Real‐time visualization and quantitation of cell death and cell cycle progression in 2D and 3D cultures utilizing genetically encoded probes

Abstract

Cancer cells grown as 3D‐structures are better models for mimicking in vivo conditions than the 2D‐culture systems employable in drug discovery applications. Cell cycle and cell death are important determinants for preclinical drug screening and tumor growth studies in laboratory conditions. Though several 3D‐models and live‐cell compatible approaches are available, a method for simultaneous real‐time detection of cell cycle and cell death is required. Here we demonstrate a high‐throughput adaptable method using genetically encoded fluorescent probes for the real‐time quantitative detection of cell death and cell cycle. The cell‐cycle indicator cdt1‐Kusabira orange (KO) is stably integrated into cancer cells and further transfected with the Fluorescence Resonance Energy Transfer‐based ECFP‐DEVD‐EYFP caspase activation sensor. The nuclear cdt1‐KO expression serves as the readout for cell‐cycle, and caspase activation is visualized by ECFP/EYFP ratiometric imaging. The image‐based platform allowed imaging of growing spheres for prolonged periods in 3D‐culture with excellent single‐cell resolution through confocal microscopy. High‐throughput screening (HTS) adaptation was achieved by targeting the caspase‐sensor at the nucleus, which enabled the quantitation of cell death in 3D‐models. The HTS using limited compound libraries, identified two lead compounds that induced caspase‐activation both in 2D and 3D‐cultures. This is the first report of an approach for noninvasive stain‐free quantitative imaging of cell death and cell cycle with potential drug discovery applications.

Differential Migration and Proliferation Potential of the Hydrogel Aided 3D Tumoroid

“…In the field of drug discovery, there is a shift in the usage from the 2D in vitro DOI: 10.1002/mabi.202200196 high-throughput screening (HTS) platforms using immortalized cell lines to more physiologically relevant 3D models for HTS. [2,3] The 3D cell culture system provides cancer spheroids imitating the spatiotemporal organization and physiological properties like metabolism, gene expression, cellular signaling, and topography alike to the cancer cell of the tumor. [4][5][6] As an in vitro lung adenocarcinoma or non-small cell lung cancer model, A549, a cell line has been frequently preferred by researchers.…”