Effect of the microenvironment on osteoclast driven bone remodelling

Abstract

Despite their rigid appearance, bones are dynamic organs going through continuous remodelling cycles. The cells responsible for bone resorption are osteoclasts. Activation of osteoclasts is regulated by hormones, cytokines, growth factors and physical properties (such as oxygen tension and pH) of the bone microenvironment. Alterations to the local microenvironment in which osteoclasts and their precursors reside is of paramount importance for the control of their differentiation and activation which can result in aberrant bone resorption. The focus of this project has been to gain a better understating of how an altered microenvironment influences osteoclast formation and activation. To do so, a panel of feline and canine cancers cell lines as models of disrupted microenvironment was studied. In particular, a novel bone invasive feline oral squamous cell carcinoma (FOSCC) cell line was isolated (SMG), characterized and its capacity to induce osteoclast formation and activity were investigated. Cats very commonly present with FOSCC that invade the bone causing osteoclast activation and bone resorption that worsen the tumour prognosis. However, how the cancer cells modify their surrounding environment to activate osteoclasts is only partially understood. An indirect co-culture system was designed to induce osteoclast differentiation from feline bone marrow precursors in presence of conditioned media from the cell lines. SMG conditioned media effect was compared with another FOSCC conditioned media that was clinically non-bone invasive (SCCF1). Presence of either conditioned media in the presence of CSF-1 and RANKL enhanced osteoclast formation on plastic but only SMG conditioned media enhanced resorption on mineralized plates. Moreover, the presence of SMG conditioned media enabled feline bone marrow cells to survive in hypoxic conditions and differentiate into osteoclast. Finally the effect of SMG conditioned media on a murine late osteoblast/early osteocyte cell line (MLO-A5) was also examined. This effect was investigated as it is possible that cancer cells induce osteoclast activation via other cells in the bone microenvironment, such as osteoblasts and osteocytes. No significant effect of SMG conditioned media on MLO-A5 cells transcription of relevant genes was detected. A similar co-culture system was set up using canine bone marrow and initial studies established a method for canine bone marrow osteoclast differentiation. Canine osteosarcoma is common, especially in large breed dogs, and it causes local bone resorption hence conditioned media from four osteosarcoma cell lines (KTOSA, CSCOS, OSA-31 and D-17) were tested for their ability to alter osteoclast formation and resorption activity. Only conditioned media from KTOSA was able to enhance osteoclast formation on plastic. The proteins present in canine and feline conditioned media from all cell lines (representing the cell lines secretome) were analyzed by mass spectrometry and proteins were identified that may be involved in osteoclast differentiation and activation, such as AnnexinA2 and Insulin-like growth factor binding proteins. In summary, in this study a method to study the influence of an altered microenvironment on osteoclast differentiation and activation was established using companion animal cancer cells. Moreover, analysis of the protein secreted by selected cancer cell lines revealed potential therapeutic targets that might aid our understanding of osteoclast activation and bone resorption in disease.