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

Abstract

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. 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. 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)]. 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.