Single-cell analysis of anchorage-independent growth ability in pancreatic ductal adenocarcinoma cell lines

OBJECTIVE: Anchorage-independent growth is a critical feature of cancer cells, reflecting their ability to survive and proliferate without attachment to the extracellular matrix. Spheres—cancerous masses formed in a three-dimensional (3D) anchorage-independent culture—contain a high level of cancer stem cells. This anchorage-independent proliferative capacity closely relates to tumorigenicity, anoikis resistance, and metastatic capability. Pancreatic ductal adenocarcinoma (PDAC) is a heterogeneous group comprising epithelial and mesenchymal features, and these subtypes exhibit different biological characteristics in 3D cultures. This study examines whether these PDAC subtypes differ in their anchorage-independent proliferative capability at the single-cell level. METHODS: Eight PDAC cell lines, including five epithelial-type and three mesenchymal-type lines, were cultured as single cells in poly (2-methacryloyloxyethyl phosphorylcholine) (MPC) polymer–coated low-attachment microwell plates, and time-lapse imaging was performed every 15 min for 60 h. RESULTS: Three phenotypes were observed: non-proliferating single cells, cells dividing into two, and those forming clusters of three or four cells. In single-cell analysis, KP4 and MIA PaCa-2 mesenchymal PDAC cells exhibited a high number of cells proliferating into two or more cells. CONCLUSION: These findings suggest that mesenchymal PDAC cells exhibit greater anchorage-independent proliferative capability, reflecting their aggressive biological behavior.