Abstract 2601: Interaction between bone marrow mesenchymal stem cells and neuroblastoma cells leads to osteoblastogenesis and VEGFA expression.
Authors: HaDuong Josephine H. , Blavier Laurence , Baniwal Sanjeev K. , Frenkel Baruch , DeClerck Yves A. 
Published: August 2014

Abstract
Bone metastasis in patients with neuroblastoma often heralds a poor prognosis despite intensive therapy. We previously reported that neuroblastoma cells interact with osteoclasts and bone marrow mesenchymal stem cells (BMSC), and that the nature of this interaction is both osteolytic and pro-tumorigenic. In many cancers, the suppression of osteoblastogenesis plays a part in increased osteolysis. However, little is known about the interaction between neuroblastoma cells and osteoblasts within the bone marrow microenvironment. We have examined this interaction within the context of BMSC differentiation to osteoblasts by bone morphogenetic protein 4 (BMP4).
To examine the effect of secreted factors from neuroblastoma cells on the BMP4 induced differentiation of murine BMSC, we cultured them in the presence or absence of neuroblastoma cells using culture inserts. We found that the mRNA encoding the osteoblast marker alkaline phosphatase (ALP) was increased by 10 fold and ALP protein level and activity were 1.5 fold higher in the presence versus absence of neuroblastoma cells. The mRNAs encoding the osteoblast master regulator Runx2 and its target osteocalcin were increased by 4 and 1.5 fold (p&lt;0.05), respectively, when BMP4 treated BMSC were co-cultured with tumor cells. Further, neuroblastoma cells stimulated the proliferation of BMSC in co-culture by &gt;2 fold. Together, these data indicate that neuroblastoma cells synergise with BMP4 to promote osteoblast growth and differentiation from BMSC.
To understand the mechanism by which neuroblastoma cells enhance osteoblastogenesis, we compared the mRNA profile of murine MSC driven to differentiate into osteoblasts by doxycycline-induced Runx2 expression (ST2Rx2dox) in the presence or absence of neuroblastoma cells. Triplicate RNA samples were labeled and hybridized to Illumina BeadChips containing probes for &gt;19,100 genes. In silico analysis identified 9 genes upregulated when co-cultured with neuroblastoma cells. Of these, two (Spondin2 and VEGFA) encode proteins with established roles in osteoblastogenesis. To validate these findings in primary murine BMSC, we showed by RT-qPCR a 2 fold increase in VEGFA mRNA in BMSC treated with BMP4 in the presence versus absence of neuroblastoma cells. ELISA analysis demonstrated a parallel 2 fold increase in intra- and extracellular VEGFA protein levels. We then found that the treatment of BMSC with BMP4 and VEGFA together led to a 1.5 fold increase (p&lt;0.05) in BMSC viability compared to VEGFA alone or no treatment, suggesting that VEGFA may provide a BMP4 dependent growth advantage.
Our data suggest that in contrast to many cancers that suppress osteoblastogenesis, neuroblastoma may promote osteoblastogenesis in part by enhancing the expression of VEGFA in BMSC. We are currently investigating the consequence of this osteoblastogenic activity on neuroblastoma bone metastasis.
Citation Format: Josephine H. HaDuong, Laurence Blavier, Sanjeev K. Baniwal, Baruch Frenkel, Yves A. DeClerck. Interaction between bone marrow mesenchymal stem cells and neuroblastoma cells leads to osteoblastogenesis and VEGFA expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2601. doi:10.1158/1538-7445.AM2013-2601