Role of Noggin in Osteolytic Bone Metastasis Figure 7. Model of osteolysis integrating a dual mechanism of stimulated bone resorption and ‘‘uncoupled’’ bone formation. The osteolytic lesion is the net result of two different mechanisms leading to uncoupling of normal bone remodeling: A. Excess of bone resorption, consequent to enhanced osteoclast recruitment and activity induced by cancer cell-derived osteolytic cytokines. The subsequent release of bone matrix-integrated growth factors further stimulates cancer cell growth. B. Suppression of bone formation, consequent to interference with osteoblast recruitment and activity induced by cancer cell-derived noggin. C. Noggin silencing in cancer cells re-establishes osteoblast recruitment and activity, and, thus, ‘‘bone coupling’’, with consequent bone formation/repair. As an effect of the rescued osteoblast activity, tumor progression is contained by yet unidentified osteoblast-derived factors. doi:10.1371/journal.pone.0016078.g007 Bern State (Permit Number: 15/07). Mice were housed in individual ventilated cages in strict accordance to the Swiss Guidelines for the Care and Use of Laboratory Animals. Autoclaved water and sterile mouse chow were provided ad libitum. Animals xenografted with human cancer cells were carefully monitored for signs of pain or distress, loss of body weight and radiological signs of severe osteolysis and imminent risk fracture. When any of these signs appeared or at the end of the experimental period, the mice were sacrificed by CO2 euthanasia. Animals BALB/c nude mice were purchased from Charles River France (L’Arbresle, France) and they were 7-8 weeks old when used for the intra-osseous inoculation of tumor cells and 5 weeks old for the intra-cardiac injection. For surgical manipulation, mice were anesthetized as described previously [18]. Cell lines and cell culture The osteolytic human CaP cell line PC-3 (ATCC/LGC Promochem, Molsheim, France) was grown in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS (Biochrom AG, Berlin, Germany) and antibiotics. Cells were shown to be free of mycoplasma by PCR (Venor GeM, Minerva Biolabs GmbH, Berlin, Germany). PC-3 cell-derived clones For in vivo tracking, PC-3 cells were permanently transduced with the firefly luciferase (Fluc) using a self-inactivating lentiviral vector [19] and cloned by limiting dilution. The PC-3/Fluc clone was electroporated (Nucleofector, Amaxa, Lonza, Verviers, Belgium) with either the combination of three vectors containing shRNA targeting the noggin transcript or a non-targeting sequence (SureSilencing, SABiosciences, LucernaChem, Lucerne, Switzerland) to generate Noggin knock-down (Nog-KD) and negative control (mock) clones, respectively. The sequences of the shRNA used are listed in Supporting Information (Table S1). Individual stably transfected clones were derived by selection with Puromycin (1 ug/ ml) and Neomycin (200 ug/ml) (Sigma-Aldrich, Buchs, Switzerland). Generation of conditioned media Cells were seeded at a density of 2.56104 cells/cm2 in 10 cm culture dishes. After 1 day, the medium was replaced with 10 ml serum-free medium and the cells were cultured for further 48 hours. The cell-conditioned media (CM) were centrifuged and stored at –20uC for later use. The cell number was determined and the volume of CM was adjusted for differences in cell density between samples. CM were dialyzed against serum-free medium and concentrated 10-fold by lyophilisation. Cell proliferation assay Cells were seeded at a density of 104 cells/cm2 in microtiter plates and cultured for 4 days. Cell proliferation was determined by measuring bromodeoxyuridine (BrdU) incorporation with a colorimetric ELISA (Cell Proliferation ELISA, BrdU; Roche Diagnostics, Rotkreuz, Switzerland) according to the manufactur- er’s protocol. PLoS ONE | www.plosone.org 7 January 2011 | Volume 6 | Issue 1 | e16078