Real-time visualization and quantitation of cell death and cell cycle progression in 2D and 3D cultures utilizing genetically encoded probes.
J Cell Biochem
; 123(4): 782-797, 2022 04.
Article
em En
| MEDLINE
| ID: mdl-35106828
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.
Palavras-chave
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Apoptose
/
Transferência Ressonante de Energia de Fluorescência
Idioma:
En
Revista:
J Cell Biochem
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
Índia