ER+ Breast Cancer Mammosphere Formation and Analysis.

Mammosphere formation assays are a popular and convenient technique in the study of breast cancer by providing an in vitro mechanism by which to study breast cancer stem cell (BCSC) contribution to tumorigenesis, as well as more closely mimicking the three-dimensional tumor microenvironment. In these assays, BCSCs are stimulated to proliferate in low adherence tissue culture dishes and the resulting mammospheres exhibit activation of stem cell-related signaling pathways. Here we describe the process for generating and analyzing mammospheres under varying conditions.

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.

Assessment of Cell Viability with Single-, Dual-, and Multi-Staining Methods Using Image Cytometry.

The ability to accurately measure cell viability is important for any cell-based assay. Traditionally, viability measurements have been performed using the trypan blue exclusion method on a hemacytometer, which allows researchers to visually distinguish viable from nonviable cells. While the trypan blue method can work for cell lines or primary cells that have been rigorously purified, in more complex samples such as PBMCs, bone marrow, whole blood, or any sample with low viability, this method can lead to errors. In recent years, advances in optics and fluorescent dyes have led to the development of automated benchtop image-based cell counters for rapid cell concentration and viability measurement. In this work, we demonstrate the use of image-based cytometry for cell viability detection using single-, dual-, or multi-stain techniques. Single-staining methods using nucleic acid stains such as EB, PI, 7-AAD, DAPI, SYTOX Green, and SYTOX Red, and enzymatic stains such as CFDA and Calcein AM, were performed. Dual-staining methods using AO/PI, CFDA/PI, Calcein AM/PI, Hoechst/PI, Hoechst/DRAQ7, and DRAQ5/DAPI that enumerate viable and nonviable cells were also performed. Finally, Hoechst/Calcein AM/PI was used for a multi-staining method. Fluorescent viability staining allows exclusion of cellular debris and nonnucleated cells from analysis, which can eliminate the need to perform purification steps during sample preparation and improve efficiency. Image cytometers increase speed and throughput, capture images for visual confirmation of results, and can greatly simplify cell count and viability measurements.