Understanding transformation and tumor initiation in the context of mesenchymal stem cells and osteosarcoma: Comparative studies in dogs and humans

Osteosarcoma (OS) is the most common primary bone tumor of human and dogs. This tumor is highly resistant to conventional chemo-and radiotherapies, and is composed of a varying proportion of undifferentiated and differentiated cell types of mesenchymal lineages. These attributes make OS as a plausible candidate for being a cancer driven by stem cells. Increasing evidence suggests that OS is a disease of blocked differentiation, caused by genetic and epigenetic changes that interrupt the process of osteoblastic differentiation. However, the origin of this tumor is still unknown. The issue that whether adult stem cells such as mesenchymal stem cells (MSCs) may require fewer or different steps than more differentiated cells to acquire a transformed phenotype is still unresolved.;The current set of studies were undertaken to gain insights into OS-relevant tumorigenic events in the appropriate cellular context as well as to study the functionally relevant markers for identification of tumor initiating fraction of this disease. Canine OS has been shown to be an appropriate and informative model for human OS. We first established the experimental system of canine MSCs by using a novel approach of modulation of cellular redox state, and extensively characterized these cells. We then incorporated defined and regulatable genetic elements into both canine and human MSCs by means of retroviral vectors. These incorporated genes were turned on at different time points of osteogenic differentiation, and evaluated for tumorigenic phenotypes in vitro . On the other hand, canine OS cells were sorted on the basis of expression of pluripotency-associated transcription factor-OCT4, and efflux of Hoechst dye (side population assay), which were then evaluated for tumorigenic potential in vitro and/or in vivo.;Our results indicate that alterations of multiple pathways seem to be necessary for tumorigenic transformation of MSCs or their differentiated descendants. We found that overexpression of exogenous MET and/or BMI1 was not sufficient to transform MSCs at any stage of differentiation. Undifferentiated MSCs underwent senescence after modest expansion of life-span. Although MSCs in early stage of differentiation were prevented from further differentiation, these cells also succumbed to senescence. On the other hand, MSCs at late stage of differentiation were completely refractory to oncogenic transformation, and underwent terminal differentiation. We found that although both OCT4-positive and OCT4-negative cell populations of a canine OS cell line were capable of giving rise to tumors, only OCT4-positive OS cells recapitulated the original tumor phenotype. Furthermore, cellular heterogeneity seemed to accelerate tumor development. We were also successful in isolating side population (SP) cells from canine OS, and we found that SP did not appear to be a major contributor of putative cancer stem cell phenotype in this tumor. Our results underscore the complexity of OS biology, and these findings will be useful to understand the disease process and design more effective strategies to treat OS in future.