Journal: PLoS ONE

Article Title: Involvement of the CXCR7/CXCR4/CXCL12 Axis in the Malignant Progression of Human Neuroblastoma

doi: 10.1371/journal.pone.0043665

Figure Lengend Snippet: (A) Semi-quantitative assessment of CXCR7 and CXCL12 expression levels in neural, endothelial and stromal cell compartments of NB primary tumors. Median score represents the average of the immunostaining score (0–4). Percentage (%) indicates percentage of CXCR7-or CXCL12-positive tumor tissues. (B) Immunohistochemical analysis of CXCR7 in undifferentiated tumor (UnNB), ganglioneuroblastoma (GGNB), ganglioneuroma (GGN) and control normal sympathetic ganglion (SG) tissues. Black arrow: ganglion cell. (C) CXCR7 expression levels (median score) in UnNBs, in differentiated tumor tissues, (D) and in tumors of patient according to the age of patient at diagnosis. (E) Immunohistochemical analysis of CXCL12 in UnNBs, GGNBs, GGNs and SG tissues. Red arrow: endothelial cell. (F) CXCL12 expression levels (median score) in the endothelial compartment of tumors according to the age of patient at diagnosis, (G) and in the stroma of UnNBs, GGNBs and GGNs. Student’s t-test: *p<0.05, **p<0.01.

Article Snippet: TMA sections were incubated overnight at 4°C with mouse anti-human CXCL12 (clone 79018, R&D systems, Minneapolis, MN, USA) and mouse anti-human CXCR7 (clone 9C4, kind gift from Dr. M. Thelen, IRB, Bellinzona, Switzerland) antibodies diluted in Dako REALTM antibody diluent (Dako, Glostrup, Denmark).

Techniques: Expressing, Immunostaining, Immunohistochemical staining, Control, Biomarker Discovery

Journal: PLoS ONE

Article Title: Involvement of the CXCR7/CXCR4/CXCL12 Axis in the Malignant Progression of Human Neuroblastoma

doi: 10.1371/journal.pone.0043665

Figure Lengend Snippet: Expression of CXCR7 and CXCL12 in NB primary tumors, metastases and control tissues.

Article Snippet: TMA sections were incubated overnight at 4°C with mouse anti-human CXCL12 (clone 79018, R&D systems, Minneapolis, MN, USA) and mouse anti-human CXCR7 (clone 9C4, kind gift from Dr. M. Thelen, IRB, Bellinzona, Switzerland) antibodies diluted in Dako REALTM antibody diluent (Dako, Glostrup, Denmark).

Techniques: Expressing, Control

Journal: PLoS ONE

Article Title: Involvement of the CXCR7/CXCR4/CXCL12 Axis in the Malignant Progression of Human Neuroblastoma

doi: 10.1371/journal.pone.0043665

Figure Lengend Snippet: (A) Qualitative RT-PCR analyses for CXCR7 and CXCR4 mRNA expression in a panel of N-, I-and S-type NB cell lines. GAPDH was used as gene of reference. The prostate cancer cell line PC-3 and the breast cancer cell line MCF-7 were used as positive controls for CXCR7 expression. (B) Flow cytometry analyses of CXCR7 and CXCR4 cell surface expression in NB cell lines. Percent represents CXCR7-or CXCR4-positive cells. Grey line: cells stained without the primary Ab. Black line: cells stained with anti-CXCR7 or anti-CXCR4. (C) Semi-quantitative real-time PCR analyses of CXCR7 mRNA expression level in the IGR-NB8 cell line after 3 days of treatment with either 10 µM RA or BrdU (left panel). Expression levels of CXCR7 transcripts were calculated relatively to the level of the housekeeping gene HPRTI . Untreated (unt.) cells and DMSO-treated NB cells represented control conditions. Columns indicate results in triplicates, and were representative of two independent experiments. Error bars indicate S.D. Student’s t-test: *p<0.05, **p<0.01. Images (right panel) represent immunofluoresence staining of β 3 -tubulin (red) and DAPI (blue) in treated or untreated IGR-NB8 cells. (D) CXCR7, CXCR4, or a combination of the two CXCL12 receptors was overexpressed in the IGR-NB8 and the SH-SY5Y cell lines. As all vectors encoded for EGFP, transduced GFP-expressing NB cells were sorted by FACS to control transfection efficiency. Both NB8pMigr and SHSYpMigr cell lines represented control cells, transduced with the pMigr empty vector. Percent of CXCR7-and CXCR4-positive transduced cells are indicated as well as the mean fluorescent intensity (brackets) for CXCR7 and CXCR4 staining. Dark and grey lines: cells stained without anti-CXCR7 and anti-CXCR4, respectively; Green and blue lines: cells stained with anti-CXCR7 and anti-CXCR4, respectively. (E) Semi-quantitative real-time PCR analyses for CXCR7 mRNA expression levels in NB transduced cell lines. Experiment was performed in triplicates. Error bars indicate S.D.

