title: Evidence for epithelial-mesenchymal transition in cancer stem cells of head and neck squamous cell carcinoma.
abstract: Initiation, growth, recurrence, and metastasis of head and neck squamous cell carcinomas (HNSCC) have been related to the behavior of cancer stem cells (CSC) that can be identified by their aldehyde-dehydrogenase-isoform-1 (ALDH1) activity. We quantified and enriched ALDH1(+) cells within HNSCC cell lines and subsequently characterized their phenotypical and functional properties like invasion capacity and epithelial-mesenchymal transition (EMT). Spheroid culture enriched CSC from five HNSCC cell lines by up to 5-fold. In spheroid-derived cells (SDC) and the parental monolayer-derived cell line ALDH1, CD44, CD24, E-Cadherin, α-SMA, and Vimentin expression was compared by flow-cytometry and immunofluorescence together with proliferation and cell cycle analysis. Invasion activity was evaluated by Matrigel assay and expression of stemness-related transcription factors (TF) Nanog, Oct3/4, Sox2 and EMT-related genes Snail1 and 2, and Twist by real-time PCR. All cell lines formed spheroids that could self-renew and be serially re-passaged. ALDH1 expression was significantly higher in SDC. ALDH1(+) cells showed increased colony-formation. The proportion of cells with a putative CSC marker constellation of CD44(+)/CD24(-) was highly variable (0.5% to 96%) in monolayer and spheroid cultures and overlapped in 0%-33% with the CD44(+)/CD24(-)/ALDH1(+) cell subset. SDC had significantly higher invading activity. mRNA of the stemness-related genes Sox2, Nanog, and Oct3/4 was significantly increased in SDC of all cell lines. Twist was significantly increased in two while Snail2 showed a significant increase in one and a significant decrease in SDC of two cell lines. SDC had a higher G0 phase proportion, showed high-level expression of α-SMA and Vimentin, but significantly decreased E-Cadherin expression. HNSCC-lines harbor potential CSC, characterized by ALDH1 and stemness marker TF expression as well as properties like invasiveness, quiescence, and EMT. CSC can be enriched by anchorage-independent culture techniques, which may be important for the investigation of their contribution to therapy resistance, tumor recurrence and metastasis.
authors.affiliation: Department of Otolaryngology, Head and Neck Surgery, Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Chao
authors.initials: C
authors.lastname: Chen
authors.forename: Yan
authors.initials: Y
authors.lastname: Wei
authors.forename: Michael
authors.initials: M
authors.lastname: Hummel
authors.forename: Thomas K
authors.initials: TK
authors.lastname: Hoffmann
authors.forename: Manfred
authors.initials: M
authors.lastname: Gross
authors.forename: Andreas M
authors.initials: AM
authors.lastname: Kaufmann
authors.forename: Andreas E
authors.initials: AE
authors.lastname: Albers
mesh_terms: D000080924:Aldehyde Dehydrogenase 1 Family; D002294:Carcinoma, Squamous Cell / Q000473:pathology*; D045744:Cell Line, Tumor; D058750:Epithelial-Mesenchymal Transition; D006258:Head and Neck Neoplasms / Q000473:pathology*; D006801:Humans; D007527:Isoenzymes / Q000032:analysis; D014411:Neoplastic Stem Cells / Q000473:pathology*; D050697:Retinal Dehydrogenase / Q000032:analysis; D014157:Transcription Factors
chemical_list: D007527:Isoenzymes; D014157:Transcription Factors; D000080924:Aldehyde Dehydrogenase 1 Family; C510223:ALDH1A1 protein, human; D050697:Retinal Dehydrogenase