human ewing sarcoma ews cell lines  (ATCC)

Journal: American Journal of Cancer Research

Article Title: Acid microenvironment promotes cell survival of human bone sarcoma through the activation of cIAP proteins and NF-κB pathway

doi:

Figure Lengend Snippet: Pharmacological inhibition of the proton pump V-ATPase causes tumor necrosis in a Ewing’s sarcoma xenograft model. A. A673 cells were subcutaneously injected in NOD/SCID mice. Mice were then treated with different concentration of omeprazole, a V-ATPase inhibitor (0 mg/kg n = 14, 2.5 mg/kg n = 7, 10 mg/kg n = 14), to reduce intratumoral acidosis. H&E staining of tumor sections, representative images of the necrotic area, scale bar, 100 µm; B. Box plot of the tumor volume. Mean ± SE; C. Box plot of the tumor necrosis. Mean ± SE (*P < 0.05, **P < 0.01).

Article Snippet: HOS, Saos2, MG-63 (osteosarcoma), and A673 (Ewing sarcoma) human cell lines were purchased from the American Type Culture Collection, cultured in IMDM plus 20 unit/mL penicillin, 100 μg/mL streptomycin, and 10% FBS at pH 7.4, and incubated at 37°C in a humidified 5% CO 2 atmosphere.

Techniques: Inhibition, Injection, Concentration Assay, Staining

Journal: American Journal of Cancer Research

Article Title: Acid microenvironment promotes cell survival of human bone sarcoma through the activation of cIAP proteins and NF-κB pathway

doi:

Figure Lengend Snippet: Extracellular acidosis promotes cell survival in bone sarcoma via the NF-κB pathway. (A) Heat map representation of the fold increase of the expression of apoptosis and stress related-genes of osteosarcoma cells (MG63, HOS, Saos-2) after short-term acidosis. mRNA were analyzed by deep-sequencing after that cells were cultured at pH 6.5 compared to physiological medium (pH 7.4) for 24 h. Colors on the heat map indicate the log2 ratios of expression (representing normalized read counts). Red, upregulation; green, downregulation. (B) Cells were cultured under different pH (6.5 and 7.4) for 24 h as described in (A), and NF-κB mRNA expression and activation were quantified in cell lysates. (C) NF-κB1 (p50) and RelB nuclear protein concentration of osteosarcoma and Ewing sarcoma cells. Mean ± SE (n = 3 for HOS and MG63, and n = 5 for A673, *P < 0.05). (D) Signaling pathways of the tumor necrosis factor receptor (TNFR) family that mediates both survival and apoptotic pathways. The draw represents the pathways and adaptor molecules that are involved in the signaling that follows the acid stress: the NF-κB activation pathway via Akt (1), via the canonical pathway (2), via the non-canonical pathway (3), the activation of the AP1 complex transcriptional factor (4), and the TRAF-mediated death signaling with the activation of caspase cascade (5).

Article Snippet: HOS, Saos2, MG-63 (osteosarcoma), and A673 (Ewing sarcoma) human cell lines were purchased from the American Type Culture Collection, cultured in IMDM plus 20 unit/mL penicillin, 100 μg/mL streptomycin, and 10% FBS at pH 7.4, and incubated at 37°C in a humidified 5% CO 2 atmosphere.

Techniques: Expressing, Sequencing, Cell Culture, Activation Assay, Protein Concentration, Protein-Protein interactions

Journal: American Journal of Cancer Research

Article Title: Acid microenvironment promotes cell survival of human bone sarcoma through the activation of cIAP proteins and NF-κB pathway

doi:

Figure Lengend Snippet: Targeting of cIAP protein or of TRAF1 inhibit cell viability of A673 cells when cultured in acid condition. A. Inhibition of cell viability as verified by cell counting after the treatment with LCL161 (25 mM). The inhibitor was more effective at pH 6.5 than at pH 7.4. Mean ± SE (n = 3, *P < 0.05); B. Induction of apoptosis as verified by the counting of apoptotic bodies after the treatment with LCL161 (25 mM) of cells cultured at low pH (6.5). Mean ± SE (n = 3, *P < 0.05); C. mRNA analysis of TRAF1 expression by Q-RT-PCR after the treatment with specific siRNA at low pH (pH 6.5); D. Inhibition of cell viability as verified by acid phosphatase indirect assay after the treatment with specific siRNA at low pH (pH 6.5). Mean ± SE (n = 19, ****P < 0.001); E. Correlation of mRNA level between different proteins of the TRAF-cIAP destruction complex in Ewing sarcoma xenografts with acid-related markers V-ATPase V1B2 or LAMP2 (n = 6, *P < 0.05, **P < 0.01).

