High-content confocal analysis of tumorigenesis, cancer stem cells, and drug response in 3D cholangiocarcinoma cultures.

Three-dimensional (3D) culture models, particularly multi-spheroid models, are becoming increasingly essential in cancer drug discovery, particularly in stem cell and cancer stem cell (CSC) research. However, analytical methods for 3D multi-spheroid models, especially for single-cell and single-spheroid analysis in CSC research, remain limited. To address this gap we developed 3D multi-spheroid cholangiocarcinoma models that incorporate a CSC live-cell biosensor and a novel analysis method, 3D Surface Integrative Spheroid Profiling (3D-SiSP), utilizing high-content confocal imaging. 3D-SiSP quantifies spheroid area, allowing for both high- and low-throughput analyses. We demonstrate three key applications of 3D-SiSP. First, it outperformed traditional length-based methods for in vitro tumorigenesis measurements, offering greater precision. Second, 3D-SiSP enabled the calculation of individual spheroid areas along with real-time CSC biosensor signals, revealing larger spheroids had more undifferentiated cells. Lastly, 3D-SiSP facilitated simultaneous, real-time quantification of CSC content during anti-cancer drug testing in individual spheroids, providing evaluation of drug responses. Drug response differences across treatments were also quantified. Overall, 3D-SiSP provides a flexible and effective methodology for characterizing cancer cells and CSCs while evaluating anti-cancer drugs, applicable in both high- and low-throughput contexts. This approach enhances our understanding of CSC dynamics and supports the development of anti-CSC therapies.