Osteosarcoma (OS) is the most common primary malignant bone tumor in children and young adults. It remains unclear at what point in the pathway of differentiation between human mesenchymal stem cells (hMSCs) and osteoblasts (OBs), OS originates. Since high grade OS frequently demonstrates histologic variability, as well as the potential for multi-lineage differentiation, some consider the hMSC as the cell of origin in OS, whereas others believe the osteoblast to be the most likely cell of origin. Identifying the primary cell of origin is crucial in understanding the molecular pathogenesis of OS. To address the potential for hMSCs or OBs to transform into OS, we have performed insertional mutagenesis in hMSCs and OBs with defined genetic elements which have the potential to lead to tumorigenesis and whose pathways have been altered in OS patient specimens. Serial introduction of the viral constructs hTERT (T), SV40Tag (S), and H-RAS (R), led to spindle cell tumor formation in mice. However, MSC-TSR tumors did not form osteoid, whereas OB-TSR tumors showed only scant production of osteoid. Also the addition of β-catenin to MSC-TS cells failed to lead to tumor formation in mice. Based on these findings, MSC-TS and OB-TS cell lines may be ideal platforms for further analysis of the impact of the genetic transformation of hMSCs and OBs into OS. c-Myc has been shown to be overexpressed in OS cells and hence has been suggested as an oncogene. It has been also suggested as a novel target of RUNX2 through rescue from the apoptosis.

MSC-TS and OB-TS cells were transformed with a retrovirus containing human c-Myc. Drug resistant colonies were picked up 21 days after selection to obtain stably transformed cell lines. Quantitative PCR and western blots were carried out to detect both gene and protein expression patterns in transformed cell lines, respectively. Furthermore, in order to determine the expression pattern of c-Myc in OS, quantitative PCR and western blots were also performed on human osteosarcoma primary samples, xenografts and cell lines. All functional assays outlined below will be performed to determine the tumorigenic properties of transformed cell lines.

Six out of seven (86%) osteosarcoma primary samples showed a significantly greater c-Myc gene expression level compared to the positive control cell lines. Selection of stably transformed clones and comparison between cell lines and characterizations are underway. Further ongoing characterization includes: soft agar assays, in-vivo tumorigenic assays, histological examination for osteoid production and other OS specific immunohistochemical markers, proliferation, invasion and migration, and differentiation assays. It is our hope that this study will highlight the model that closely recapitulates the human osteosarcoma phenotype by forming a malignant spindle cell tumor that produces aberrant osteoid, and has the potential for multi-lineage differentiation.

Citation Format: Sajida Piperdi (Thein), Wendong Zhang, Daria Ivenitsky, Yidan Zhang, Yunjia Zhang, David S. Geller, Bang Hoang, Rui Yang, Jonathan B. Gill, Michael Roth, Richard Gorlick. Introducing c-Myc into transformed human mesenchymal stem cells and osteoblasts to recapitulate the osteosarcoma phenotype [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 1950. doi:10.1158/1538-7445.AM2017-1950