Anti-TGFβ antibody decreased osteoclast numbers and <i>in vitro</i> osteoclastogenesis.

<p><b>Panel a:</b> Boxplot of number of TRAP positive osteoclasts per millimeter of bone surface in MBA-MB-231 tumor-bearing tibiae, showing significantly decreased osteoclasts upon 1D11 treatment, compared to 13C4. Wilcoxon rank-sum p-value = 0.027. Mean ± standard deviation = 13C4: 0.7733±0.3002, 1D11: 0.4539±0.1141. N = at least 6). <b>Panel b:</b> To assess the effect of anti-TGFβ treatment directly on osteoclast population, an <i>ex vivo</i> osteoclastogenesis assay was performed using bone marrow mononuclear cells. Bone marrow mononuclear cells were isolated and cultured in presence of 13C4 (control antibody), 1D11(anti-TGFβ antibody), TGFβ+13C4 or TGFβ+1D11 in presence of both RANKL and MCSF. Both 13C4 and 1D11 was used at a concentration of 25 µg/ml. TGFβ as used at a concentration of 5 ng/ml. Osteoclasts were stained using using a Leucocyte acid phosphatase (TRAP) kit as per manufacturer's instruction (Sigma-Aldrich) and TRAP positive cells (reddish brown) were counted under microscope. Boxplots of number of osteoclasts by group for osteoclastogenesis assay show that treatment with 1D11 significantly reduced TGFβ-mediated osteoclast formation. Wilcoxon rank-sum p-value for TGFβ and TGFβ + 1D11 groups is 0.01. Mean ± standard deviation = TGFβ: 26.5±3.83, TGFβ + 1D11: 17.83±4.17. Data presented here is representative of two independent experiments. <b>Panel c:</b> To assess the effect of anti-TGFβ antibody on osteoblast-mediated osteoclastogenesis, bone marrow mononuclear cells were cultured on a layer of primary mouse calverial osteoblasts in the presence of either control antibody (13C4) or the anti-TGFβ antibody (1D11). After 7–10 days, TRAP staining was performed to identify mature osteoclasts (indicated by arrow). <b>Panel d:</b> Osteoblast mediated osteoclastogenesis increases significantly upon 1D11 treatment compared to control, Wilcoxon rank-sum p-value = 0.006. Mean ± standard deviation = 13C4: 26.5±11.31, 1D11: 4.83±2.48. Data presented here is representative of two independent experiments.</p

Suppression of TGFβ by anti-TGFβ antibody 1D11 increased the mineral-to-collagen ratio.

<p>Confocal raman spectroscopy was performed on mice bearing MDA-MB-231 tumors in bone treated for 4 weeks with 1D11 or 13C4 antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027090#s2" target="_blank">materials and methods</a> section. At least nine spectra were analyzed per specimen and the mean mineral-to-collagen ratio, Type-B carbonate substitution, and crystallinity were scored. Both mineral-to-collagen ratio and carbonate substitution increased significantly upon 1D11 treatments compared to control. Mean ± standard deviation is shown, p value was determined using Wilcoxon test.</p

Anti-TGFβ antibody improves trabecular architecture in tumor bearing mice tibia and femur.

<p>MicroCT analysis of the tibiae from MDA-MB-231tumor-bearing mice treated for 4 weeks, starting one day after tumor cell inoculation, revealed that suppression of TGFβ by the antibody 1D11 increased trabecular bone volume through increases in trabecular number, and this improved the connectivity of the trabeculae (lack of fenestrations), compared to isotype control. Wilcoxon rank-sum test was used for this analysis. Means and standard deviations by group for the MDA-MB-231 four week data with p-values from Wilcoxon rank-sum tests. Quantitative analysis of microCT data from MDA-MB-231 tumor-bearing mice treated with 13C4 or 1D11 for weeks. Trabecular bone volume (BV/TV), trabecular thickness (Tb.Th*), trabecular number (Tb.N*), and connectivity density (Conn.D), and mean volumetric density of the mineralized tissue (Tb.TMD) were calculated using the Scanco evaluation software.</p

Anti-TGFβ antibody increases bone volume in tumor bearing mice.

