Real-Time Apoptosis and Viability High-Throughput Screening of 3D Multicellular Tumor Spheroids Using the Celigo Image Cytometer

Abstract

Three-dimensional tumor spheroid models have been increasingly used to investigate and characterize cancer drug compounds. Previously, the Celigo image cytometer has demonstrated its utility in a high-throughput screening manner for evaluating potential drug candidates in a 3D multicellular tumor spheroid (MCTS) primary screen. In addition, we have developed real-time kinetic caspase 3/7 apoptosis and propidium iodide viability 3D MCTS assays, both of which can be used in a secondary screen to better characterize the hit compounds. In this work, we monitored the kinetic apoptotic and cytotoxic effects of 14 compounds in 3D MCTS produced from the glioblastoma cell line U87MG in 384-well plates for 9 days. The kinetic results allowed the categorization of the effects from 14 drug compounds into early and late cytotoxic, apoptotic, cytostatic, and no effects. The real-time apoptosis and viability screening method can serve as an improved secondary screen to better understand the mechanism of action of these potential drug candidates identified from the primary screen, allowing one to identify a more qualified drug candidate and streamline the drug discovery process of research and development.

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

“…The cells were allowed to culture at 37 C and 5% CO 2 . On day 2, the working concentrations of the four cancer drugs were added at 100 mL, producing final concentrations for doxorubicin (10,5, and 1 mM), paclitaxel (10, 5, and 2.5 nM), 8-quinolinol (10, 5, and 1 mM), and salinomycin (5, 1, and 0.1 mM) at 200 mL/well. Controls were set up for adherent and nonadherent cell cultures in the absence of drug compounds.…”

“…In order to determine or validate the cytotoxic effects of the drug compounds, fluorescent staining using calcein AM, PI, and Hoechst was employed to measure viability. This method was developed in a previous publication 10,12 to determine the fitness of tumor spheroids. Essentially, the ratio between calcein and PI fluorescent intensities can indicate the fitness of the mammospheres.…”

“…Previously, the Celigo Image Cytometer has demonstrated endpoint or kinetic measurement of 3D tumor spheroid formation, viability, and apoptosis using bright-field and fluorescence image analysis in ultra-low-attachment U-bottom plates, as well as Z-prime calculation for high-throughput assays. [9][10][11] Image cytometry was used to analyze and characterize single tumor spheroids formed in the U-bottom plates, where a cancer drug compound scoring method was developed for a high-throughput drug screening assay. 12 In this work, the Celigo Image Cytometer is employed to characterize mammosphere formation, morphological changes, and drug efficacy in ultra-low-attachment flat-bottom plates that allowed the formation of multiple mammospheres or clusters.…”

“…Furthermore, fluorescent assays can characterize drug-treated mammospheres by investigating viability, apoptosis, necrosis, and hypoxia. 10,12,36…”

A Perspective on Expanding Our Understanding of Cancer Treatments by Integrating Approaches from the Biological and Physical Sciences

“…Confocal and widefield imaging methods coupled with vital dyes (e.g., propidium iodide and trypan blue) have been widely used to characterize cell death directly. 7,69 Garvey et al have applied these techniques using a high-content imaging platform to screen drug responsive and resistant populations within co-culture systems. 7 They were able to distinguish each cell type and quantify multiple parameters such as birth and death rates, cell morphology, area, and percent viability over multiple time points and across large populations of cells.…”