[Enrichment and functional characterization of cancer stem cells from squamous cell carcinoma of head and neck by suspension culture].

OBJECTIVE: Initiation, growth, recurrence, and metastasis of head and neck squamous cell carcinoma (HNSCC) have been related to the cancer stem cells (CSC) that can be identified by their aldehyde-dehydrogenase-isoform-1 (ALDH-1) activity. In this study, we try to prove that suspension culture can enrich ALDH-1 high expression cells within HNSCC cell lines and the enriched cells possess cancer stem cell properties.

METHODS: Cells from five HNSCC cell lines were cultured in ultra-low attachment plates in serum-free Quantum 263 medium supplemented with 10 ng/ml EGF and 10 ng/ml bFGF, and ALDH-1 expression level was evaluated by ALDEFLUOR assay. ALDH-1 high expression cells were separated by FACS sorting, and their phenotypical and functional properties were characterized.

RESULTS: Spheroids can be formed from all five HNSCC cell lines (UD-SCC1, UT-SCC22, UM-SCC11B, UT-SCC9 and UT-SCC24A) under anchorage independent culture condition. The proportion of ALDH1 high expression cells were highly increased in speroids derived cells (SDCs) compared with their monolayers (P < 0.05). The clones formed by ALDH1 high expression cells on average contained 197 (197 ± 47) cells compared with 33 (33 ± 16) cells in clones generated from ALDH1 low expression cells (P < 0.01). Single ALDH1 high expression cell could significantly better regenerate a spheroid (UT-SCC9: 17.1%, UD-SCC1:19.3%), whereas under the same conditions single ALDH1 low expression cells regenerated only in one case a spheroid (P < 0.01). SDCs from all five tested cell lines also showed a significantly increased invasion capacity (P < 0.05). We also found that the mRNA levels of Oct-4, Sox2, and Nanog were all significantly increased in the SDC. The reactive oxygen species (ROS) levels in SDCs from UD-SCC1 and UT-SCC24A were significantly increased compared with their monolayer counterpart [(26.3 ± 4.9)% vs (8.6 ± 1.7)% and (72.1 ± 6.1)% vs (23.7 ± 7.5)%, P < 0.05)].

CONCLUSION: Cancer stem cells can be enriched by suspension culture, which may be of importance in investigation of their contribution to therapy resistance, tumor recurrence and metastasis.