Quantitative identification of senescent cells in aging and disease.

Anat Biran,Ezra Vadai,Valery Krizhanovsky,Lior Roitman,Lior Zada,Ziv Porat,Yossi Ovadya,Paula Abou Karam

doi:10.1111/acel.12592

Abstract

SummarySenescent cells are present in premalignant lesions and sites of tissue damage and accumulate in tissues with age. In vivo identification, quantification and characterization of senescent cells are challenging tasks that limit our understanding of the role of senescent cells in diseases and aging. Here, we present a new way to precisely quantify and identify senescent cells in tissues on a single‐cell basis. The method combines a senescence‐associated beta‐galactosidase assay with staining of molecular markers for cellular senescence and of cellular identity. By utilizing technology that combines flow cytometry with high‐content image analysis, we were able to quantify senescent cells in tumors, fibrotic tissues, and tissues of aged mice. Our approach also yielded the finding that senescent cells in tissues of aged mice are larger than nonsenescent cells. Thus, this method provides a basis for quantitative assessment of senescent cells and it offers proof of principle for combination of different markers of senescence. It paves the way for screening of senescent cells for identification of new senescence biomarkers, genes that bypass senescence or senolytic compounds that eliminate senescent cells, thus enabling a deeper understanding of the senescent state in vivo.

Highlights

Cellular senescence is a multifaceted phenomenon that functions in tumor suppression, wound healing, embryonic development, biological aging, and development of pro-inflammatory age-related diseases (Campisi, 2013; Burton & Krizhanovsky, 2014; Munoz-Espin & Serrano, 2014; Salama et al, 2014)
Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd
The results are in line with those obtained when we quantified SA-b-gal-positive cells by microscopy (Fig. S1C,D, Supporting information). These findings demonstrate that SA-b-gal staining can be detected and quantified using ImageStreamX

Summary

Introduction

Cellular senescence is a multifaceted phenomenon that functions in tumor suppression, wound healing, embryonic development, biological aging, and development of pro-inflammatory age-related diseases (Campisi, 2013; Burton & Krizhanovsky, 2014; Munoz-Espin & Serrano, 2014; Salama et al, 2014). Our understanding of the role of cellular senescence in different biological contexts has been impeded in part by the difficulty of detecting their presence within tissues Such detection is currently performed mainly by evaluation of senescence-associated beta-galactosidase (SA-b-gal) (Dimri et al, 1995; Debacq-Chainiaux et al, 2009; Kuilman et al, 2010). The SA-bgal and each of the markers are usually evaluated separately in consecutive sections This procedure is laborious and expensive and does not allow multiple senescence biomarkers to be detected within the same cells, limiting the possibility of quantitative evaluation of senescent cells derived from tissues. SA-b-gal activity within cells can be quantified by flow cytometry using 5-dodecanoylaminofluorescein di-b-D-galactopyranoside as a substrate (DebacqChainiaux et al, 2009) This method can be performed only on intact cells and does not allow identification of intracellular markers in the same cells. Current methods do not allow detection and quantification of senescent cells in tissues based on combination of markers that is essential for their confident identification

Results

Discussion

Conclusion