Algorithmic assessment of cellular senescence in experimental and clinical specimens

Abstract

The development of genetic tools allowed for the validation of the pro-aging and pro-disease functions of senescent cells in vivo. These discoveries prompted the development of senotherapies-pharmaceutical interventions aimed at interfering with the detrimental effect of senescent cells-that are now entering the clinical stage. However, unequivocal identification and examination of cellular senescence remains highly difficult because of the lack of universal and specific markers. Here, to overcome the limitation of measuring individual markers, we describe a detailed two-phase algorithmic assessment to quantify various senescence-associated parameters in the same specimen. In the first phase, we combine the measurement of lysosomal and proliferative features with the expression of general senescence-associated genes to validate the presence of senescent cells. In the second phase we measure the levels of pro-inflammatory markers for specification of the type of senescence. The protocol can help graduate-level basic scientists to improve the characterization of senescence-associated phenotypes and the identification of specific senescent subtypes. Moreover, it can serve as an important tool for the clinical validation of the role of senescent cells and the effectiveness of anti-senescence therapies.

SARS-CoV-2 infects lung epithelial cells and induces senescence and an inflammatory response in patients with severe COVID-19

“…A proportion of SARS-CoV-2 infected AT2 cells (range 8 to 21%) displayed a senescent phenotype, with positive staining for SenTraGor and p16 INK4A ( Figure 2A-C ) (12,26–28). By contrast lung tissues from age-matched non-COVID-19 cases with analogous co-morbidities ( Suppl Table 1 ) showed significantly lower senescence (range 1-2%, p<0.01, Wilcoxon paired non-parametric test) ( Figure 2A-C ), suggesting that SARS-CoV-2 infection may induce senescence.…”

Pulmonary infection by SARS-CoV-2 induces senescence accompanied by an inflammatory phenotype in severe COVID-19: possible implications for viral mutagenesis

“…SenTraGor TM (trademark of GL13 compound) staining and double staining experiments were performed and evaluated as previously described [ 27 , 35 ]. The mean percentage of SenTraGor positive alveolar cells in at least 10 high power fields (×400) per patient was quantified.…”

“…Given the implication of an inflammatory response in the progression of COVID-19 and the SASP secretion by senescent cells, we investigated whether cellular senescence occurs in COVID-19. Since individual markers are not adequate to unequivocally detect senescence, especially in vivo , as they may also be present in non senescent conditions, we followed in a clinical setting a detailed multi-marker algorithmic approach that we and others recently published and was approved by the senescence community [ 12 , 27 ]. We were the first to provide evidence supporting the proof for senescence in COVID-19 infected lung tissue by applying this algorithm in a previous, preprint (bioRvix), version of the current manuscript [ 28 ], which subsequently has been confirmed by others [ 29 ].…”

Telomere Shortening and Its Association with Cell Dysfunction in Lung Diseases

“…To better understand the role of cell senescence in the development of pathologies, new models of premature cell senescence based on the deletion of genes involved in the regulation of the cell cycle have been developed [ 40 ]. On the other hand, lysosomal activity, expression of Ki67, RPS6, and beta-galactosidase, and soluble molecules related to the SASP have been explored as biomarkers to discriminate senescent subtypes [ 41 , 42 , 43 ]. Moreover, these molecules are identified at a different cellular level, from the genetic to protein level, and they provide information regarding signaling pathways of senescence [ 44 , 45 ].…”