Epithelial-mesenchymal transition in cancer stem cells and during metastasis of a squamous cell carcinoma

Scientific background: Current understanding of carcinogenesis supposes a heterogeneous composition of malign tumors from cancer stem cells (CSCs) and stromal cells. CSCs most likely play an important role during treatment failure and metastasis. A possibly associated phenomenon is the phenotype of cancer cells undergoing an epithelial-mesenchymal transition (EMT). This study investigated the role of EMT in CSCs of a HPV-negative pharyngeal squamous cell carcinoma during metastasis. Methods: A cell line was established from a HPV-negative pharyngeal squamous cell carcinoma. Enrichment of cancer stem cells was performed by stem cell-enriching conditions in spheroid colonies. Gene expression profiling was performed (Human 8 × 60 K design array, Agilent Technologies) and the phenotype EMT was assessed by a gene set (MSigDB: M5930, Hallmark_epithelial_mesenchymal_transition) applying Gene set expression analysis (GSEA). A validation of the results was carried out by immunohistochemical staining and flow cytometry of CD44 und E-Cadherin. Results: Primary tumor and metastasis were highly positive for CD44. E-Cadherin was shown in primary tumor and the cell line derived from it. Flow cytometry showed a heterogeneous composition of the stem cell-enriched spheroid colonies with the appearance of a distinct population with the loss of E-Cadherin. GSEA showed significant enrichment of the EMT-phenotype in the primary tumor (FDR < 25 %, p < 5 %) compared to the other phenotypes. Conclusions: In this study, CSCs showed mesenchymal characteristics. EMT may be a dynamic state in HPV-negative pharyngeal squamous cell carcinoma that is active in CSCs during metastasis and at least partially turned back later. 6 different phenotypes were analysed, 2 of which (u45, H170) were for internal reference only. 4 phenotypes were included into further analyses (T, M, NZK, SphK) and consisted from 3 biological replicates each.