC71 (RRID: CVCL_2,213, DSMZ, Braunschweig, Germany) was cultivated in Iscove’s modified Dulbecco’s medium (IMDM, Euroclone, Milan, Italy) supplemented with 10% FBS (Carlo Erba Reagents Srl, Cornaredo, Italy), 1% L-glutamine (200 mM; BioWhittaker, Lonza, Verviers, Belgium), and 1% penicillin-streptomycin (104 UI/ml penicillin and 10 mg streptomycin/ml; Carlo Erba Reagents Srl).

Techniques: Modification, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: A 3D Platform to Investigate Dynamic Cell-to-Cell Interactions Between Tumor Cells and Mesenchymal Progenitors

doi: 10.3389/fcell.2021.767253

Figure Lengend Snippet: TC71 cell line and MSCs effectively express reporter genes. (A) , Gating strategy for the analysis of the GFP expression on MSCs transduced with GFP-encoding pCCL PGK WPRE lentiviral vector and sorted by FACS. Gate 1: forward scatter (FSC) and side scatter (SSC) area was used to enrich for intact cells (P1). Gate 2: GFP fluorescence recorded in the logarithmic scale was used to identify MSC GFP+ (P2; 93.9 ± 0.4%). (B) , Representative picture of GFP-expressing MSCs by fluorescence microscopy (original magnification ×100). (C) , Gating strategy for the analysis of the DsRed expression on TC71 Luc cells transduced with DsRed-encoding MIGR1 vector. Gate 1: FSC and SSC area was used to enrich for intact cells (P1). Gate 2: DsRed fluorescence assessed in the logarithmic scale allowed the identification of DsRed-expressing TC71 Luc cells (P2; 98.5 ± 0.7%). (D) , Representative picture of DsRed-expressing TC71 Luc cells by fluorescence microscopy (original magnification ×100).

Article Snippet: The human ES cell line TC71 (RRID: CVCL_2,213, DSMZ, Braunschweig, Germany) was cultivated in Iscove’s modified Dulbecco’s medium (IMDM, Euroclone, Milan, Italy) supplemented with 10% FBS (Carlo Erba Reagents Srl, Cornaredo, Italy), 1% L-glutamine (200 mM; BioWhittaker, Lonza, Verviers, Belgium), and 1% penicillin-streptomycin (104 UI/ml penicillin and 10 mg streptomycin/ml; Carlo Erba Reagents Srl).

Techniques: Expressing, Transduction, Plasmid Preparation, Fluorescence, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: A 3D Platform to Investigate Dynamic Cell-to-Cell Interactions Between Tumor Cells and Mesenchymal Progenitors

doi: 10.3389/fcell.2021.767253

Figure Lengend Snippet: The VITVO50-based fluidic circuit allows to investigate dynamic cell-to-cell interactions between tumor nodules and MSCs. (A) , Scan of nine representative fields of the VITVO50 matrix colonized by TC71 Luc cells (red; Objective 4×). (B) , Representative picture taken at a higher magnification showing the formation of in vivo-like nodules of tumor cells in the VITVO50 matrix (red cell aggregates; Objective 10×) (C) Mean (SD) number of tumor cells colonizing VITVO50 bioreactor derived from escape data. Numbers of independent experiments n = 3. (D) , Bioluminescent signal after luciferin administration was used to quantify the viability of TC71 Luc cells seeded on the matrix. Data are expressed as the mean (SD). Numbers of independent experiments n = 3. (E) , Scan of nine representative fields of the VITVO50 matrix to evaluate interaction between TC71 Luc cells (red) and MSCs (green; Objective 4×). (F) , Representative picture taken at a higher magnification showing MSCs trapped between the fibers of VITVO50 matrix and interacting with tumor nodules (Objective 10×). (G) , Mean (SD) of the percentage of tumor cells (red) and MSCs (green) within the VITVO50 matrix obtained using the 3-plex ddPCR assay. Numbers of independent experiments n = 4. (H) , Ratio between the number of MSCs and tumor cells on VITVO50 matrix expressed as mean (SD). Numbers of independent experiments n = 4.

Article Snippet: The human ES cell line TC71 (RRID: CVCL_2,213, DSMZ, Braunschweig, Germany) was cultivated in Iscove’s modified Dulbecco’s medium (IMDM, Euroclone, Milan, Italy) supplemented with 10% FBS (Carlo Erba Reagents Srl, Cornaredo, Italy), 1% L-glutamine (200 mM; BioWhittaker, Lonza, Verviers, Belgium), and 1% penicillin-streptomycin (104 UI/ml penicillin and 10 mg streptomycin/ml; Carlo Erba Reagents Srl).

