Abstract 3692: Development and validation of high content screening assay for identification of compounds based on cytotoxicity and cell cycle analysis using FUCCI probe
Authors: Znojek Pawel , Gurska Sona , Dzubak Petr , Hajduch Marian 
Published: August 2015

Abstract
Introduction
High content screening (HCS) is a method that uses automatic microscopy and image analysis techniques to extract multiple phenotypically relevant measurements at cellular level. By now for high content screening there is a few technigues to evaluate cell cycle. This include BrdU staining and/or mitosis-specific marker phospho-histone H3 immunostaining. In our endeavors we used HeLa cells stably expressing FUCCI (Fluorescent Ubiquitination-based Cell-Cycle Indicator) probe. The FUCCI probe was generated by fusing red and green fluorescent proteins with ubiquitination domains of Cdt1 and Geminin respectively. As a means of tracking cell cycle progression FUCCI exploits cyclical expression and degradation of the ubiquitination oscillators Cdt1 and Geminin to specifically mark cell cycle phases in living cells. Cell cycle perturbation can be evaluated by measuring mean fluorescence intensity of Cdt1-Red and Geminin-Green in single cell and eventually based on fluorescence intensity range cells can be classified as one of G1, G1/S, S/G2/M and M cell cycle phase. The FUCCI technology can be used as alternative method for FACS and it is capable to procces more data than FACS. The goal was to set up robust assay to screen large compound collections to search for potential cell cycle modifiers.
Brief description
The assay was designed for full automatization on robotic station which has liquid dispenser, incubators and Yokogawa CV7000 microscope connected. This means that cell plating, addition of compounds, cytotoxicity measurement and high content microscopy is processed by robotic system. Images are exported to Columbus image analysis system and final results are processed in Tibco Spotfire software. For setting up and validation of the assay, dose response curve was generated for 8 reference compounds with known cell cycle activity.
Summary of the new, unpublished data
We have observed good assay reproducibility and robustness with Z’ value of above 0.5 for G1, S/G2/M and M phases. The assay methodology was used to test how commercially available LOPAC library, which includes 90 drug-like molecules from the fields of cell signaling and neuroscience. The results of cytotoxicity assay allowed to validate 26 compounds as active based on dose response curve fitting. For the first time FUCCI probe was used in high content screening experiments. The results demonstrates that several agents from LOPAC library (e.g. bosutinib, crizotinib, torcetrapib) were identified as cell cycle modulators.
Statement of the conclusions
The results we obtained shows that our robotic station is fully capable for primary screen for cell cycle modulators. This newly developed assay is simple to set up, robust, sensitive and inexpensive.
Acknowledgement: Postup II CZ.1.07/2.3.00/30.0041
Citation Format: Pawel Znojek, Sona Gurska, Petr Dzubak, Marian Hajduch. Development and validation of high content screening assay for identification of compounds based on cytotoxicity and cell cycle analysis using FUCCI probe. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3692. doi:10.1158/1538-7445.AM2015-3692