Recent advancements in cholangiocarcinoma (CCA) research have been marked by the innovative use of three-dimensional multicellular spheroid (3D MCS) models. These models, which replicate the 3D architecture and multicellular arrangement of human tissues, are proving to be more physiologically relevant than traditional 2D cultures in studying this form of cancer.

Key findings from this research reveal that the ability of CCA cell lines to form 3D MCSs varies based on their differentiation level. Poorly differentiated CCA cell lines struggle to form 3D MCSs due to a deficiency in cell adhesion molecules and a higher expression of mesenchymal markers. In contrast, well-differentiated CCA and cholangiocyte cell lines successfully develop 3D MCSs characterized by round shapes and smooth perimeters, facilitated by the presence of cell adhesion molecules.

A pivotal discovery in this study is the formation of a hypoxic and oxidative microenvironment within these 3D structures. This was particularly evident in the spheroids formed by MMNK-1, KKU-213C, and KKU-213A cell lines. Proteo-metabolomic analysis of these 3D MCSs revealed significant differences in protein expression and metabolic products compared to their 2D cultured counterparts. Notably, there were alterations in cell-cell adhesion molecules, enzymes related to energy metabolism, and metabolites associated with oxidative stress.

These findings underscore the physiological differences between 3D MCSs and traditional 2D cultures, highlighting the former’s potential in providing a more accurate representation of the in vivo state. The study suggests that the 3D MCS model could lead to the identification of alternative biochemical pathways, which may be crucial for developing more effective treatments for CCA.

This research paves the way for a better understanding of the complex molecular and metabolic landscape of cholangiocarcinoma, and holds promise for the development of targeted therapies that could significantly improve patient outcomes.