Emerging evidence indicates the presence of tumor-initiating cells (TIC) or cancer stem cells (CSCs) in osteosarcoma. However, no study has demonstrated specific markers to identify osteosarcoma TICs with in vivo tumor formation ability. Additionally, there has been a lack of investigations gauging the contribution of osteosarcoma TICs to metastatic and drug-resistant properties. In this study, we have identified mouse and human osteosarcoma TICs using mesenchymal stem cell (MSC) markers CD117 and Stro-1. These markers were preferentially expressed in spheres and doxorubicin-resistant cells. Both mouse and human cells expressing these markers were sorted and analyzed for their abilities of tumor formation with as few as 200 cells, self-renewability, multipotency, drug resistance, metastatic potential, and enrichment of a metastasis-associated marker CXCR4 and a drug-resistance marker ABCG2. CD117+Stro-1+ cells efficiently formed serially transplantable tumors, whereas CD117−Stro-1− cells rarely initiated tumors. Upon orthotopic injections, CD117+Stro-1+ cell-derived tumors metastasized at a high frequency. Further, CD117+Stro-1+ cells showed high invasive and drug-resistant properties and were efficiently enriched for CXCR4 (20–90%) and ABCG2 (60–90%). These results suggest possible mechanisms for the high metastatic and drug-resistant properties of osteosarcoma TICs. In summary, CD117 and Stro-1 identify osteosarcoma TICs associated with the most lethal characteristics of the disease - metastasis and drug resistance - and these markers offer candidates for TIC-targeted drug delivery aimed at eradicating osteosarcoma.

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Osteosarcoma, the most common bone cancer, is the second highest cause of cancer-related death in children and adolescents. Approximately 90% of cases show micro-metastasis at diagnosis, making systematic chemotherapy the first choice of treatment. Despite intensive chemotherapy, the survival rate for high-grade osteosarcomas remains only 50-80%. The inadequacy of current treatments may result from the inability to effectively target cancer stem cells (CSCs) or tumor initiating cells (TI-Cs) of osteosarcoma. Thus, novel therapies targeting cancer stem cells (CSCs) in osteosarcoma is an urgent requirement for eradicating this dreadful disease. Here, we have identified mouse and human osteosarcoma CSCs using mesenchymal stem cell markers CD117 and Stro-1. These markers were preferentially expressed in spheres and doxorubicin-resistant cells. CD117+Stro-1+ cells efficiently formed serially transplantable tumors, whereas CD117-Stro-1-cells rarely initiated tumors. Upon orthotopic injections, CD117+Stro-1+-derived tumors metastasized at a high frequency. Further, CD117+Stro-1+ cells were capable of differentiating into adipogenic lineage, and were enriched for CXCR4 and ABCG2, respectively associated with metastasis and drug-resistance. Thus, CD117 and Stro-1 identify osteosarcoma CSCs with the most lethal characteristics of the disease—metastasis and drug-resistance—proposing these markers as potential candidates for CSC-targeted drug delivery towards eradication of osteosarcoma. Citation Information: Cancer Res 2009;69(23 Suppl):C11.

Supplementary Materials and Methods from CD117 and Stro-1 Identify Osteosarcoma Tumor-Initiating Cells Associated with Metastasis and Drug Resistance