(1070-A) Evaluation of the bioactivity of compounds using Cell Painting assay and alternative High-Content assays

Abstract: Cell Painting is a high-content, image-based technique that allows visualising changes in the cell morphology induced by studied chemical compounds. This assay, developed in 2016 [1], uses a specific set of fluorescent probes that stain various cell components. As a result, a large plethora of features and properties can be assessed, like multiple numeric parameters describing signal intensity, area, shape, texture etc. From obtained multiparametric data, hundreds to thousands of cell features can then be extracted that are used to describe the phenotypic profile of the cells. These profiles of cells treated with chemical compounds (e.g. chemical libraries), positive controls (compounds with known mechanism of action (MOA)) and negative control (DMSO) are then compared. This allows to predict the MOA of studied compounds, group compounds based on the MOA as well as to detect even subtle cytotoxicity (even at sublethal doses) at an early stage. In our Centre, we optimised Cell Painting protocol for high-content screening in different cell lines such as A549, PC3, HepG2 and U2-OS. We have used EU-OPENSCREEN Bioactives library (approx. 2.5k compounds) as a certified partner site and Cell Profiler software for automatic image analysis followed by additional data analysis to create morphological profiles.
Despite widespread use and many advantages of the Cell Painting method, an increasing amount of in-depth analysis indicated that not all dyes and cellular features they stain contribute significantly to distinguishing different MOA and at the same time, not all toxicity mechanisms can be visualised by monitoring the selected features. Therefore, there is room for improvement both in the dye selection and in switching to other sets of imaged parameters. Currently, our research focuses on finding alternatives to dyes used in typical Cell Painting protocol: staining compartments that are not stained in the classic Cell Painting assay as well as using other approaches to simultaneously monitor multiple cytotoxic mechanisms of studied compounds. Some of the modifications included the use of staining cytoskeleton by non-actin targeted dyes, the application of a single dye for simultaneous compartmentalization of cytoplasm and nucleus and the use of apoptosis-visualising dyes. These modified assays can be used to confirm the cytotoxicity of tested compounds. We are also testing other dyes to create an offer of broad panels of dyes that can be used in different high-content screenings according to the information that is sought by the user.
ACKNOWLEDGEMENTS – funding from Polish Ministry of Science and Higher Education (decision DIR/WK/2018/06, Polish Platform of Screening Infrastructure for Chemical Biology, POL-OPENSCREEN – part of EU-OPENSCREEN Consortium) & Foundation for Polish Science (project no: POIR.04.04.00-00-441F/17-00 (Homing/2017-4/33 for “MultiGATE: dual-analyte responsive fluorescent probes for a real-time multi-parametric sensing in cellular models” (JLK)).

References: M.A. Bray et al. (2016), Nat. Protoc. 11, 1757-1774.