Anti-Transforming Growth Factor ß Antibody Treatment Rescues Bone Loss and Prevents Breast Cancer Metastasis to Bone
Figure 3
Anti-TGFβ antibody decreased osteoclast numbers and in vitro osteoclastogenesis.
Panel a: 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). Panel b: To assess the effect of anti-TGFβ treatment directly on osteoclast population, an ex vivo 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. Panel c: 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). Panel d: 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.