Article Snippet: HOS, Saos2, MG-63 (osteosarcoma), and A673 (Ewing sarcoma) human cell lines were purchased from the American Type Culture Collection, cultured in IMDM plus 20 unit/mL penicillin, 100 μg/mL streptomycin, and 10% FBS at pH 7.4, and incubated at 37°C in a humidified 5% CO 2 atmosphere.

Techniques: Cell Culture, Inhibition, Cell Counting, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular Therapy Oncolytics

Article Title: Myelolytic Treatments Enhance Oncolytic Herpes Virotherapy in Models of Ewing Sarcoma by Modulating the Immune Microenvironment

doi: 10.1016/j.omto.2018.10.001

Figure Lengend Snippet: Clodrosome Depletion of Macrophages Enhances OV Efficacy (A) Treatment regimen of A673 tumor xenograft-bearing mice given 400 μg of liposome or Clodrosome intravenously (i.v.) on day −3 and intraperitoneally (i.p.) on days −2, −1, and 0 followed by weekly i.p. treatments. Oncolytic herpes simplex virus rRp450 was given at 10 7 pfu intratumorally (i.tu.) on days 0 and 2. (B) A673 flank tumors treated with the above regimen were measured until tumors reached an endpoint volume of 2,000 mm 3 . Both tumor growth curves with SEMs and Kaplan-Meier plots of survival are shown. Error bars indicate SEM. (C) Virus replication in A673 xenografts treated with the above Clodrosome regimen and a single 10 7 pfu dose of rRp450 given intratumorally (i.t.) at the time points indicated prior to tumor homogenization and plaque assay. Statistical analyses of survival curves were performed using Mantel-Cox test, and bar graphs were done using unpaired multiple two-tailed t test; *p < 0.05, **p < 0.005, ***p < 0.0005. Error bars indicate SD.

Article Snippet: The A673 human Ewing sarcoma cell line and Vero green monkey kidney cell line were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Virus, Homogenization, Plaque Assay, Two Tailed Test

Journal: Molecular Therapy Oncolytics

Article Title: Myelolytic Treatments Enhance Oncolytic Herpes Virotherapy in Models of Ewing Sarcoma by Modulating the Immune Microenvironment

doi: 10.1016/j.omto.2018.10.001

Figure Lengend Snippet: Clodrosome Reduces Systemic and Intratumoral Macrophages (A and B) Flow cytometry quantification of total splenocytic (A) and tumor (B) macrophage infiltration and macrophage M1-like and M2-like phenotype infiltration in A673 xenografts on days 0, 5, and 25 in the single intratumorally (i.tu) rRp450 treatment regimen done in Figure 1 C. Statistical analysis of bar graphs was done using unpaired multiple two-tailed t test; *p < 0.05, **p < 0.005, ***p < 0.0005.

Article Snippet: The A673 human Ewing sarcoma cell line and Vero green monkey kidney cell line were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Flow Cytometry, Two Tailed Test

Journal: Molecular Therapy Oncolytics

Article Title: Myelolytic Treatments Enhance Oncolytic Herpes Virotherapy in Models of Ewing Sarcoma by Modulating the Immune Microenvironment

doi: 10.1016/j.omto.2018.10.001

Figure Lengend Snippet: Clodrosome Depletion of Macrophages Impairs A673 Tumorigenesis (A) Treatment regimen of athymic nude mice given 400 μg of liposome or Clodrosome intravenously (i.v.) on day −3 and intraperitoneally (i.p.) on days −2, −1, and 0 followed by weekly i.p. treatments. A673 Ewing sarcoma cells were implanted subcutaneously into the flank on day 0. (B) A673 flank tumors treated with the above regimen were measured until tumors reached an endpoint volume of 2,000 mm 3 . Flow cytometry quantification of (C) splenocytic and (D) tumor leukocyte infiltrates in A673 tumors 10 days after A673 cell implantation. Statistical analyses of survival curves were done using Mantel-Cox test, and bar graphs were performed using unpaired multiple two-tailed t test; *p < 0.05, **p < 0.005, ***p < 0.0005. Error bars indicate SD.

