Role of Noggin in Osteolytic Bone Metastasis Figure 4. Noggin silencing corrects the alterations of bone structural parameters in osteolytic lesions. A. The ratio of bone volume over total volume (BV/TV, +/2 SD) was determined by m-CT at day 25 after tumor cells inoculation; n = 6–7 animals for each experimental group. ***P,0.001, mock 5 versus Nog-KD clones and sham; **P,0.01, Nog-KD 14 versus PC-3/Fluc and mock 4; *P,0.05, sham and Nog-KD 17 versus PC-3/Fluc and mock 4. B. Total bone mineral content (TBMC; mg/mm +/2 SD) was measured by pQCT at day 25 after tumor cells inoculation; n = 6–7 animals for each experimental group. ***P,0.001, mock 5 versus Nog-KD 14; *P,0.05, sham and Nog- KD 17 versus mock 5, and Nog-KD 14 versus mock 4. doi:10.1371/journal.pone.0016078.g004 Nog-KD clones. This suggests that the difference in IL-8 mRNA expression was functionally irrelevant. Growth characteristics in vivo of Noggin-silenced PC-3/Fluc cells Tumor take was 100% for all xenografted clones. Weekly monitoring and quantification of intra-osseous tumor growth by BLI revealed that, in both experiments, noggin suppression in cancer cells had a moderate impact on their proliferation in vivo. Initially, the Nog-KD clones grew similarly to PC-3/Fluc and mock clones. However, their growth progressively slowed down and they could not reach the same tumor burden as for PC-3/Fluc and mock clones (Figure 6). In the second experiment, where tumor growth was monitored for longer periods, a growth arrest was even observed (Figure S3C). Metastatic potential of Noggin-silenced PC-3/Fluc cells In order to investigate whether noggin silencing influences the metastatic ability of PC-3 cells, systemic metastases were induced by injection into the left cardiac ventricle. The kinetics of development and the number of bone metastases per mouse 28 days after intra-cardiac injection of Nog-KD 17 cells did not differ from that induced by PC-3/Fluc and mock clones (not shown). After intra-cardiac injection of PC-3/Fluc cells systemic bone metastases develop asynchronously at variable bone Figure 5. Noggin silencing normalizes the bone histomorpho- metric indexes of bone formation and resorption in osteolytic lesions. A. Number of osteoclasts (N.Oc/BS; /mm, +/2 SD) on the endosteal surface in trabecular and cortical bone of sham-operated tibiae or on residual bone adjacent to cancer cells of tibiae xenografted with PC-3/Fluc, mock and Nog-KD clones. n = 6–7 animals for each experimental group. ***P,0.001, PC-3/Fluc and mock clones versus sham, and mock clones versus Nog-KD 17; **P,0.01, Nog-KD 17 versus PC-3/Fluc, and Nog-KD 14 versus PC-3/Fluc and mock clones. B. Number of osteoblasts (N.Ob/BS; /mm, +/2 SD) on the endosteal surface in trabecular and cortical bone of sham-operated tibiae or on residual bone adjacent to cancer cells of tibiae xenografted with PC-3/Fluc, mock and Nog-KD clones. n = 6–7 animals for each experimental group. ***P,0.001, PC-3/Fluc and mock clones versus Nog-KD clones, and mock 4 versus sham; **P,0.01, sham versus PC-3/Fluc and mock 5; *P,0.05, Nog-KD 14 versus sham. C. Percentage of endosteal surface in cortical and trabecular bone occupied by osteoclasts (Oc.S/BS; %, +/2 SD) in sham-operated and cancer cell-xenografted tibiae. n = 6–7 animals for each experimental group. ***P,0.001, mock clones versus sham and Nog-KD clones, and PC-3/Fluc versus sham and Nog-KD 17; **P,0.01, Nog-KD 14 versus PC-3/Fluc. D. Percentage of endosteal surface in cortical and trabecular bone occupied by osteoblasts (Ob.S/ BS; %, +/2 SD) in sham-operated and cancer cell-xenografted tibiae. n = 6–7 animals for each experimental group. ***P,0.001, PC-3/Fluc and mock clones versus sham and Nog-KD clones. doi:10.1371/journal.pone.0016078.g005 sites, making difficult a direct comparison of the radiographic aspect and growth progression of metastatic lesions at the same bone site among different animals. Furthermore, PC-3/Fluc cells, like the parental PC-3, almost invariably metastasize to the jaw, thereby impairing the nutritional status of the animals and, consequently, limiting the length of the experimental observation. These drawbacks prevented us to verify whether in this model the bone response and the tumor growth induced by the Nog-KD clone progress equally to those observed in the intra-osseous model. Discussion Here we show for the first time that in the PC-3 xenograft model of osteolytic bone metastasis the increase in osteoclast number is associated with impairment in osteoblast number and activity. This indicates that the osteolytic lesion is not only the result of increased bone resorption, but also of an additional PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e16078