Recently, there has been renewed interest in the role of tumor stem cells (TSCs) 
in tumorigenesis, chemoresistance, and relapse of malignant tumors including 
osteosarcoma. The potential exists to improve osteosarcoma treatment through 
characterization of TSCs and identification of therapeutic targets. Using 
transcriptome, proteome, immunophenotyping for cell-surface markers, and 
bioinformatic analyses, heterogeneous expression of previously reported TSC or 
osteosarcoma markers, such as CD133, nestin, POU5F1 (OCT3/4), NANOG, SOX2, and 
aldehyde dehydrogenase, among others, was observed in vitro. However, 
consistently significantly lower CD326, CD24, CD44, and higher ABCG2 expression 
in TSC-enriched as compared with un-enriched osteosarcoma cultures was observed. 
In addition, consistently higher CBX3 expression in TSC-enriched osteosarcoma 
cultures was identified. ABCA5 was identified as a putative biomarker of TSCs 
and/or osteosarcoma. Lastly, in a high-throughput screen we identified 
epigenetic (5-azacytidine), anti-microtubule (vincristine), and anti-telomerase 
(3,11-difluoro-6,8,13-trimethyl- 8H-quino [4,3,2-kl] acridinium methosulfate; 
RHPS4)-targeted therapeutic agents as candidates for TSC ablation in 
osteosarcoma.