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Expression of an Exogenous Human Oct-4 Promoter Identifies Tumor-Initiating Cells in Osteosarcoma

¹ Department of Orthopaedics and Rehabilitation, and ² McKnight Brain Institute, Department of Neuroscience, University of Florida College of Medicine; ³ Flow Cytometry Core Laboratory, University of Florida Interdisciplinary Center for Biotechnology Research, Gainesville, Florida; ⁴ Department of Orthopaedic Surgery and Rehabilitation, University of Nebraska Medical Center, Lincoln, Nebraska; and ⁵ Department of Statistics and Actuarial Sciences, University of Central Florida, Orlando, Florida

Requests for reprints: C. Parker Gibbs, Department of Orthopaedics and Rehabilitation, University of Florida College of Medicine, P.O. Box 112727, Gainesville, FL 32611. Phone: 352-273-7365; Fax: 352-273-7388; E-mail: gibbscp{at}ortho.ufl.edu.

Key Words: Oct-4 • Osteosarcoma • Cancer Stem Cell • Embryonic Stem Cell • Tumorigenicity

We explored the nature of the tumor-initiating cell in osteosarcoma, a bone malignancy that predominately occurs in children. Previously, we observed expression of Oct-4, an embryonal transcriptional regulator, in osteosarcoma cell cultures and tissues. To examine the relationship between Oct-4 and tumorigenesis, cells from an osteosarcoma biopsy (OS521) were stably transfected with a plasmid containing the human Oct-4 promoter driving a green fluorescent protein (GFP) reporter to generate the transgenic line OS521Oct-4p. In culture, only 24% of the OS521Oct-4p cells were capable of activating the transgenic Oct-4 promoter; yet, xenograft tumors generated in NOD/SCID mice contained 67% GFP⁺ cells, which selectively expressed the mesenchymal stem cell–associated surface antigens CD105 and ICAM-1. Comparison of the tumor-forming capacity of GFP-enriched and GFP-depleted cell fractions revealed that the GFP-enriched fractions were at least 100-fold more tumorigenic, capable of forming tumors at doses of <300 cells, and formed metastases in the lung. Clonal populations derived from a single Oct-4/GFP⁺ cell were capable of forming tumors heterogeneous for Oct-4/GFP expression. These data are consistent with the cancer stem cell model of tumorigenesis in osteosarcoma and implicate a functional link between the capacity to activate an exogenous Oct-4 promoter and tumor formation. This osteosarcoma tumor-initiating cell appears highly prolific and constitutes a majority of the cell population in a primary xenograft tumor, which may provide a biological basis for the particular virulence of this type of cancer. [Cancer Res 2009;69(14):5648–55]