Bone invasion in oral squamous cell carcinoma (OSCC)

Abstract

Background: Bone invasion in OSCC has poor outcomes. We hypothesise stimulation of osteoclasts directly by cancer cells; indirectly via osteoblasts. Methods and Results: Cross-over co-culture of OSCC and bone cell-lines shows: matrix-metalloproteinases (MMPs) from OSCC affect osteoblast differentiation and osteoclastogenesis; conditioned medium (CM) from osteoblasts increases MMPs in OSCC cells. Western blotting showed increased Twistl protein (not Runx2). RANKL/OPG ratio, zymogen and protein expression of MMP-9 were increased in bone cells cultured with CM from OSCC cell lines; rtPCR shows mRNA consistent with protein. All these molecules were expressed in invading malignant keratinocytes; all but OPG in osteoclasts. OSCC cell-lines were induced to epithelial-mesenchymal-transition (EMT) by TGF-β. Whilst this did not affect proliferation, Vimentin was induced and EMT markers Twist1 and N-cadherin up-regulated; Snaill and E-cadherin down-regulated. Of factors associated with invasion, MMP-2,9 were increased/unchanged in OSCC cell-lines. For osteo-clast-related molecules, membrane-type-1-MMP and RANKL were up-regulated; OPG down-regulated in all cells. CM from OSCC cells pre-treated with TGF-β prolonged survival of mature osteoclasts. For future biotherapies, we asked whether inhibition of monocyte-chemotactic-protein (MCP)-l, a chemokine which recruits/activates osteoclasts, might inhibit bone invasion. MCP-1 protein was observed in tumour cells and osteoclasts from 10 patients. Real-time-PCR demonstrated MCP-1 mRNA highly expressed by OSCC cell-lines. A plasmid with inhibitor of MCP-1 (7ND vector) was transfected into SCC25 cells: CM of SCC25-7ND efficiently inhibited formation of human osteoclasts from a CD14+ monocyte subpopulation. Conclusion: Extensive crosstalk occurs between cancer and bone cells, suggesting pathways for inhibition of bone invasion.