Article Snippet: The A673 human Ewing sarcoma cell line and Vero green monkey kidney cell line were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Flow Cytometry, Two Tailed Test

Journal: Molecular Therapy Oncolytics

Article Title: Myelolytic Treatments Enhance Oncolytic Herpes Virotherapy in Models of Ewing Sarcoma by Modulating the Immune Microenvironment

doi: 10.1016/j.omto.2018.10.001

Figure Lengend Snippet: Bone Marrow-Derived Macrophages Alter Phenotypes in Response to Exogenous Stimuli But Do Not Affect Viral Oncolysis (A) F4/80 + bone marrow-derived macrophages were isolated and grown in 10F DMEM unstimulated or stimulated with LPS, IFNγ, LPS+IFNγ, IL-4, IL-10, or TGF-β and analyzed for gene expression. (B) A673 Ewing sarcoma cells were cultured alone or cocultured with the different macrophage populations and infected with rRp450 for 6 days before performing MTS viability assays. Statistical analysis of viability curves was performed using ANOVA; *p < 0.05, **p < 0.005, ***p < 0.0005. Error bars indicate SD.

Article Snippet: The A673 human Ewing sarcoma cell line and Vero green monkey kidney cell line were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Derivative Assay, Isolation, Gene Expression, Cell Culture, Infection

Journal: Molecular Therapy Oncolytics

Article Title: Myelolytic Treatments Enhance Oncolytic Herpes Virotherapy in Models of Ewing Sarcoma by Modulating the Immune Microenvironment

doi: 10.1016/j.omto.2018.10.001

Figure Lengend Snippet: Clodrosome Plus Virus Alter the Phenotypes of Tumor-Associated Macrophages qPCR array of macrophage activity in F4/80 + tumor macrophages isolated from A673 xenografts treated under the regimen in Figure 1 A at 5 days after the first rRp450 infection. Gene expression was normalized within each sample to actin; then the expression of each gene was normalized to that gene’s expression in the control liposome/PBS group. Genes were sorted based on their expression in the combination Clodrosome/oHSV group.

Article Snippet: The A673 human Ewing sarcoma cell line and Vero green monkey kidney cell line were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Virus, Activity Assay, Isolation, Infection, Gene Expression, Expressing, Control

Journal: Molecular Therapy Oncolytics

Article Title: Myelolytic Treatments Enhance Oncolytic Herpes Virotherapy in Models of Ewing Sarcoma by Modulating the Immune Microenvironment

doi: 10.1016/j.omto.2018.10.001

Figure Lengend Snippet: Clodrosome Plus Virus Alter the Tumor Stromal Microenvironment qPCR array with mouse-specific primers of whole A673 tumor powder treated under the regimen in Figure 1 A at 5 days after first rRp450 infection. Gene expression was analyzed as in the legend of Figure 5 .

Article Snippet: The A673 human Ewing sarcoma cell line and Vero green monkey kidney cell line were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Virus, Infection, Gene Expression

Journal: Molecular Therapy Oncolytics

Article Title: Myelolytic Treatments Enhance Oncolytic Herpes Virotherapy in Models of Ewing Sarcoma by Modulating the Immune Microenvironment

doi: 10.1016/j.omto.2018.10.001

Figure Lengend Snippet: Trabectedin Enhances Oncolytic Herpes Virus Efficacy (A) Treatment regimen of A673 tumor xenograft-bearing mice given 0.15 mg/kg trabectedin intravenously (i.v.) on days 0 and 7. Oncolytic herpes simplex virus rRp450 was given at 10 7 pfu intratumorally (i.tu.) on days 0 and 2. (B) Flow cytometry quantification of tumor leukocyte infiltrates in A673 tumors on day 3. (C) A673 flank tumors treated with the above regimen were measured until tumors reached an endpoint volume of 2,000 mm 3 . Statistical analyses of survival curves were performed using the Mantel-Cox test, and bar graphs were done using unpaired multiple two-tailed t test; *p < 0.05, **p < 0.005. (D) Virus replication in A673 xenografts treated with or without trabectedin and a single 10 7 pfu dose of rRp450 given i.t. at the time points indicated prior to tumor homogenization and plaque assay. Error bars indicate SD.

Article Snippet: The A673 human Ewing sarcoma cell line and Vero green monkey kidney cell line were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Virus, Flow Cytometry, Two Tailed Test, Homogenization, Plaque Assay