#Sclerostin induced #tumor growth,bone metastasis and osteolysis in #breast cancer

Expression of sclerostin (SOST) in tumor tissues. (A) Representative expression of sclerostin by immunohistochemical staining (100 × magnification). Left: strongly positive expression of sclerostin in breast cancer bone metastasis (BCBM) tumor cells; middle: weakly positive expression of sclerostin in localized breast cancer (BC); right: negative expression of sclerostin in benign breast tumor (NC) tissue. (B) Quantification of sclerostin in the plasma of NC, BC and BCBM patients by ELISA. Expression of sclerostin mRNA (C), protein (D) and protein (E) were quantified in NC, BCBM and BC tissues. Each bar represents mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.005.

Expression of sclerostin in human breast cancer cell lines. (A)Expression of sclerostin protein. GAPDH was used as an internal control. Sclerostin protein was extracted from supernatant culture medium after centrifugation and concentration. (B) Quantification of sclerostin are normalizing to GAPDH. (C) Expression of sclerostin mRNA (RT-PCR: 45 cycles of amplification). GAPDH mRNA was used as an internal control. Each bar represents mean ± SEM.

Effect of sclerostin inhibition on cell proliferation, migration and invasion. MTT assays were performed at 1d, 2d, and 3d after treatment with sclerostin antibody (at 0, 1 and 4 µg/ml, respectively) in (A) MCF-7 and (B) MDA-MB-231 cells. (C) Cell migration assays were performed using Transwell system. MDA-MB-231 and MCF-7 cells were treated with sclerostin antibody (at 1 µg/ml and 4 µg/ml, respectively) for 24 h. Migrated cells were stained with crystal violet and (D) migration capability was evaluated by counting migrated cells. (E) Invasion assays were performed and (F) invasiveness was quantified by counting invading cells. NC, non-treated control group; 1 µg/ml, 1 µg/ml sclerostin antibody-treated group; 4 µg/ml, 4 µg/ml sclerostin antibody-treated group. Each bar represents mean ± SEM. *P < 0.05, **P < 0.01. All experiments were performed at least three times with duplication within each individual experiment.

Effect of Scl-Ab on tumor growth in vivo. A breast cancer xenograft model was established using MDA-MB-231 cells (5 × 106cells in 30 μl PBS) implanted into the femur of female nude mice (7–8 weeks old). (A) Tumor size was measured using Vernier caliper once a week until the animals were sacrificed after 40 days of treatment. (B) Tumor weight was measured at the last time point. (C) Kaplan-Meier survival plot of xenografted control, PBS-treated and Sci-Ab treated groups. (D) Sclerostin protein expression and quantification (E) in tumor tissues of the above three groups. NC, non-treated control group; PBS, PBS-treated group; Sci-Ab, sclerostin antibody-treated group (1 μg antibody in 20 μl PBS). Each bar represents mean ± SEM. NS, non-significant.

Analyses of tumor characteristics and serum of xenografted mice. (A) Representative H&E staining and immunohistochemistry for sclerostin (100 × magnification) in tumor sections obtained from different groups. (B) Quantification of osteocalcin (OCN), (C) osteoprotegerin (OPG), and (D) sclerostin in serum of mice from different groups by ELISA. NC, non-treated control group; PBS, PBS-treated group; Scl-Ab, sclerostin antibody-treated group (1 μg antibody in 20 μl PBS). Each bar represents mean ± SEM. **P < 0.01, NS, non-significant.

In vivo micro-CT analysis. (A) Presence of tumor-induced osteolytic lesions detected by micro-CT scans. Representative 3-D reconstruction of micro-CT images of femurs from non-treated control, PBS- and Sci-Ab treated mice. Three-dimensional images reconstructed from micro-CT analysis on the cortical and trabecular bone microarchitecture of whole left femur (B, longitudinal section) and of distal femoral metaphysis (C, cross section) in different groups. On Micro-CT, NC and PBS mice showed increased (D) trabecular thickness (Tb.Th), (E) trabecular number (Tb.N), (F) bone volume/tissue volume ratio (BV/TV), (G) bone mineral density (BMD), and (H) cortical bone volume/tissue volume ratio (cortical BV/TV), whereas decreased (I) trabecular separation (Tb.Sp) and (J) bone surface/bone volume ratio (BS/BV) compared with Scl-Ab treated mice. NC, non-treated control group; PBS, PBS-treated group; Scl-Ab, sclerostin antibody-treated group (1 μg antibody in 20 μl PBS). Each bar represents mean ± SEM. *P < 0.05, **P < 0.01; NS, non-significant.