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.
Highlights
head and neck squamous cell carcinomas (HNSCC) accounts for approximately 6% of all cancer cases and for about 650,000 new cases and 350,000 deaths worldwide each year [1,2,3]
The ability of cancer stem cells (CSC) to rebuild the tumor from a single cell could explain many of the differences that discriminate tumor cells from differentiated somatic cells like immortality, quiescence, invasion leading to metastasis, and recurrence after treatment
Mack and colleagues questioned the use of CD44 as a specific CSC marker in HNSCC since CD44 was abundantly expressed in most tumor cells within HNSCC (60%-100%) and could not be used to distinguish normal from benign or malignant epithelia of the head and neck [10]
Summary
HNSCC accounts for approximately 6% of all cancer cases and for about 650,000 new cases and 350,000 deaths worldwide each year [1,2,3]. Advances in therapy have improved quality of life, but survival rates have remained unchanged over the past decades. Mortality from this disease remains high because of the development of distant metastases and the emergence of local and systemic recurrences resistant to chemo- and radiotherapy. It is essential to develop a deeper understanding of the biology of this disease in order to develop more effective therapeutic approaches. Evidence has recently been accumulating to support the hypothesis that tumors contain a small subpopulation of cells called cancer stem cells (CSC), which exhibit self-renewing capacities and are responsible for tumor maintenance and metastasis [4]. CD44+/CD242cells have been firstly proposed to exhibit CSC properties in breast cancer [5]
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