Epithelial-Mesenchymal Transition during Metastasis of HPV-Negative Pharyngeal Squamous Cell Carcinoma

Abstract

In epithelial tumors, a shift towards a mesenchymal phenotype has been associated with increased invasiveness andmetastasis. It is assumed that this phenomenon plays amajor role in disease progression and ultimately prognosis.This study investigated epithelialmesenchymal transition (EMT) in human papillomavirus- (HPV-) negative pharyngeal squamous cell carcinoma. Tissue was obtained from one hypopharyngeal primary tumor and a regional lymph nodemetastasis during surgery with curative intention. A cell culture was established fromthe primary tumor andmesenchymal growth conditions were emulated.Gene expression profiling was performed (Human 8 × 60K design array, Agilent Technologies) and EMT was assessed by a gene set (MSigDB: M5930, Hallmark epithelial mesenchymal transition), applying gene set expression analysis (GSEA). Immunohistochemical staining and flow cytometry of CD44 and E-cadherin were compared in primary tumor, metastasis, and cell cultures. Primary tumor and metastasis were highly positive for CD44.Aloss of E-cadherin occurred in the metastasis. Flowcytometry showed the appearance of a population without E-cadherin in spheroid colonies. In GSEA, the EMT phenotype was enriched in the primary tumor compared to metastasis and cell cultures (FDR < 25%, 𝑝 < 5%). EMT showed variable expression during metastasis. It may thereby be a dynamic state in HPV-negative pharyngeal squamous cell carcinoma that is active only during the process of metastasis itself. Thereby, the primary tumor as well as the metastasis may exhibit fewer EMT properties.