Journal: Cancer research

Article Title: G-CSF promotes neuroblastoma tumorigenicity and metastasis via STAT3-dependent cancer stem cell activation

doi: 10.1158/0008-5472.CAN-14-2946

Figure Lengend Snippet: (A) Cell viability assay of different NB cell lines (NGP, SH-SY5Y, IMR-32, CHLA-255) in response to various concentrations of STAT3 inhibitor (Stattic) treatment for 24 h. Cell viability was measured using MTS assay. Experiment was repeated two times with six replicates for each condition and represented as mean ± SD. (B) Representative pictures of colony formation assay for different NB cell lines treated with various doses of Stattic (μM). (C) Quantitation of relative inhibition of colony formation is shown as mean ± SD (t-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). (D) Measurement of apoptosis in CD114+ and CD114- NB subpopulations of NGP and SH-SY5Y cell lines using Annexin-V staining along with CD114 staining in response to increasing doses of Stattic for 2 h. Data are represented as means ± SD for three independent experiments. DMSO was used as control in “0” groups. (t-test, *p<0.05, **p<0.01, ****p<0.0001). (E) Cell cycle analysis using BrdU incorporation assay in NGP NB subpopulations with 1μM Stattic treatment for 2 h. Data is represented as relative percentage and representative of experiment repeated three times. (F) Representative flow cytometry analysis plots for cell cycle analysis on NGP NB subpopulations in response to Stattic treatment (1μM). Values shown on each dot plot are representing the percentage of cells.

Article Snippet: Generation of stable STAT3 knockdown cell lines Lentiviral shRNA vectors pSIH1-puro-STAT3 (26596, Addgene) and pSIH1-puro-control (26597, Addgene) ( 15 ) were used to transduce NB cell lines as described previously ( 11 ).

Techniques: Viability Assay, MTS Assay, Colony Assay, Quantitation Assay, Inhibition, Staining, Control, Cell Cycle Assay, BrdU Incorporation Assay, Flow Cytometry

Journal: Cancer research

Article Title: G-CSF promotes neuroblastoma tumorigenicity and metastasis via STAT3-dependent cancer stem cell activation

doi: 10.1158/0008-5472.CAN-14-2946

Figure Lengend Snippet: (A) Cell viability assay of different NB cell lines (NGP, SH-SY5Y, IMR-32, CHLA-255) were tested by MTS assay in response to etoposide alone and in combination of 0.5 μM and 1.0 μM Stattic treatment for 24 h. Experiments were repeated two times with six replicates for each condition and represented as mean ± SD. (B) Schematic representation of experimental plan to analyze the in vivo effect of STAT3 inhibitor and dual treatment strategy on neuroblastoma. Orthotopic mouse model was used to develop xenografts of NB NGP and SH-SY5Y cell lines and treated with either etoposide (12 mg/kg/day, i.p. injection, three times/week) and Stattic (25 mg/kg/day, i.p. injection, five days/week) alone or combined. Implanted mouse were treated two weeks post-implantation for two weeks. Vehicle (DMSO) was used as control in all experiments. (C) NGP xenografts showing significant decrease in tumor weights in response to Stattic treatment (p=0.03), etoposide treatment and combo treatment (p=0.001) in comparison to controls. Combo treatment also significantly reduced tumor burden in comparison to etoposide alone (p=0.008). (Mann-Whitney test, *p<0.05, **p<0.01, ***p<0.001). The percentage of CD114+ cells determined by flow cytometry analysis on individual tumors showed as mean ± SEM. Human MYCN mRNA was detected by qPCR in bone marrow of xenotransplanted mice. (D) SH-SY5Y xenografts showing significant decrease in tumor weights in response to Stattic treatment (p=0.02), etoposide treatment (p=0.0002) and combo treatment (p=<0.0001) in comparison to controls. Combo treatment also significantly reduced tumor burden in comparison to etoposide (p=0.02) and Stattic alone (p=0.0002). (Mann-Whitney test (*p<0.05, **p<0.01, ***p<0.001). The percentage of CD114+ cells determined by flow cytometry analysis on individual tumors showed as mean ± SEM. Detection of human MYCN mRNA by qPCR in bone marrow of xenotransplanted mice shows trend of reduction of metastasis with Stattic (p=ns).

