Fully automated viability and toxicity screening—A reliable all‐in‐one attempt

• Published in: Cancer medicine (Malden, MA) Vol. 13; no. 12
• Main Authors: Liedtke, Victoria, Weiss, Romano, Skifov, Anastasia, Rödiger, Stefan, Schenk, Lysann
• Format: Journal Article
• Language: English
• Published: Bognor Regis John Wiley & Sons, Inc 01.06.2024; John Wiley and Sons Inc; Wiley
• Subjects: Adherent cells; Antineoplastic drugs; automated system; Automation; bioimage informatics; Biosensors; Cancer; Cell proliferation; Cells; Chemoresistance; Cloning; CRISPR; CRISPR/Cas9; Cytotoxicity; Data acquisition; Data processing; DNA damage; DNA repair; Drinking water; Fluorescence microscopy; Fluorescent indicators; Genes; Genetic disorders; Genome editing; Informatics; Metabolism; Propidium iodide; Proteins; viability; France; United States > US; Texas; Massachusetts; Germany
• DOI: 10.1002/cam4.7392
• ISSN: 2045-7634

Background The CRISPR/Cas9 technology is nowadays a common tool for genome editing to achieve new insights into, for example, diagnostics and therapeutics in cancer and genetic disorders. Cell proliferation and anticancer drug response studies are widely used to evaluate the impact of editing. However, these assays are often time‐consuming, expensive, and reproducibility is an issue. To overcome this, we developed a fast and cheap assay that combines a fully automated multispectral fluorescence microscopy platform with a nuclei staining and open‐source software analysis. Methods Here, we generated different LEDGF/p75 model cell lines to validate the effect on proliferation and chemosensitivity. Therefore, a fast protocol for an optimized all‐in‐one attempt for cytotoxicity screenings and proliferation analysis of adherent cells in a 96‐well plate format was established using differential staining with two fluorescent dyes (Hoechst 33342 and propidium iodide) for live/dead cell discrimination. Subsequently, an automated cell nuclei count and analysis were performed using bioimage informatics. Results With the new established assay technology, up to 50,000 cells/well can be detected and analyzed in a 96‐well plate, resulting in a fast and accurate verification of viability and proliferation with consistency of 98% compared to manual counting. Our screening revealed that LEDGF depletion using CRISPR/Cas9 showed a diminished proliferation and chemosensitivity independent of cell line origin. Moreover, LEDGF depletion caused a significant increase in 𝛾H2AX foci, indicating a substantial increase in DNA double strand breaks. LEDGF/p75 overexpression enhanced proliferation and chemoresistance underlining the role of LEDGF in DNA damage response. Conclusion Independent of cancer cell type, LEDGF/p75 is a central player in DNA damage repair and is implicated in chemoresistance. Moreover, our automated fluorescence biosensor technology allowed fast and reliable data acquisition without any fixation or additional washing steps. Additionally, data analysis was implemented using the modular open‐source software that can be adapted as needed. In this manuscript, we provide a fast, easy and cost‐efficient all‐in‐one attempt for analyzing proliferation and cytotoxicity in a wide range of human cancer cell lines. Additionally, it is a reliable, nontoxic, and metabolism‐independent method for comparing genetically modified cell lines or different chemotherapeutic drugs.