Abstract 3087: Forced induction of differentiation in osteosarcoma tumor initiating cells
Authors: White Margaret Ellen , Guijarro Maria V. , Ghivizzani Steven , Gibbs Charles P. 
Published: July 2017

Osteosarcoma (OS) is a highly malignant bone cancer that is defined histologically by the secretion of immature osteoid. The chemotherapeutic regimen has not changed in the last 40 years and similarly, the five-year survival rate remains at ~60%, with the majority of fatalities arising from metastases in the lungs. OS is believed to originate from osteogenic committed progenitors, involving disruption of extracellular matrix synthesis in favor of proliferation. In previous work, our lab has developed a method to selectively identify Tumor Initiating Cells (TICs) in OS xenografts based on their ability to activate a transcriptional reporter comprised of a human OCT4 promoter linked to green fluorescent protein (GFP) coding sequence. Clonally derived, stably transfected OS Oct4/GFP+ TICs are capable of initiating and maintaining the growth of heterogeneous tumors and driving disease progression. The loss of GFP expression and tumorigenic capacity occurs during tumor formation in response to cues within the tumor microenvironment, resulting in adoption of a specialized secretory phenotype as an adaptive survival mechanism: a phenotypic change similar to that seen in physiological differentiation. During skeletal formation, Bone Morphogenetic Proteins (BMP) play key roles in osteogenic maturation. Because the OS lineage of origin suggests an innate sensitivity to BMP proteins, we hypothesize that BMP stimulation will force the induction of differentiation in OS TICs and will impair the ability of these cells to initiate and maintain tumor growth. To first probe that OS TICs respond to BMP stimulation, we detected by flow cytometry the expression of BMP Receptors Type II and 1A in two primary OS-derived cell lines (OS521 and OS156), with similar expression seen in Mesenchymal Stem Cells. Furthermore, by treating OS TICs with BMP heterodimers - BMP2/7 and BMP4/7 - we observed an activation of canonical BMP signaling, suggesting a functional and intact signaling network. BMP4/7 stimulation of OS521 TICs in vitro showed an increase in cells in the G1 phase of the cell cycle, visualized by DAPI, and higher p21 and p27 expression than controls, shown in immunoblotting. On the other hand, BMP4/7 treatment of OS156 TICs reduced its invasion capacity through a complex of Fibronectin and Bovine Serum Albumin by 33% (p= 0.00144) compared to untreated controls. Strikingly, in vivo studies revealed that time to tumor onset of BMP4/7 pretreated OS521 TICs in NSG mice at a dose of 3x104 cells was significantly longer than in controls (p=0.0003). In summary, our data suggests a reduction of OS tumorigenicity due to a BMP4/7 induced differentiation-based response. Such a differentiation-based therapy could become a more effective and safer alternative to cytotoxic chemotherapy. Future studies include TIC treatment with hypoxia, adenosine, and calcium, to investigate the underlying mechanisms responsible for BMP involvement in the tumorigenic capacity of osteosarcoma.

Citation Format: Margaret Ellen White, Maria V. Guijarro, Steven Ghivizzani, Charles P. Gibbs. Forced induction of differentiation in osteosarcoma tumor initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3087. doi:10.1158/1538-7445.AM2017-3087