Abstract LB-298: The bone marrow microenvironment increases osteosarcoma tumour cell migration by signaling through uPA/uPAR
Authors: Endo-Munoz Liliana B. , Cumming Andrew , Loo Dorothy , Thang Sarah , Hill Michelle , Sommerville Scott , Saunders Nicholas 
Published: August 2014

Abstract
Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. Pulmonary metastasis is the major complication of OS and can occur in up to 50% of cases, resulting in just 10–20% long-term survival for these patients. We have shown that the bone microenvironment contributes to the metastatic potential of osteosarcoma (Endo-Munoz et al. Cancer Res 70:7063–72, 2010). Specifically, loss of osteoclasts increases the metastatic potential of OS in vitro and in vivo, and bone marrow cells (BMC) increase the migration of OS cells in vitro. However, the factors mediating this increase in migration has not been elucidated. Using a combined transcriptomic and proteomic (SILAC) approach, we found very high levels of mRNA and protein expression of the urokinase plasminogen activator (uPA) and its receptor (uPAR) in metastatic, but not in non-metastatic OS. Furthermore, examination of the cytokines secreted in the conditioned medium of metastatic and non-metastatic OS cells in an antibody array showed high and selective secretion of uPA by metastatic OS cells only. Addition of this conditioned medium to metastatic OS cells significantly (P &lt; 0.007) increased their in vitro migration to levels similar to those observed with recombinant human uPA. No effect was observed for non-metastatic cells. Overall migration was significantly (P &lt; 0.005) inhibited by addition of a neutralizing monoclonal antibody against uPAR, indicating that uPA/uPAR signaling acts in a positive feedback loop in the regulation of inherent OS cell migration. To investigate whether the uPA/uPAR system could also be involved in signaling by BMC, we performed a comparative transcriptomic analysis of metastatic and non-metastatic OS cell lines before and after treatment with BMC conditioned medium. We found uPA to be the most significantly upregulated gene after BMC treatment (FC = 7.97, B = 18.82), suggesting that increased uPA gene expression induced by BMC could contribute to enhanced secretion of uPA by OS cells, which in turn could augment signaling through uPAR to increase migration. Finally, we explored the possibility that BMC may also secrete uPA into the microenvironment to further increase migration of OS tumours in close proximity. Immunohistochemistry on femurs of mice bearing OS tumours showed high expression of uPA in the bone marrow as well as in the leading edges of the tumour.
These data indicate that BMC increases OS cell migration not only by increasing uPA gene expression in the tumour, but also by secreting uPA which serves to amplify the migration-inducing signaling of tumour-secreted uPA. This opens the possibility that uPA inhibitors, which are already in clinical trials for a number of cancers, may also be a useful therapeutic in the treatment for OS metastasis.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-298. doi:10.1158/1538-7445.AM2011-LB-298