Abstract 1412: Utilizing cell surface markers to define osteosarcoma and the stages of osteoblast differentiation
Authors: Koirala Pratistha , Poon Vincent , Piperdi Sajida , Park Amy , Fremed Michael , Roth Michael , Gill Jonathan , Gorlick Richard 
Published: August 2015

Abstract
Background
Osteosarcoma (OS) is the most common primary malignant bone tumor in children. Despite advances in OS treatment, survival rates have remained stagnant over the past three decades. This may in part be reflective of the complex nature of OS—although all OS’ are pleomorphic, spindle shaped cells that are capable of producing osteoid, this is often where the similarities between tumors ends. Tumor phenotypes can be used to classify OS into multiple groups including the most common form, conventional, which can be further subdivided into osteoblastic, chondroblastic, or fibroblastic OS. Although these tumors display unique phenotypes, they have similar response rates to standard treatments such as chemotherapy and surgery suggesting that they may have a common progenitor, which we seek to identify.
Methods
In order to determine the OS cell of origin, we first performed flow cytometry analysis using cell surface markers that are differentially expressed on mesenchymal stem cells (MSC) and osteoblasts (OB)—CD44, CD105, CD54, CD49b, CD325, and GD2. MSCs were differentiated into OBs using induction media and cells were collected for analysis at multiple time points in differentiation; day (d)0, d3, d5, d10, d15, and d20. MSCs became fully differentiated OBs by d20, as determined by alizarin red staining. Similar flow cytometry analysis was performed on four OS standard cell lines, SaOS, U2OS, HOS, and HOS-143B, and marker profiles were compared to MSC to OB differentiation.
Results
CD44 and CD105 expression, both of which are known MSC markers, was high in MSCs and immediately dropped off during differentiation, while decrease in CD325 expression was more gradual. CD49b expression increased over time and GD2 expression varied greatly. CD54 expression peaked at d10 and decreased as differentiation continued. Expression of these markers was varied in the OS standard cell lines. These markers can be used to sort out specific populations of cells at different time points during differentiation.
Conclusions and Future Directions
In this study we have identified potential markers of OB progenitor populations and compared their expression to OS standard cell lines. We will use these prospective makers to sort progenitor populations and drive their differentiation into OBs, chondroblasts, and fibroblasts in order to determine branch points in MSC differentiation. Finally, in order to assess the potential of these cells to form OS, we plan to transform candidate progenitor cells with human telomerase reverse transcriptase, simian virus 40 large T antigen, and lentivirus containing oncogenic H-Ras serially.
Citation Format: Pratistha Koirala, Vincent Poon, Sajida Piperdi, Amy Park, Michael Fremed, Michael Roth, Jonathan Gill, Richard Gorlick. Utilizing cell surface markers to define osteosarcoma and the stages of osteoblast differentiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1412. doi:10.1158/1538-7445.AM2015-1412