Osteosarcoma Stem Cells Have Active Wnt/β‐catenin and Overexpress SOX2 and KLF4

Osteosarcoma is a bone tumor, displaying significant cellular and histological heterogeneity and a complex genetic phenotype. Although multiple studies strongly suggest the presence of cancer stem cells in osteosarcoma, a consensus on their characterization is still missing. We used a combination of functional assays (sphere-forming, Aldefluor, and side-population) for identification of cancer stem cell populations in osteosarcoma cell lines. Expression of stemness-related transcription factors, quiescent nature, in vivo tumorigenicity, and Wnt/β-catenin activation were evaluated. We show that different cancer stem cell populations may co-exist in osteosarcoma cell lines exhibiting distinct functional properties. Osteosarcoma spheres are slowly-proliferating populations, overexpress SOX2, and KLF4 stemness-related genes and have enhanced tumorigenic potential. Additionally, spheres show specific activation of Wnt/β-catenin signaling as evidenced by increased nuclear β-catenin, TCF/LEF activity, and AXIN2 expression, in a subset of the cell lines. Aldefluor-positive populations were detected in all osteosarcoma cell lines and overexpress SOX2, but not KLF4. The side-population phenotype is correlated with ABCG2 drug-efflux transporter expression. Distinct functional methods seem to identify cancer stem cells with dissimilar characteristics. Intrinsic heterogeneity may exist within osteosarcoma cancer stem cells and can have implications on the design of targeted therapies aiming to eradicate these cells within tumors. J. Cell. Physiol. 231: 876-886, 2016. © 2015 Wiley Periodicals, Inc.