Techniques: In Vivo, Derivative Assay

Journal: Cancer cell

Article Title: STAG2 mutations alter CTCF-anchored loop extrusion, reduce cis-regulatory interactions and EWSR1-FLI1 activity in Ewing sarcoma.

doi: 10.1016/j.ccell.2021.04.001

Figure Lengend Snippet: Figure 1. STAG2 knockout profoundly alters the transcriptomic landscape (A) Representative western blotting in cellular extracts from isogenic STAG2 knockout (KO) (generated with two independent sgRNAs: SA2m#1 and SA2m#2), STAG1 KO (sgRNA: SA1m#1), STAG2 rescued (sgRNA: SA2r) and STAG2 knockdown (KD) at 48 h (generated with two independent siRNAs: siSA2#6 and siSA2#8) Ewing sarcoma cells. Color code for sgRNA isogenic models is indicated for each model and kept identical throughout the article. (B) Scaled Venn diagram for modulated genes between STAG2 WT and KO conditions (n = 3); total modulated genes for each condition represent the sum of intra- circle numbers; universe includes expressed genes (n = 13,780). p value for intersection was calculated with the SuperExact test. (C) Boxplots of log2 fold change for up-, un-, and downregulated genes in STAG2 KO and STAG2 rescued cells as compared with A673 or TC71 parental cells (n = 3 for each model); number of genes is indicated for each category. p values: two-tailed paired Wilcoxon test. Boxes represent the central 50% of data points (interquartile range). Upper and lower whiskers represent the largest and smallest observed values within 1.5 times the interquartile range from the ends of the box. (D) Heatmap for the core set of commonly upregulated (left panel) and downregulated (right panel) genes identified in (B and C) for STAG2 KO and KD Ewing cell lines. Time after siRNA transfection is indicated at the top, sgRNA and siRNA identifiers at the bottom. See also Figures S1 and S2, Table S1.

Article Snippet: The Ewing sarcoma A673 cell line was obtained from the American Type Culture Collection (ATCC) and the Ewing sarcoma TC71 cell line was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ).

Techniques: Knock-Out, Western Blot, Generated, Knockdown, Two Tailed Test, Transfection

Journal: Cancer cell

Article Title: STAG2 mutations alter CTCF-anchored loop extrusion, reduce cis-regulatory interactions and EWSR1-FLI1 activity in Ewing sarcoma.

doi: 10.1016/j.ccell.2021.04.001

Figure Lengend Snippet: Figure 5. STAG2 mutation is globally associated with decreased cis-promoter-enhancer and enhancer-enhancer interactions within loops (A–D) Color-coded boxplots of comparative analysis of (A–B) H3K27ac ChIP-seq peak intensities and (C–D) H3K27ac HiChIP interactions along promoter- enhancer chains between STAG2 WT and KO as well as between STAG2 KO and rescued cells. p values: two-tailed Wilcoxon test. P (promoter) and E (enhancer) positions in the chain are shown for ranks 1 to 5. (E) Color-coded boxplots of comparative analysis of intra super-enhancer interactions in H3K27ac HiChIP data between STAG2 WT and KO or rescued cells. p values: two-tailed t test. (A–E) Boxes represent the central 50% of data points (interquartile range). Upper and lower whiskers represent the largest and smallest observed values within 1.5 times the interquartile range from the ends of the box. The n value for each condition is indicated. (F) Top, curve of cumulative percentage of loop presence upon genomic distance (log10 scale) in A673 and TC71 (STAG2 WT). Loop size threshold: 75% of cumulative loops corresponding to 595 kb for A673 (red line) and 340 kb (green line) for TC71. Numbers of loop and median loop size for each cell line is indicated. Bottom, percentage of cis-interaction read pairs upon genomic distance between STAG2 WT, KO, and rescued conditions in CTCF and H3K27ac HiChIP data. A threshold of 20 kb used for H3K27ac chain detection is displayed (blue dashed line). Right, zoom in CTCF and H3K27ac HiChIP plots flanking lower and top thresholds (highlighted in gray). See also Figures S7 and S8, Tables S4, S5, and S6.

Article Snippet: The Ewing sarcoma A673 cell line was obtained from the American Type Culture Collection (ATCC) and the Ewing sarcoma TC71 cell line was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ).

