Abstract
Head and neck squamous cell carcinoma (HNSCC) has a poor 5-year survival rate of 50%. One potential reason for treatment failure is the presence of cancer stem cells (CSCs). Several cell markers, particularly CD44, have been used to isolate CSCs. However, isolating a pure population of CSC in HNSCC still remains a challenging task. Recent findings show that normal oral stem cells were isolated using CD271 as a marker. Thus, we investigated the combined use of CD271 and CD44 to isolate an enriched subpopulation of CSCs, followed by their characterization in vitro, in vivo, and in patients' tissue samples. Fluorescent-activated cell sorting was used to isolate CD44+/CD271+ and CD44+/CD271- from two human HNSCC cell lines. Cell growth and self-renewal were measured with MTT and sphere/colony formation assays. Treatment-resistance was tested against chemotherapy (cisplatin and 5-fluorouracil) and ionizing radiation. Self-renewal, resistance, and stemness-related genes expression were measured with qRT-PCR. In vivo tumorigenicity was tested with an orthotopic immunodeficient mouse model of oral cancer. Finally, we examined the co-localization of CD44+/CD271+ in patients' tissue samples. We found that CD271+ cells were a subpopulation of CD44+ cells in human HNSCC cell lines and tissues. CD44+/CD271+ cells exhibited higher cell proliferation, sphere/colony formation, chemo- and radio-resistance, upregulation of CSCs-related genes, and in vivo tumorigenicity when compared to CD44+/CD271- or the parental cell line. These cell markers showed increased expression in patients with the increase of the tumor stage. In conclusion, using both CD44 and CD271 allowed the isolation of CSCs from HNSCC. These enriched CSCs will be more relevant in future treatment and HNSCC progression studies.