Fig 2: (A–F). The reduction of mRNA and protein expression of osteogenic molecules, OSP (A,B), BSP (C,D), and BMP-2 (E,F) in VIC exposed to HGOM for 7 days and transfected with a different type of nanocarriers on the fifth day. Western blots: NM (lane 1); HGOM (lane 2); V-LPP/shRunx2 (lanes 3, 4); Scr-LPP/shRunx2 (lane 5); C60-PEI/shRunx2 (lane 6); V-LPP/shCTR (lanes 7, 8); Scr-LPP/shCTR (lane 9). (G–I) The gene expression of OSP, BSP, and BMP-2 in VIC exposed to HGOM for 14 days and subjected to a second transfection on the 12th day. As controls, transfection using V-LPP/shCTR and Scr-LPP/shCTR were employed. Results, normalized to ß-actin, were expressed as mean ± S.D. of two independent experiments made in duplicate (n = 4) and represented as fold change relative to HGOM condition (considered as 1). The samples considered statistically different were marked with * p < 0.05, ** p < 0.01, *** p < 0.001 when compared to HGOM, # p < 0.05, ## p < 0.01, ### p < 0.001 when compared to NM.

Fig 3: CGA blocks cooperative function of miR-19a and IBSP in ER+ BCBM.a The mice were treated with 2 µg exosomes prepared from MCF7BoM2 and 0.5 µg recombinant IBSP by injecting them into the tibias every two days. The exosomes from MCF7BoM2/miR-19aKO and PBS were used as controls. After two weeks, the bones were removed, and the TRAP staining of the bones was performed. The OC surface of the bone surface was quantified by ImageJ and compared by the two-sided student’s t-test. p = 0.0181 (PBS + Exo19aKO vs IBSP + Exo), p = 0.2436 (PBS + Exo19aKO vs PBS + Exo), p = 0.4594 (PBS + Exo19aKO vs IBSP + Exo19aKO). n = 5 in all groups. b The ratio of OC surface relative to the bone surface was calculated after TRAP staining. p < 0.0001 (MCF7BoM2 vs MCF7BoM2 + CGA, n = 13 vs 8), p = 0.0001 (MCF7BoM2 vs MCF7BoM2 + GW4869, n = 13 vs 5). Representative H&E staining of the same field was shown (left panel) together with the TRAP staining (right panel). Scale bar, 100 µm. Two-sided student’s t-tests were performed. Data are presented as mean values ± SEM. c The legs of tumor-bearing mice were imaged by X-ray, and the bone density was measured by ImageJ. Two-sided student’s t-tests were performed. p < 0.0001 (MCF7BoM2 vs MCF7BoM2 + CGA, n = 13 vs 8), p = 0.0002 (MCF7BoM2 vs MCF7BoM2 + GW4869, n = 13 vs 5). Data are presented as mean values ± SEM. d T47D, T47DBoM, and T47D with ectopic expression of miR-19a or/and IBSP were intra-cardially injected into mice. One group of mice injected with T47DBoM also received CGA treatment. The growth of bone metastasis was monitored by IVIS Bioimager, and the Kaplan–Meier analysis and log-rank tests were performed for bone metastasis-free survival. p = 0.000036 (T47D vs T47DBoM); p = 0.0078 (T47D vs T47D/IBSP/miR-19a); p = 0.0003 (T47DBoM vs T47DBoM + CGA). e The legs of tumor-bearing mice were imaged by X-ray, and the bone density was measured by ImageJ. p = 0.0027 (T47D vs T47D/IBSP/miR-19a, n = 4 vs 12); p = 0.0111 (T47D vs T47DBoM, n = 4 vs 14); p = 0.0009 (T47DBoM vs T47DBoM + CGA, n = 14 vs 7). Two-sided student’s t-tests were performed. Data are presented as mean values ± SEM. f The ratio of OC surface relative to the bone surface was calculated and compared after TRAP staining. H&E staining of the same field was shown (left panels) together with the TRAP staining (right panels). Scale bar, 100 µm. p = 0.0028 (T47D vs T47D/IBSP/miR-19a, n = 4 vs12); p = 0.0023 (T47D vs T47DBoM, n = 4 vs 14); p = 0.0003 (T47DBoM vs T47DBoM + CGA, n = 14 vs 7). Two-sided student’s t-tests were performed. Data are presented as mean values ± SEM. g One week after intra-tibia injection of MCF7BoM2, mice were treated with CGA or DMDP. The growth of bone tumors was monitored by measuring the luciferase activity by IVIS Bioimager. Two-sided student’s t-tests were performed. p = 0.0215 (MCF7BoM2 vs MCF7BoM2 + CGA, n = 8 vs 6), p = 0.1920 (MCF7BoM2 vs MCF7BoM2 + DMDP n = 8 vs 6). Data are presented as mean values ± SEM. h After 24 days, the legs were removed and the bioluminescence of each leg was quantified. Two-sided student’s t-tests were performed. p = 0.0144 (MCF7BoM2 vs MCF7BoM2 + CGA, n = 8 vs 6), p = 0.1747 (MCF7BoM2 vs MCF7BoM2 + DMDP n = 8 vs 6). Data are presented as mean values ± SEM.