Article Snippet: TMA sections were incubated overnight at 4°C with mouse anti-human CXCL12 (clone 79018, R&D systems, Minneapolis, MN, USA) and mouse anti-human CXCR7 (clone 9C4, kind gift from Dr. M. Thelen, IRB, Bellinzona, Switzerland) antibodies diluted in Dako REALTM antibody diluent (Dako, Glostrup, Denmark).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Flow Cytometry, Staining, Real-time Polymerase Chain Reaction, Control, Transfection, Transduction, Plasmid Preparation

Journal: PLoS ONE

Article Title: Involvement of the CXCR7/CXCR4/CXCL12 Axis in the Malignant Progression of Human Neuroblastoma

doi: 10.1371/journal.pone.0043665

Figure Lengend Snippet: Immunobloting of phospho-ERK (pERK) and total ERK (ERK) in transduced cells, treated with (A, B) 100 ng/ml human recombinant CXCL12, in presence or in absence of 1 µM of the CXCR4 blocker TN14003, (C) 100 ng/ml human recombinant CXCL11.

Article Snippet: TMA sections were incubated overnight at 4°C with mouse anti-human CXCL12 (clone 79018, R&D systems, Minneapolis, MN, USA) and mouse anti-human CXCR7 (clone 9C4, kind gift from Dr. M. Thelen, IRB, Bellinzona, Switzerland) antibodies diluted in Dako REALTM antibody diluent (Dako, Glostrup, Denmark).

Techniques: Western Blot, Recombinant

Journal: PLoS ONE

Article Title: Involvement of the CXCR7/CXCR4/CXCL12 Axis in the Malignant Progression of Human Neuroblastoma

doi: 10.1371/journal.pone.0043665

Figure Lengend Snippet: (A) Clonogenic growth of NB transduced cell lines was evaluated in a soft agar assay, after at least two weeks of incubation at 37°C. Columns: mean values ± SEM of two independent experiments. When stipulated, 100 ng/mL CXCL12 were added to the culture medium. (B) Chemotaxis of transduced NB cells toward 100 ng/ml CXCL12. Columns: mean values ± SEM of at least two independent experiments. Student’s t-test was used for all experiments: *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: TMA sections were incubated overnight at 4°C with mouse anti-human CXCL12 (clone 79018, R&D systems, Minneapolis, MN, USA) and mouse anti-human CXCR7 (clone 9C4, kind gift from Dr. M. Thelen, IRB, Bellinzona, Switzerland) antibodies diluted in Dako REALTM antibody diluent (Dako, Glostrup, Denmark).

Techniques: Soft Agar Assay, Incubation, Chemotaxis Assay

Journal: PLoS ONE

Article Title: Involvement of the CXCR7/CXCR4/CXCL12 Axis in the Malignant Progression of Human Neuroblastoma

doi: 10.1371/journal.pone.0043665

Figure Lengend Snippet: (A) In vivo tumor take (number of sites with tumor/total sites) and growth (mean tumor volume ± SEM) after s.c implantation of transduced NB cells in nude mice. Two-way ANOVA: **p<0.01. (B) Production of CXCL12 was measured by ELISA in transduced NB cell lines, and derived s.c tumors. Normal nude mouse adrenal gland (AG) tissue was used as positive control. Results are expressed in triplicates as pg of CXCL12 per mg of extracted protein. Error bars indicate S.D. of triplicates. (C) In vivo orthotopic implantation of NB8×4 and NB8×4×7 cell lines in nude mice. Upper panel: tumor take represented as fraction and percentage of tumor-bearing mice from week 3 to week 6 after NB cell implantations. Lower panel: kinetics of tumor volume. Mann-Whitney test: *p<0.05.

Article Snippet: TMA sections were incubated overnight at 4°C with mouse anti-human CXCL12 (clone 79018, R&D systems, Minneapolis, MN, USA) and mouse anti-human CXCR7 (clone 9C4, kind gift from Dr. M. Thelen, IRB, Bellinzona, Switzerland) antibodies diluted in Dako REALTM antibody diluent (Dako, Glostrup, Denmark).

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Derivative Assay, Positive Control, MANN-WHITNEY