Category: Assay Development and Screening

1046-B - Real-time kinetic viability and apoptosis detection and screening of 3D multicellular tumor spheroids using the Image Cytometer

Monday, February 5, 2018
5:00 PM - 6:00 PM

In the recent years, three-dimensional (3D) tumor spheroid models have been increasingly used for small molecule or antibody-based cancer therapy research. The use of 3D multicellular tumor spheroid (MCTS) models may have better representation of the complex in vivo tumor microenvironments for cancer research. Previously, we have published a novel MCTS screening method using the Celigo Image Cytometer, where we described a method for measuring of end point viability and apoptosis of MCTS in 384-well ultra-low attachment (ULA) U-bottom microplates. The spheroid size was measured kinetically over a 9-day time frame using bright field imaging, showing different growth inhibition patterns for drug-treated MCTS. It would be important to also obtain real-time viability and apoptosis MCTS results, however, there are no established methods. Herein we demonstrate the use of PI and caspase 3/7 as a method to detect viability and apoptosis in 3D MCTS. The method was initially validated by comparing the bright field kinetic growth rates of MCTS in the presence or absence of PI and caspase 3/7 stains for 16 and 21 days. Furthermore, the potential toxicity of PI was analyzed by digesting the tumor spheroids that were exposed to the reagent into single cell suspension and comparing their viabilities to untreated MCTS. By monitoring PI and caspase 3/7 fluorescent intensities in combination with spheroid size over time, the growth inhibition, viability, and apoptosis can be used to characterize MCTS in real-time. Furthermore, PI and caspase 3/7 fluorescent intensities can be correlated to the formation of a necrotic core and this can be combined with spheroid size to determine an optimal time frame for spheroid formation and cancer drug treatment. Finally, we performed a high-throughput 3D MCTS screening experiment to screen the real-time kinetic viability and apoptosis effects of 14 drug compounds (NIH/NCAT) on U87MG spheroids at different concentrations. Real-time kinetic viability and apoptosis assays are highly important for developing proper 3D cancer models, which can allow researchers to determine time-dependent drug effects that usually are not captured by end point assays. This would allow the improvement MCTS analysis method to better identify more qualified drug candidates for cancer drug discovery research.

Leo L. Chan

Technology R&D Manager
Nexcelom Bioscience
Lawrence, MA

Leo Chan currently serves as the Technology R&D Manager at Nexcelom Bioscience LLC, Lawrence, MA. His research involves in the development of instrument and applications for the Cellometer and Celigo image cytometry system for detection and analysis of cells in immuno-oncology, regenerative medicine, toxicology, and the brewing industry. He received his B.S., M.S., and Ph.D. in Electrical and Computer Engineering from the University of Illinois at Urbana-Champaign (2000-2008).