Introduction: Tumor-initiating cells (TICs) are a subpopulation of therapy resistant cells in osteosarcoma. To date, TICs have been experimentally defined based on the chosen isolation technique. Common to all isolation methods, TICs demonstrate a high degree of the multi-drug resistance phenotype, are more invasive, metastasize readily, and have increased capacity for tumorigenesis. Given this phenotype TICs represent a viable therapeutic target. Cultivating cells in low-adherence, serum-deprived conditions results in spherical colony formation. Certain growth factors present in serum drive epithelial-to-mesenchymal transition, and typically confound TIC models of carcinoma. The effect of serum on mesenchymal-based sarcoma TIC models is less clear. Recent literature suggests that merely the presence of low-adherence conditions can enrich in TICs. We have shown that osteosarcoma cells cultured in low-adherence conditions in the presence of 10% fetal bovine serum (FBS) form spheres, exhibit an increased expression of genes implicated in developmental programs and stem cells, are more resistant to chemotherapy, and more readily initiate tumors than adherent cells when injected into mice.

Methods: The osteosarcoma cell lines 143B, mHOS, and MG-63 were cultured in low-adherence plates using DMEM or MEM media supplemented with 10% FBS. Spheres were routinely grown for 4-6 days between passages and dissociated using 0.05% trypsin. Relative mRNA expression levels of Nanog, Sox2, Oct4 and Axin2 was assessed using quantitative-PCR (SYBR Green). Resistance to chemotherapy (doxorubicin and cisplatin) was determined by comparing both adherent cells and spheres (in low adherence 96-well plates) grown to at least the second passage. After 72 hours IC50 values were calculated using the CellTitre Glo luminescence assay. To assess in vivo tumorgenicity, adherent and sphere cells were dissociated and injected into the flanks of nude mice at a density of 10,000 cells. Upon tumor formation the mice were euthanized and the tumors re-implanted into a second set of mice to test their ability to serially transplant.

Results: Sarcospheres can be successfully and reproducibly grown and passaged in low-adherent culture conditions in media supplemented with 10% FBS. Expression profiling demonstrates increased expression of Sox2, Oct4, Nanog, and Axin2, all genes that have been identified to be associated with developmental programs and stem cells. Sarcospheres also displayed increased chemoresistance compared to adherent cultures to both cisplatin (IC50 change of 1.9, 6.9, and 6.2-fold in 143B, mHOS and MG-63, respectively) and more so to doxorubicin (IC50 change of 12.7, 35 and greater than 74-fold in 143B, mHOS, and MG-63, respectively). Additionally, in a pilot subcutaneous xenograft study, using sphere and adherent cells, differential growth was observed within the TIC-enriched populations demonstrating (1) earlier tumor initiation and (2) a lower overall proliferation rate among the primary and serially transplanted tumors confirming a persistence of the TIC phenotype through in vivo passaging.

Conclusions: Osteosarcoma cells cultured in low-adherence conditions in the presence of serum display many of the characteristics of putative tumor-initiating cells including robust sphere formation, upregulation of key stemness mediators, enhanced chemoresistance, and in vivo tumor initiation and serial transplantability. Using this system, we aim to further evaluate these stem-like populations in order to elucidate potential therapies targeting this specialized, chemoresistant niche.

Citation Format: Jessica M. Foley, Noel R. Monks, Donald J. Scholten, II, David J. Monsma, Dawna Dylewski, Paula J. Davidson, Matthew R. Steensma. Chemotherapy-resistant subpopulations in a tumor-initiating cell model of human osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A70.