onwards. Cancer-induced changes in osteoblast and osteoclast numbers were evident from day 15, but differed substantially depending on whether the cells were in direct contact with the tumour. Compared to naïve controls, areas of bone in direct contact with the tumour had significantly reduced osteoblast but increased osteoclast numbers. Conversely, in areas of bone away from the tumour, numbers of osteoblasts were increased and numbers of osteoclasts decreased. Conclusions: The spatial and temporal relationship between bone and cancer cells may have a pronounced effect on osteoblasts and osteoclasts prior to the formation of osteolytic lesions. Our data suggest that early treatment may be required to prevent cancer-induced bone disease. Conflict of interest statement: I. Holen, research funding from Novartis and speakers fee from Novartis. doi:10.1016/j.bone.2010.10.114 P-59 Molecular characterization of the stromal response to osteoblastic bone metastasis by tissue-compartment specific transcriptional profiling B. Ozdemir a, *, C. Secondini a , A. Wetterwald a , R. Schwaninger a , W. Raffelsberger b , O. Poch b , M. Delorenzi c , G. van der Pluijm d , G.N. Thalmann a , M.G. Cecchini a a Department of Urology and Department of Clinical Research, University of Bern, Bern, Switzerland b Department of Structural Biology and Genomics, Institure of Genetics and Molecular and Cellular Biology, Illkirch-Strasbourg, France c Bioinformatic Core Facility, Swiss Institute of Bioinformatics, Lausanne, Switzerland d Department of Urology, Leiden University Medical Center, Leiden, The Netherlands Progression of primary and metastatic cancer depends on tissue- specific stromal support. Gene expression profiling may identify key molecular determinants and biomarkers of the stromal response in bone metastasis. However, the relative contribution of the stromal compart- ment to the transcriptome cannot be readily assessed by gene exression profiling of bulk tumour tissue. To obviate this obstacle, we derived an osteoblastic bone metastasis-associated stroma transcriptome (ObBMST) from mouse bone xenografted with osteoinductive, human prostate cancer (PCa) cell lines, either C4-2B4 or VCaP by tissue compartment-specific transcriptional profiling (TCTP). This novel meth- od exploits the divergence between human and mouse transcriptomes in combination with a highly mouse-specific gene array platform, and the exclusion of cross-hybridizing cDNA probes by a computational mask. C4-2B4- and VCaP-xenografted bones shared a common ObBMST list of 323 differentially regulated (≥2-fold change) genes, as compared to sham-operated bones. Gene ontology clusters of the 206 up-regulated genes related them to extracellular matrix (ECM), cell–matrix interac- tions, osteo- and angio-genesis. The stroma-specificity and the up- regulation of an ObBMST-derived gene cluster were validated by quantitative RT-PCR and immunohistochemistry. Induction of this gene cluster in cultured osteoblasts was dependent on direct contact with cancer cells. Notch-specific inhibitors blocked this induction. Human primary and bone metastatic PCa and mammary carcinome (MCa) showed equivalent stromal expression of selected proteins encoded by the ObBMST-derived gene cluster. Meta-analysis of a public repository of gene expression studies revealed that the ObBMST-derived gene cluster marks the transition from localized to invasive carcinoma in multiple, human cancer types and may negatively correlate with patient survival. TCTP reliably provides the gene profile of bone stroma reactive to osteoinductive PCa cells. An ObBMST-derived gene cluster generated a prognostic signature of cancer progression. Proteins encoded by this gene cluster are potential biomarkers of bone metastasis. Conflict of interest statement: None declared. doi:10.1016/j.bone.2010.10.115 P-60 Combination vs single agent treatment: The effects of zoledronic acid +/-doxorubicin on breast tumour growth in bone prior to development of osteolytic bone disease H.K. Brown*, P.D. Ottewell, C.A. Evans, R.E. Coleman, I. Holen Oncology, University of Sheffield, Sheffield, UK Background: Administration of doxorubicin (dox) followed 24 h later by zoledronic acid (zol) reduces tumour burden and bone destruction in models of advanced breast cancer with extensive lytic disease. However, little is known about the bone cell–cancer cell interactions associated with early stages of tumour growth in bone, and the effects of administration of combination therapy at this point. Aims: This project investigates the effects of sequential treatment with dox followed by zol on the early stages of breast tumour growth in bone, prior to the appearance of osteolytic lesions. Methods: 1× 105 MDA-231-GFP breast cancer cells were injected i.c. in 5–6-week old female balb/c nude mice 2 days prior to administration of a single dose of PBS, zol (100 μg/kg), dox (2 mg/kg), or dox followed 24 h later by zol (n= 10/group). Animals were sacrificed on day 15 and hind legs were processed for X-ray, uCT and histology. Serum was collected for bone resorption marker (CTX) analysis. Tumour area, osteoclasts and osteoblasts were assessed on histological slides following H&E/TRAP staining. Findings: There was no evidence of lytic bone lesions in any group at this early disease stage. In agreement with this, trabecular bone volume was unchanged in tumour bearing control (PBS) compared to naïve animals, and CTX serum levels were unchanged in all groups compared to naïves. Trabecular bone volume was significantly increased in animals treated with zol (alone or in combination with dox), compared to PBS control. Initial evaluation of tumour area indicated a decrease in animals treated with dox and dox- then-zol, compared to PBS and zol only. Conclusion: Our data demonstrate that zoledronic acid alone does not eliminate breast tumour growth in bone, even when administered at very early stages of disease (day 2). In contrast, a single sequential administration of doxorubicin followed by zoledronic acid reduces osseous tumour growth. Conflict of interest statement: R.E. Coleman, research funding; Novartis, consultancy: Novartis and Amgen, speaker fees: Novartin, Amgen. I. Holen, research funding: Novartis and speaker fee: Novartis. doi:10.1016/j.bone.2010.10.116 P-61 CpG-ODN inhibits Smad-dependent BMP signaling; Effects on myeloma cell apoptosis and in vitro osteoblastogenesis N. Nørgaard a , T. Holien a , S. Jönsson b , H. Hella a , T. Espevik a , A. Sundan a , T. Standal a, * a Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway b Section of Hematology, Department of Internal medicine, Sahlgrenska University Hospital, Gothenburg, Sweden The TLR9 agonist CpG-oligodeoxynucleotide with a phosphorothioate backbone (PTO-CpG-ODN) is evaluated in clinical trials as a vaccine adjuvant or as treatment of cancers. Bone morphogenetic proteins (BMPs) regulate growth and differentiation of several cell types, and also induce apoptosis of cancer cells. Cross talk between BMP- and Toll-like receptor (TLR)-signaling has been reported, and we aimed to investigate Abstracts / Bone 48 (2011) S22–S55 S41