Bone Abstracts

Osteosarcoma is a heterogeneous tumor from the mesenchymal lineage, and is the most common form of primary bone cancer. Normally, lesions contain undifferentiated cancer stem cells (CSCs) that support uncontrolled growth/proliferation, and pre-osteoblasts that form excessive amounts of immature bone. CSCs are characterized by high expression of the transcriptional regulators Sox2 and yes-associated protein (YAP) that are essential for tumorigenicity. YAP is restrained by the Hippo pathway, via its phosphorylation and nuclear exclusion, whereas Sox2 acts as an inhibitor of this pathway while also maintaining cell “stemness.” Interestingly, YAP activity is also mechanosensitive, and the main transcriptional activator that responds to changes in substrate stiffness. In healthy bone physiology, mechanical signals are vital for homeostasis, and the primary source of mechano-stimulation is shear-stress induced via interstitial fluid flow. Our objective was to establish the effect of fluid flow induced shear-stresses (FSS) on osteosarcoma tumorigenicity and heterogeneity. Specifically, we investigated whether FSS regulates YAP activity and osteogenic gene expression in osteosarcoma derived cells. Tumor cells (mOS482) were obtained from spontaneous osteosarcoma occurring in mice with bone specific knockouts of pRb and p53. Monolayer cell-cultures were exposed to physiological FSS in a parallel-plate flow device, while controls experienced no flow. Cells were isolated and subcellular location of YAP and osteogenic gene expression were quantified using immunocytochemistry and qPCR, respectively. Nuclear YAP and Osterix expression increased due to FSS (P=0.01), while two other osteogenic genes (Runx2, -Osteocalcin) increased slightly, but did not reach significance. FSS also increased osteoprotegerin expression (OPG, P=0.03), however the ratio between OPG and RANKL did not change. These findings demonstrate that osteosarcoma cells are mechano-sensitive and mechanical signals increase their expression of osteogenic genes. This concept may present novel paradigms where tumor cell behavior and fate could be modulated by targeting its physical micro-environment.