<p>MDA-MB-231 cells were injected via intra-cardiac route in 4 week old female nude mice and 4T1 cells were injected in 4–5 week old female Balb/C mice. Mice were treated either with control antibody (13C4, 10 mg/kg) or anti-TGFβ antibody (1D11, 10 mg/kg) for 4 weeks, starting 1 day after tumor cell inoculation. Trabecular bone volume in the tibial metaphysis of tumor-bearing mice was analyzed by microCT. <b>Panel a:</b> Representative three dimensional reconstitutions of microCT images from both 13C4 and 1D11 treated groups from mice injected with MDA-MB-231 cells. <b>Panel b:</b> Boxplots of average BV/TV (bone volume/total volume) by group for the MDA-M<b>B</b>-231 tumor- bearing mice show significant increase in bone mass after treatment with anti-TGFβ antibody. Wilcoxon rank-sum p-value = <0.001. Mean ± standard deviation = 13C4: 0.06±0.04, 1D11: 0.32±0.09. N = at least 10. <b>Panel c.</b> Boxplots of average BV/TV (bone volume/total volume) by group for the 4T1 tumor- bearing mice show a significant increase in bone mass as a result of treatment with 1D11 as measured by BV/TV. Wilcoxon rank-sum p-value = 0.036. Mean ± standard deviation = 13C4: 0.09±0.01, 1D11: 0.11±0.01, N = at least 5. <b>Panel d:</b> Mouse calverial osteoblasts were isolated and cultured for 7–10 days as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027090#s2" target="_blank">Materials and Methods</a>, either in presence of anti-TGFβ antibody (1D11) or isotype control (13C4) and mineralized matrix formation was measured using Von Kossa staining as a surrogate for osteoblast differentiation. <b>Panel e:</b> Boxplot analysis reveals treatment with anti-TGFβ antibody (1D11) significantly increased percent areas of mineralized matrix. Images were taken from representative fields and quantified using Metamorph software. Wilcoxon rank-sum p-value = 0.005. Mean ± standard deviation = 13C4: 16±3.7, 1D11: 32.3±1. Data presented here is representative of two independent experiments.</p

Anti-TGFβ antibody reduces osteolytic lesions in MDA-MB-231 breast cancer bone metastasis cardiac injection model.

<p>Mice were inoculated with MDA-MB-231 human breast cancer cells in the left cardiac ventricle and were treated with either isotype control (13C4, 10 mg/kg) or anti-TGFβ antibody (1D11, 10 mg/kg) for 4 weeks, starting from 1 day post tumor cell injection. At the end of the experiment, whole body X-ray images of mice from both control and anti-TGFβ antibody treated group were taken and osteolytic lesion area and osteolytic lesion counts were analyzed using image analysis software (Metamorph, Molecular Device). <b>Panel a:</b> Representative X-ray images of osteolytic bone lesions in the hind leg of mice treated for 4 weeks either with control antibody (13C4, left panel) or anti-TGFβ antibody (1D11, right panel). White arrows indicate presence of osteolytic lesions. <b>Panel b:</b> A boxplot representing the average lesion counts in mice inoculated with MDA-MB-231 cells in the left cardiac ventricle, treated with either control antibody (13C4, 10 mg/kg) or anti-TGFβ antibody (1D11, 10 mg/kg) for 4 weeks, starting 1 day after tumor cell injection shows decrease in lesion numbers after anti-TGFβ treatment (6.9±1.7 for control and 1.9±0.7 for 1D11; Wilcoxon rank-sum p-value = <.001, N = 9). <b>Panel c:</b> A boxplot representing the lesion area from the same experiment shows decrease in the lesion area after anti-TGFβ treatment (20520±6000 for control and 1497±888 for 1D11; Wilcoxon rank-sum p-value = <.001, N = at least 9). Lesion areas were measured using arbitrary pixel unit.</p