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.
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.|Pharmacology and Experimental Therapeutics, Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal.|CNC.IBILI, University of Coimbra, Coimbra, Portugal.|Center of Investigation in Environment, Genetics and Oncobiology, CIMAGO - Faculty of Medicine, University of Coimbra, Coimbra, Celas, Portugal.
Sara R
SR
Martins-Neves
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
Willem E
WE
Corver
Pharmacology and Experimental Therapeutics, Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal.|CNC.IBILI, University of Coimbra, Coimbra, Portugal.
Daniela I
DI
Paiva-Oliveira
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
Brendy E W M
BE
van den Akker
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
Inge H
IH
Briaire-de-Bruijn
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
Judith V M G
JV
Bovée
Pharmacology and Experimental Therapeutics, Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal.|CNC.IBILI, University of Coimbra, Coimbra, Portugal.|Center of Investigation in Environment, Genetics and Oncobiology, CIMAGO - Faculty of Medicine, University of Coimbra, Coimbra, Celas, Portugal.
Célia M F
CM
Gomes
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
Anne-Marie
AM
Cleton-Jansen
D000818:Animals; D063646:Carcinogenesis / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002999:Clone Cells; D053595:Embryonic Stem Cells / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism*; D008819:Mice, Nude; D014411:Neoplastic Stem Cells / Q000378:metabolism* / Q000473:pathology; D010006:Osteoblasts / Q000378:metabolism / Q000473:pathology; D012516:Osteosarcoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D039904:Pluripotent Stem Cells / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000378:metabolism*; D018874:Spheroids, Cellular / Q000378:metabolism / Q000473:pathology; D060449:Wnt Signaling Pathway; D051176:beta Catenin / Q000378:metabolism*
C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D055748:SOXB1 Transcription Factors; D051176:beta Catenin