Quantitative Cellular AGing Evaluation system (qCAGEs): a dual-parameter platform for high-throughput senescence...

Area of Science:

• Cellular biology
• Biotechnology
• Pharmacology

Background:

• Accurate identification of senescence-modulating compounds requires advanced screening.
• Current methods using single parameters cannot differentiate specific drug responses from cytotoxicity.
• Distinguishing senolytic and anti-aging effects necessitates multi-parameter analysis.

Purpose of the Study:

• To develop a dual-parameter high-throughput screening platform for precise identification and quantification of senescence-modulating compounds.
• To establish a standardized method for classifying drug responses based on cellular aging status.
• To enable systematic phenotypic classification of drug responses in therapeutic development.

Main Methods:

• Developed the quantitative Cellular AGing Evaluation system (qCAGEs) measuring α-L-fucosidase activity (4ME) and cell viability.
• Established the Cell count-Aging activity Reference Line (CARL) for baseline relationship definition.
• Validated the platform using human dermal fibroblast senescence models and 3D culture formats.

Main Results:

• The 4ME/cell count ratio effectively discriminated senescent from young cells with a >sixfold difference.
• Assay precision met high-throughput screening criteria (CV < 15%).
• Platform adaptation to 3D cultures showed strong concordance with 2D results (cosine similarity > 0.5 for >75% of compounds).

Conclusions:

• qCAGEs provides a standardized, quantitative approach for evaluating drug-induced changes in cellular aging.
• The platform enables accurate classification of drug responses into anti-aging, pro-aging, senolytic, and cytotoxic categories.
• qCAGEs has broad applications in senolytic and anti-aging therapeutic development.