Article Snippet: Generation of stable STAT3 knockdown cell lines Lentiviral shRNA vectors pSIH1-puro-STAT3 (26596, Addgene) and pSIH1-puro-control (26597, Addgene) ( 15 ) were used to transduce NB cell lines as described previously ( 11 ).

Techniques: Viability Assay, MTS Assay, In Vivo, Injection, Control, Comparison, MANN-WHITNEY, Flow Cytometry

Journal: Cancer research

Article Title: G-CSF promotes neuroblastoma tumorigenicity and metastasis via STAT3-dependent cancer stem cell activation

doi: 10.1158/0008-5472.CAN-14-2946

Figure Lengend Snippet: (A) Relative CSF3R expression analysis in NB subpopulations of NGP, SH-SY5Y, and IMR-32 in response to G-CSF (20 ng/ml), Stattic (1μM) and combination treatment (G+S; represents G-CSF + Stattic) for 2 hr. Data are mean ± SEM of three replicates of experiment repeated twice. (t-test, *p<0.05, **p<0.01, ***p<0.001). (B) Schematic representation of CSF3R gene promoter showing 3.8 kb long 5′ untranslated region (5′ UTR) and 2.5 kb long promoter region. A potential STAT3 binding site was determined in 5′ UTR region at -200 bp (S) from translation start site (arrow). EGFP reporter driven by 5′UTR contacting STAT3 binding site (S) shows significant increase in percentage of CD114+/GFP+ cells in response to G-CSF treatment (20 ng/ml for 2 h) in contrast to untreated or reporter driven by promoter region. CHIP-qPCR primers were designed for STAT3 binding site (S). (C) CHIP-qPCR analysis showing direct binding enrichment of STAT3 and pSTAT3 (Y705) at S site of CSF3R 5′ UTR. NGP NB subpopulation CD114+ cells showing enriched binding of STAT3 by 2.2 fold (p=0.007) pSTAT3 by 3.1 fold (p=0.002) in comparison to CD114- cells. G-CSF treatment (20ng/ml for 2 h) in CD114+ cells further enriched the binding of STAT3 by 2.7 fold (p<0.001) and pSTAT3 (Y705) by 2.4 fold (p<0.01) in comparison to baseline untreated while Stattic treatment (1μM for 2 h) blocks the binding of STAT3 or pSTAT3. Data are mean ± SEM of three replicates of experiment repeated twice. (t-test *p<0.05, **p<0.01, ***p<0.001).

Article Snippet: Generation of stable STAT3 knockdown cell lines Lentiviral shRNA vectors pSIH1-puro-STAT3 (26596, Addgene) and pSIH1-puro-control (26597, Addgene) ( 15 ) were used to transduce NB cell lines as described previously ( 11 ).

Techniques: Expressing, Binding Assay, ChIP-qPCR, Comparison

Journal: Cancer research

Article Title: G-CSF promotes neuroblastoma tumorigenicity and metastasis via STAT3-dependent cancer stem cell activation

doi: 10.1158/0008-5472.CAN-14-2946

Figure Lengend Snippet: Schematic representation of conclusions drawn from current study showing upregulation of JAK/STAT3 pathway in response to binding of G-CSF to G-CSFR. Active STAT3 translocate to nucleus and upregulate the expression of key target genes that are know to be involved in tumorigenicity, metastasis and drug resistance. CSF3R (gene coding for G-CSFR) is also a direct transcriptional target of STAT3. Binding of STAT3 on CSF3R promoter further upregulate surface expression of the G-CSFR (CD114), making a feedback loop mechanism for tumor progression in NB. STAT3 inhibition and anti-G-CSF antibody are shown to block this positive feedback loop and therefore decrease the cancer stem cell functions in NB.

Article Snippet: Generation of stable STAT3 knockdown cell lines Lentiviral shRNA vectors pSIH1-puro-STAT3 (26596, Addgene) and pSIH1-puro-control (26597, Addgene) ( 15 ) were used to transduce NB cell lines as described previously ( 11 ).

Techniques: Binding Assay, Expressing, Inhibition, Blocking Assay