A High-Throughput Image Cytometry Method for the Formation, Morphometric, and Viability Analysis of Drug-Treated Mammospheres.

The nonadherent mammosphere assay has been commonly used to investigate cancer stem cell activities in breast cancers that have the ability to form tumorspheres and maintain tumor growth. The sphere formation step is critical, in that it enables the construction of the mammosphere models for downstream assays. The mammosphere assay has also been used to assess the effects of drug treatment on the tumorspheres formed from primary cancer cells or cell lines. Traditionally, the mammosphere formation has been evaluated by standard microscopy systems that required external software for additional analyses. However, this method can be time-consuming and low-throughput, thus impractical for high-throughput characterization of mammosphere models and screening for potential therapeutic cancer drugs. To overcome these challenges, we developed a plate-based high-throughput method to rapidly analyze mammospheres in whole wells using the Celigo Image Cytometer. The method is employed to characterize mammosphere formation and morphology for adherent and nonadherent propagation of four breast cancer cell lines (MCF7, MDA-MB-436, MDA-MB-231, and SKBR3). Next, the dose-dependent effects of four small molecule drugs (doxorubicin, paclitaxel, 8-quinolinol, and salinomycin) are characterized based on sphere formation and viability stained with calcein AM and propidium iodide. We observed growth and morphometric differences between adherent and nonadherent propagation of the four cell lines. Furthermore, drug treatments induced various effects on mammosphere formation, morphology, and viability. The proposed image cytometry method provides a useful tool suitable for high-throughput characterization and analysis of mammospheres, which can improve assay efficiency when investigating the formation capabilities and drug-induced cytotoxicity effects.