Journal: FEBS letters

Article Title: Differential effects of the N-terminal helix of FGF8b on the activity of a small-molecule FGFR inhibitor in cell culture and for the extracellular domain of FGFR3c in solution.

doi: 10.1002/1873-3468.14976

Figure Lengend Snippet: Fig. 1. Activity of SSR against FGF2 and FGF8b. (A–D) Fluorescence intensity analysis of U-2 OS cells immunolabeled for pMAPK (pY202/ 204) (A, B) or pPLCγ1 (pY783) (C, D) after stimulation with FGF2 (A, C) and FGF8b (B, D) and in the presence and absence of 25 μM SSR. The statistical significance was evaluated using the Mann–Whitney U test (*P < 0.05; **P < 0.01; ***P < 0.001; n.s., non-significant). Number of biologically independent replicates is n = 4 for A, B, and D; and 3 for panel C. (E) Representative microscopic images of proliferating bEnd.3 cells (EdU+, green; upper panel) and total bEnd.3 cells (nuclear marker DAPI, gray; bottom panel). Length of the scale bar equals 100 μM. (F) Percentage of proliferating human bEnd.3 cells upon stimulation by FGF2 and FGF8b in the presence and absence of either 50 nM or 100 μM SSR. BSA was used as a negative control. Error bars represent the SEM. Number of biologically independent replicates is n = 3.

Article Snippet: The human osteosarcoma cell line U-2 OS (CLS Cell Lines Service GmbH, Eppelheim, Germany; cat. no. 300364) was grown in growth medium consisting of high glucose DMEM/F-12 (Gibco, Grand Island, NY, USA; cat. no. 21041025) and supplemented with 1% GlutaMAX (Gibco; cat. no. 35050061), 1 unit mL 1 penicillin, 1 μg mL 1 streptomycin (Gibco; cat. no. 10378016), and 10% FBS (Satorius, Goettingen, Germany) at 37 °C and 5% CO2.

Techniques: Activity Assay, Fluorescence, Immunolabeling, MANN-WHITNEY, Marker, Negative Control

Journal: Cell Death & Disease

Article Title: Nanoparticle enhanced MRI can monitor macrophage response to CD47 mAb immunotherapy in osteosarcoma

doi: 10.1038/s41419-018-1285-3

Figure Lengend Snippet: a CD47 gene expression in a normal bone cell line, a normal patient bone, and osteoma patient specimen, three different osteosarcoma cell lines and eight osteosarcoma patient specimens. CD47 expression was measured with quantitative real-time PCR (qPCR), using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control, and compared between osteosarcomas and osteomas and normal bone using exact two-sided Wilcoxon rank-sum tests, p < 0.001. b Representative confocal CD47, CD68, and CD163 stains of three human osteosarcoma samples (scale bar 25 μm), a human osteoma sample (scale bar 25 μm), and a human bone sample (scale bar 50 μm) as negative control. c Corresponding quantitative area of CD47, CD68, and CD163 stains of one bone, one osteoma, and five osteosarcoma samples was measured over four high-power fields with Image J. Differences in CD47-, CD68-, and CD163-stained area of osteosarcoma, osteoma, and bone specimens were tested with exact two-sided Wilcoxon rank-sum tests, p < 0.001

Article Snippet: In vitro studies were performed in three human osteosarcoma cell lines (Saos-2, U-2 OS, MNNG/HOS, ATCC, Manassas, VA, USA), and one murine osteosarcoma cell line (K7M2, ATCC, Manassas, VA, USA).