Fig 4: IBSP and miR-19a cooperatively induce osteoclastogenesis and promote cancer cell growth in the bone.a, b MCF7 cells were injected into the tibias of mice. These mice also received 2 µg exosomes prepared from MCF7BoM2 and 0.5 µg recombinant IBSP injected into the tibias every 2 days. The exosomes prepared from MCF7BoM2/miR-19aKO and PBS were used as controls. After three weeks, the bioluminescence signals of tibial bones were measured. Two-sided student’s t-tests were performed. p = 0.4670 (Exo19aKO + PBS vs Exo19aKO + IBSP, n = 4 vs 6), p = 0.1949 (Exo19aKO + PBS vs Exo + PBS, n = 4 vs 6), p = 0.0446 (Exo19aKO + PBS vs Exo + IBSP, n = 4 vs 6). Data are presented as mean values ± SEM (a). The bones were then removed and bone densities were measured by X-ray. Two-sided student’s t-tests were performed; p = 0.9280 (Exo19aKO + PBS vs Exo19aKO + IBSP, n = 4 vs 6), p = 0.9143 (Exo19aKO + PBS vs Exo + PBS, n = 4 vs 6), p = 0.0257 (Exo19aKO + PBS vs Exo + IBSP, n = 4 vs 6) Data are presented as mean values ± SEM (b). c The removed bones were decalcified, sectioned, and examined for the amount of OC surface by TRAP staining followed by quantification using the ImageJ. Two-sided student’s t-tests were performed; p = 0.7224 (Exo19aKO + PBS vs Exo19aKO + IBSP, n = 4 vs 6), p = 0.8773 (Exo19aKO + PBS vs Exo + PBS, n = 4 vs 6), p = 0.0002 (Exo19aKO + PBS vs Exo + IBSP, n = 4 vs 6). Data are presented as mean values ± SEM. d Integrin aVß3 was seeded on the bottoms of 96-well plates. IBSP was then added to the well in the presence or absence of the library of natural compounds. The bound IBSP was measured by colorimetric substrates of alkaline phosphatase (AP). The OD 405 was compared between CGA-treated groups and the DMSO-treated group. Two-sided student’s t-tests were performed; p < 0.0001 (n = 3) in all groups. CGA was found to most significantly inhibit the binding of IBSP to integrin aVß3. Data are presented as mean values ± SEM. e The role of IBSP as a chemoattractant of osteoclast cells, and the inhibitory effect of CGA on this chemoattraction were examined by the µ-Slide Chemotaxis assay. The movement of 20 OC cells with the highest mobility was tracked and the directionality was calculated by Chemotaxis and Migration Tool. p-value was calculated with the Rayleigh test (left: p = 0.0000002, right: p = 0.194763).

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.