Techniques: Mutagenesis, ChIP-sequencing, HiChIP, Two Tailed Test

Journal: Translational Oncology

Article Title: Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma

doi: 10.1016/j.tranon.2021.101240

Figure Lengend Snippet: BF MSCs establish strong and stable connections with GD2-expressing ES cells. GD2 tCAR-mediated binding of MSCs to ES cell lines was investigated using cell-to-cell interaction assays. a-c , The number of MSC-ES cell aggregates, reported as the fold change versus EV MSCs, for all three ES cell lines. For TC71 * p < .001, ° p < .001; for A673 * p < .05, ° p < .001. d , The stability of the GD2 tCAR-mediated binding was examined by comparing the interactions of EV and GD2 tCAR MSCs with the TC71 ES line. MSC-TC71 aggregates were maintained at 4 °C on a rotating support for 2 and 4 h, and the number of aggregates was quantified by FACS. The number of MSC-TC71 aggregates at 2 and 4 h is reported as the fold change relative to the respective baseline number at the time of detachment (T0). * p < .05. All-p values were calculated by Student's t -test. Data are expressed as the mean (SD). Numbers of independent experiments n = 2, each with three technical replicates.

Article Snippet: The human ES cell lines TC71 (RRID: CVCL_2213, DSMZ, Braunschweig, Germany) and A673 (kind gift from Dr. Nicola Baldini, Rizzoli Orthopaedic Institute, Bologna, Italy) were cultivated in Iscove's modified Dulbecco's medium (IMDM, Euroclone, Milan, Italy).

Techniques: Expressing, Binding Assay

Journal: Translational Oncology

Article Title: Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma

doi: 10.1016/j.tranon.2021.101240

Figure Lengend Snippet: BF MSCs and their conditioned supernatants exert in vitro cytotoxic effects against targeted ES cell lines. The in vitro impact of BF MSCs against the ES cell lines a , TC71 b , A673 and c , RD-ES was examined by co-cultures using multiple target:effector ratios (1:1; 1:2, and 1:5). Recombinant human TRAIL (rhTRAIL, 1 µg/ml) was used as a positive control, whereas tumour cells cultured alone were used as the negative control (CTR). Tumour cell death was examined by supravital propidium iodide (PI) after 24 h. Reported p-values represent the results of multiple comparisons between sTRAIL and BF MSC conditions and control groups, represented by EV MSCs, GD2 tCAR MSCs, rhTRAIL, or CTR. For TC71 * p < .001, ° p < .01, § p < .01; for A172 * p < .001, ° p < .001, § p < .001; for RD-ES * p < .001, ° p < .001, § p < .001. sTRAIL-mediated cytotoxicity against d , TC71 e , A673 and f , RD-ES cell lines. Tumour cells were incubated for 24 h with sTRAIL-containing supernatants (SNs) collected from sTRAIL and BF MSCs. SNs deriving from EV and GD2 tCAR MSCs were used as controls. ES cells in normal culture media (CTR) or treated with rhTRAIL (1 µg/ml) were evaluated for comparison. After 24 h, tumour cell death was assessed by supravital PI. For TC71 * p < .05, ° p < .001, § p < .05; for A673 * p < .001, ° p < .001, § p < .001; for RD-ES * p < .01, ° p < .001, § p < .001. All-p values have been calculated by Student's t -test. Data are expressed as the mean (SD). Numbers of independent experiments n = 2, each with three technical replicates.

Article Snippet: The human ES cell lines TC71 (RRID: CVCL_2213, DSMZ, Braunschweig, Germany) and A673 (kind gift from Dr. Nicola Baldini, Rizzoli Orthopaedic Institute, Bologna, Italy) were cultivated in Iscove's modified Dulbecco's medium (IMDM, Euroclone, Milan, Italy).

Techniques: In Vitro, Recombinant, Positive Control, Cell Culture, Negative Control, Control, Incubation, Comparison

Journal: Translational Oncology

Article Title: Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma

doi: 10.1016/j.tranon.2021.101240

Figure Lengend Snippet: BF MSCs display potent antitumour activities against ES spheroid models. a , Tumour spheroids were established using DsRed-expressing TC71 and A673 cells (red) and treated with MSCs labelled with CFSE (green). Tumour spheroids were monitored for MSC infiltration and cytotoxicity for up to 48 h of co-culture by fluorescence microscopy and frozen sections at deep levels obtained by cryostat serial cutting. Representative images of the TC71 cell line are shown (magnification 50x, columns 1–3, and 100x, columns 4 and 5). The 15-hour time point was identified as the optimal time to quantify the antitumour effects of BF MSCs on TC71 and A673 spheroids, in terms of both reduced cell viability ( b and d ) and caspase-8 activation ( c and e ) by luminescence-based assays. For cell viability assay, TC71 * p < .001, ° p < .01, § p < .01; A673 * p < .001, ° p < .01, § p < .01. For caspase-8 activation assay TC71 * p < .01, ° p < .001, § p < .001; A673 * p < .01, ° p < .001, § p < .001. f and g , A 3D fibre-based matrix was employed to better model the tumour architecture. Bioluminescent signal after luciferin administration was used to quantify the viability of TC71 cells seeded on the matrix. The killing capacity of BF MSCs in co-culture with TC71 Luc (T:E ratio of 3:1) was assessed in a time-course experiment ( f , * p < .01, ° p < .05) and an endpoint assay after 4 days ( g , * p < .01, ° p < .01). The BF MSC cytotoxic effect was compared to those of rhTRAIL alone (1 µg/ml) and MSCs expressing sTRAIL only. EV MSCs, GD2 tCAR MSCs, and TC71 cells alone (CTR) were used as negative controls. The basal bioluminescence of the 3D matrix loaded by TC71 Luc cells was assessed immediately before MSC seeding or rhTRAIL treatment (T0). All p-values were calculated by Student's t -test. Data are expressed as the mean (SD). Numbers of independent experiments n = 2, each with three technical replicates.

