Fig. 5: ER⁺ breast cancer cells secrete IBSP to recruit osteoclast precursors.

a Trans-well migration assays were performed to measure the migration ability of BMMs towards breast cancer cells. BMMs were seeded on the luminal side and breast cancer cells (MCF7, MCF7BoM2, and MCF7/IBSP) were seeded on the abluminal chamber. Data are presented as mean values ± SEM. b CRISPR-Cas9 mediated knockout of IBSP in MCF7BoM2. Three knockout clones were confirmed by western blot. The band intensity was quantified and normalized to that of control (left panels). c The selected clones were mixed and designated as MCF7BoM2IBSPKO. Cell proliferation was measured for MCF7BoM2 and MCF7BoM2/IBSPKO by MTS assay. A two-sided student’s t-test was performed on day 3 and p = 0.0496 (n = 3 vs 3). Data are presented as mean values ± SEM. d Cell migration ability of MCF7BoM2 and MCF7BoM2/IBSPKO was measured by wound-healing assay. Scale bar, 100 µm. e MCF7BoM2/ScrambledKO and MCF7BoM2/IBSPKO (500,000/0.1 ml PBS) were transplanted into female nude mice via intracardiac injection. The growth of bone metastasis was monitored by measuring the luciferase activity by IVIS Bioimager, and the Kaplan–Meier analysis was done for bone metastasis-free survival (p = 0.0496, HR = 4.101). f The tumor-bearing bones were imaged by X-ray, and the relative bone densities were quantified by ImageJ (p = 0.0124, n = 9 vs 4). Two-sided student’s t-tests were performed. Data are presented as mean values ± SEM. g OC surface relative to the bone surface was calculated and compared after TRAP staining. H&E staining of the same field was shown together with the TRAP staining (p = 0.0036, n = 9 vs 4). Two-sided student’s t-tests were performed. Data are presented as mean values ± SEM. Scale bar, 100 µm.