Techniques: Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Control, Negative Control, Staining

Journal: Cell Death & Disease

Article Title: Nanoparticle enhanced MRI can monitor macrophage response to CD47 mAb immunotherapy in osteosarcoma

doi: 10.1038/s41419-018-1285-3

Figure Lengend Snippet: a Schematic representation of experimental design. Tumors were initiated in NSG mice ( n = 6/group) by subcutaneous injection of human MNNG/HOS osteosarcoma cells positive for tdTomato-Luciferase expression. Treatment with control and anti-CD47 mAbs (10 mg/kg, 3× for 5 days) was initiated once the tumors were detected with bioluminescent imaging. MRI was performed on day 5, day 6, and day 10 of therapy. b Representative T2-weighted MR images of MNNG/HOS subcutaneous tumors in mice treated with control or CD47 mAbs. The tumors are hyperintense (bright) on pre-contrast MR images (white arrows) and show hypointense (dark) enhancement after ferumoxytol administration (red arrows). c Tumor MRI enhancement, quantified as T2 relaxation times, of MNNG/HOS tumors treated with control IgG or CD47 mAbs. CD47 mAb-treated tumors demonstrated significantly shortened T2 relaxation times compared to control antibody-treated tumors on ferumoxytol-enhanced MRI images. d Tumor volumes, as measured on T2-weighted MR scans, were significantly smaller on day 10 after anti-CD47 mAb treatment compared to controls. All results are represented as mean ± SD from six tumors per experimental group, p value as indicated, exact two-sided Wilcoxon rank-sum tests

Article Snippet: In vitro studies were performed in three human osteosarcoma cell lines (Saos-2, U-2 OS, MNNG/HOS, ATCC, Manassas, VA, USA), and one murine osteosarcoma cell line (K7M2, ATCC, Manassas, VA, USA).

Techniques: Injection, Luciferase, Expressing, Control, Imaging

Journal: Cell Death & Disease

Article Title: Nanoparticle enhanced MRI can monitor macrophage response to CD47 mAb immunotherapy in osteosarcoma

doi: 10.1038/s41419-018-1285-3

Figure Lengend Snippet: a Representative coronal T2-weighted MR images of intratibial K7M2 osteosarcomas at different time points after therapy with control or CD47 mAbs. White arrow points tumors on pre-contrast images, and red arrow points on post-contrast images. b T2 relaxation times of K7M2 osteosarcomas on unenhanced and ferumoxytol-enhanced MRI images at day 6 after control or CD47 mAb therapy. Only ferumoxytol-enhanced MR images show a significantly different T2 relaxation time between CD47 mAb-treated tumors and controls. c Prussian blue iron stains and F4/80, CD80, inducible nitric oxide synthase (iNOS) TAM stains, and active caspase-3 stain of K7M2 intratibial tumors treated with control and CD47 mAb. d Corresponding quantitative area of Prussian blue iron staining and F4/80, CD80, iNOS TAM staining, and caspase-3 staining in control and CD47 mAb-treated sets. e Bioluminescent in vivo images of mice with intratibial K7M2 osteosarcomas before and after therapy with IgG or CD47 mAb. f Total quantified flux in control and treated mice with K7M2 osteosarcomas. Results are represented as mean ± SD from six tumors per experimental group, p value as indicated, exact two-sided Wilcoxon rank-sum tests

Article Snippet: In vitro studies were performed in three human osteosarcoma cell lines (Saos-2, U-2 OS, MNNG/HOS, ATCC, Manassas, VA, USA), and one murine osteosarcoma cell line (K7M2, ATCC, Manassas, VA, USA).

Techniques: Control, Staining, In Vivo

Journal: Cell Death & Disease

Article Title: Nanoparticle enhanced MRI can monitor macrophage response to CD47 mAb immunotherapy in osteosarcoma

doi: 10.1038/s41419-018-1285-3

Figure Lengend Snippet: The signal-regulatory protein alpha (SIRPα) surface marker on osteosarcoma cells inhibits macrophage phagocytosis. Blocking the CD47–SIRPα interaction with CD47 monoclonal antibody (mAb) induces phagocytosis of both tumor and ferumoxytol nanoparticles. Increased uptake of ferumoxytol nanoparticles generates T2 contrast on MR scans that can serve as an imaging biomarker for monitoring responses to CD47 immunotherapy

Article Snippet: In vitro studies were performed in three human osteosarcoma cell lines (Saos-2, U-2 OS, MNNG/HOS, ATCC, Manassas, VA, USA), and one murine osteosarcoma cell line (K7M2, ATCC, Manassas, VA, USA).

Techniques: Marker, Blocking Assay, Imaging, Biomarker Discovery