Article Snippet: The human ES cell lines TC71 (RRID: CVCL_2213, DSMZ, Braunschweig, Germany) and A673 (kind gift from Dr. Nicola Baldini, Rizzoli Orthopaedic Institute, Bologna, Italy) were cultivated in Iscove's modified Dulbecco's medium (IMDM, Euroclone, Milan, Italy).

Techniques: Expressing, Co-Culture Assay, Fluorescence, Microscopy, Activation Assay, Viability Assay, End Point Assay

Journal: Translational Oncology

Article Title: Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma

doi: 10.1016/j.tranon.2021.101240

Figure Lengend Snippet: Establishment of an in vivo model that closely mimics metastatic ES. Two million TC71 Luc cells were intravenously injected into NSG mice. TC71 Luc cell engraftment was assessed by monitoring in vivo bioluminescence using the IVIS system. a , Ten minutes after TC71 Luc cell injection, the luminescence signal accumulated in the lung. b , Over the next few hours, the bioluminescence gradually disappeared as the cells dispersed and re-emerged 10 days later at various locations where tumours developed. The most common engraftment sites were the lungs, liver, and femur. c , At sacrifice, on day 13, metastases were quantified in extracted organs by IVIS. d and e , Tumour metastases (black arrows) were confirmed by H&E staining on lung ( d ) and liver ( e ) sections (magnification 100x).

Article Snippet: The human ES cell lines TC71 (RRID: CVCL_2213, DSMZ, Braunschweig, Germany) and A673 (kind gift from Dr. Nicola Baldini, Rizzoli Orthopaedic Institute, Bologna, Italy) were cultivated in Iscove's modified Dulbecco's medium (IMDM, Euroclone, Milan, Italy).

Techniques: In Vivo, Injection, Staining

Journal: Translational Oncology

Article Title: Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma

doi: 10.1016/j.tranon.2021.101240

Figure Lengend Snippet: BF MSCs are able to reduce lung metastases in the ES metastatic model. The BF MSC antitumour potential was examined in a metastatic model of ES. a , Representative therapeutic schedule: TC71 Luc cells (two million) were intravenously (i.v.) injected into NSG mice ( n = 62). Starting four days after TC71 Luc cell injection, the animals were randomly divided into five groups for treatment: control group ( n = 22) received no treatment (CTR); the EV MSC group ( n = 10), GD2 tCAR MSC group ( n = 10), sTRAIL MSC group ( n = 10), and BF MSC group ( n = 10) received multiple ( n = 3) i.v. injections of one million of the respective gene-modified MSCs, which were administered every three days. Before the infusion, the third dose of gene-modified MSCs was labelled by DiR dye (8 µM; in red). After 13 days, the animals were sacrificed. Lungs and liver were extracted, maintained on dry ice, and stored at −80 °C. 4-plex ddPCR assays were performed on organ-derived gDNA to simultaneously detect the presence of various cell types. b and c , Ratios of TC71 Luc cells per µl to the total number of cells per µl were calculated for lungs and liver. For each mouse group, the median (interquartile range; IQR) values were derived and multiplied by 1000, and groups were compared in terms of metastatic growth in lungs ( b ) and liver ( c ). For the lungs, sTRAIL MSCs vs CTR, EV MSCs or GD2 tCAR MSCs * p < .05, BF MSCs vs CTR, EV MSCs or GD2 tCAR MSCs ° p < .05. All p-values were calculated by the Wilcoxon–Mann–Whitney test.

Article Snippet: The human ES cell lines TC71 (RRID: CVCL_2213, DSMZ, Braunschweig, Germany) and A673 (kind gift from Dr. Nicola Baldini, Rizzoli Orthopaedic Institute, Bologna, Italy) were cultivated in Iscove's modified Dulbecco's medium (IMDM, Euroclone, Milan, Italy).

Techniques: Injection, Control, Modification, Derivative Assay, MANN-WHITNEY