Analysis of Research Methods for Heterogeneous Cellular Senescence Biomarkers

Cellular senescence is a fundamental mechanism underlying organismal aging and the development of numerous age-related diseases. Accurate detection of senescent cells is key for understanding and intervening in the aging process. Molecules such as p16INK4a, p21, and SA-β-gal have been widely used in senescence detection. Traditional SA-β-gal staining is popular for its simplicity, but emerging high-throughput spatial omics techniques, such as seqFISH, now enable high-resolution and dynamic multi-marker tracking of senescent cells. Using multi-omics and spatial approaches, researchers are elucidating the distribution and molecular heterogeneity of senescent cells in tissues. However, existing detection methods still lack in specificity, throughput, and clinical utility, and a universal, standardized high-precision detection system remains elusive. This review systematically analyzes the molecular mechanisms and detection methods for p16INK4a, p21, and SA-β-gal, evaluates technological developments from classical biochemical methods to innovative spatial transcriptomics (ST), discusses the potential of single-cell multi-omics and novel biomarkers for improving detection accuracy and understanding senescence heterogeneity, and summarizes limitations and challenges faced by each approach. The results suggest that integrating multi-marker and spatial omics technology can significantly enhance the accuracy and resolution of senescent cell detection, providing a technical basis for developing innovative anti-aging drugs and disease interventions. This study provides reference for optimizing senescent cell detection and biomarker screening, emphasizing the importance of a standardized multi-omics system. Future directions should focus on improving clinical translatability, exploring molecular regulation and cellular heterogeneity within the senescent microenvironment, and advancing disease diagnosis and healthy aging.