Proteomic and immunocytochemical analyses of squamous components in two‑ and three‑dimensional‑cultured pancreatic ductal adenocarcinoma cell lines

Adenosquamous carcinoma of the pancreas consists of both adenocarcinoma and squamous cell carcinoma components and shows a worse prognosis compared with pancreatic ductal adenocarcinoma (PDAC). Given that squamous cells are absent in healthy pancreatic tissue, their origin in pancreatic tumors remains unclear. The present study aimed to analyze ductal and squamous marker expression in PDAC cell lines under two-dimensional (2D) and three-dimensional (3D) culture conditions. A total of eight PDAC cell lines were examined using liquid chromatography-tandem mass spectrometry-based proteomics and immunocytochemistry to evaluate the expression of cytokeratins (CK7, CK20 and CK5/6), p63 and p40. CK7 was found to be expressed under both culture conditions in epithelial-type PDAC cell lines but not in mesenchymal-type PDAC cell lines. CK20 was not detected in any PDAC cell line under either culture condition. Among the squamous cell markers, CK5/6 was expressed in PK-59, PK-1 and T3M-4 cells in 2D culture, but not in PK-59 cells in 3D culture. Furthermore, p63 and its isoform p40 were strongly expressed in PK-8, PK-1 and T3M-4 cells in 2D culture but were not expressed in PK-8 cells in 3D culture. These squamous cell markers were not observed in mesenchymal PDAC cell lines, except for p63 in MIA PaCa-2 cells. Epithelial PDAC cells maintained their CK7⁺/CK20^- adenocarcinoma characteristics, while a number of cell lines also acquired squamous features. Overall, the present study provides new insight into the squamous cell component of PDAC and may therefore contribute to understanding the mechanism of its formation.