BACKGROUND: Bone metastasis is a main cause of morbidity in breast cancer. Since breast cancer is a heterogeneous disease, the interactions of cancer cells with the skeletal host cells might also be diverse. We hypothesized that gene expression signatures induced by heterotypic interaction of breast cancer cells and osteoblasts might be of clinical relevance. METHODOLOGY/PRINCIPAL FINDINGS: We established an ex vivo co-culture model using benign breast epithelial cells or a panel of 5 malignant breast epithelial cells in combination with primary human osteoblasts and determined associated gene expression changes with HEEBO microarrays. Pretreatment gene expression profiles of 295 early stage breast cancers published from the Netherlands Cancer Institute with a median follow up of 12.6 years allowed evaluating in vitro effects in the in vivo situation.The effects of the interaction between osteoblasts and breast cancer cell lines of different origin were very heterogeneous. Hs578T cells started to proliferate in co-culture with osteoblasts, SKBR-3 induced a TGF-ß response and MDA-MB231 cells showed two distinct sets of up-regulated genes: A set of interferon response genes associated with an up-regulation of STAT1 was in vivo remarkably coherent providing a basis for segregation of tumors into two groups. In a uni-variate analysis, early stage tumors with high expression levels (n?=?136) of this gene set had a significantly lower overall survival rate (p?=?0.005) (63% at 10 years) than tumors with low expression levels (n?=?159) (overall survival: 77% at 10 years). The second gene set was associated with IL-6 and did not significantly change the overall survival rate (p?=?0.165), but was significantly associated with a shorter time to bone metastasis (p?=?0.049; 74% vs. 83% at 10 years). CONCLUSION/SIGNIFICANCE: An IL-6 gene expression pattern induced by heterotypic interaction of breast cancer cells with osteoblasts in vitro is associated with a higher rate of bone metastasis in vivo.