Cellular Senescence: Review and Reagent Selection Guide

Science Note

[Oct. 22, 2024]                                                                                                                                                                                                                       Previous Science Note
Targeting Senescent Cells to Enhance Cancer Treatment Outcomes

Recent cancer research has focused on intratumoral senescent cancer cells and it is becoming clear that these cells contribute to immune evasion of cancer cells and create a cancer-promoting environment.  Here are some of the papers that have identified senescent cells or senescent cells with specific phenotypes in cancer treatment and have shown that removing these cells is important for cancer treatment.

Senescent cells are damaged or stressed cells that permanently stop dividing but do not undergo cell death. In the context of cancer, senescent cells can accumulate in tumors, particularly after treatments such as chemotherapy, contributing to an immunosuppressive environment. This environment can promote tumor growth, immune evasion and resistance to therapy. However, the selective targeting and elimination of senescent cells, known as senolytic therapy, is emerging as a promising strategy to improve cancer treatment by reducing tumor progression and improving patient outcomes.

The efficacy of chemotherapy is limited by intratumoral senescent cells expressing PD-L2
Click here for the original article: Selim Chaib, et. al., Nature Cancer, 2024.

Cellular senescence in malignant cells promotes tumor progression in mouse and patient Glioblastoma
Click here for the original article: Rana Salam, et. al., Nature Communications, 2023.

 Senescence drives immunotherapy resistance by inducing an immunosuppressive tumor microenvironment
Click here for the original article: Damien Maggiorani, et. al., Nature Communications, 2024.

Point of Interest
- Chemotherapy often generates senescent cancer cells, which have increased PDL-2 expression, aiding immune evasion and tumor growth.

- PD-L2 is not necessary for senescence but crucial for immune evasion by senescent cells, promoting tumor persistence after chemotherapy.

- Antibody-mediated PD-L2 blockade synergizes with chemotherapy, suggesting a potential therapeutic strategy targeting senescent cancer cells.

Point of Interest
- Senescent cells contribute to glioblastoma (GBM) progression and their elimination may improve treatment outcomes.

- Removal of p16Ink4a-expressing senescent cells from GBM tumors improves survival in mice and alters the tumour environment.

- Senolytic drugs that target senescent cells may be a promising adjuvant therapy for GBM patients, improving survival.
 

Point of Interest
- Immunotherapy resistance is associated with reduced CD8 T cell activity in tumors.

- Elimination of senescent cells restores CD8 T cell proliferation and reduces immunotherapy resistance.

- Anti-senescent cell drugs prior to immune checkpoint inhibitors may enhance the effectiveness of cancer immunotherapy.
Related Techniques
           Cellular senescence detection SPiDER-βGal for live-cell imaging or flow cytometry / microplate reader / tissue samples
NEW SPiDER-βGal Blue for fixed cell and for multiple staining with immunostaining and other methods
           Glycolysis/Oxidative phosphorylation Assay Glycolysis/OXPHOS Assay Kit
           Endocytosis Detection detection ECGreen-Endocytosis Detection
           Lysosomal function Lysosomal Acidic pH Detection Kit-Green/Red and Green/Deep Red
           First-time autophagy research Autophagic Flux Assay Kit
           Total ROS detection Highly sensitive DCFH-DA or Photo-oxidation Resistant DCFH-DA
           Apoptosis detection in multiple samples Annexin V Apoptosis Plate Assay Kit
           Cell proliferation/ cytotoxicity assay Cell Counting Kit-8 and Cytotoxicity LDH Assay Kit-WST
           Glutathione Quantification GSSG/GSH Quantification Kit
Related Applications

Co-staining with Lipid droplet and SA-β-Gal in fixed cells

 

NAD(+) levels decline during the aging process, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies*. Here, we try to redemonstrate this phenomenon in the doxorubicin (DOX)-induced cellular senescence model with a comprehensive analysis of our products.

*S. Imai, et al., Trends Cell Biol, 2014, 24, 464-471


Products in Use
① DNA Damage Detection Kit - γH2AX
② Cellular Senescence Detection Kit - SPiDER-βGal
③ NAD/NADH Assay Kit-WST
④ JC-1 MitoMP Detection Kit
⑤ Glycolysis/OXPHOS Assay Kit、Lactate Assay Kit-WST

 

Multiple staining with oxidative stress-related markers using Doxorubicin-induced senescent cells（flow cytometry)

Using A549 cells induced to senescence by doxorubicin (DOX) and normal cells (CTRL), changes in oxidative stress-related markers in senescent cells were analyzed by flow cytometry with multiple staining. SA-βGal as a senescence marker was detected by Cellular Senescence Detection Kit - SPiDER Blue, total ROS as an oxidative stress marker was detected by ROS Assay Kit - Photo-oxidation Resistant DCFH-DA-, and γH2AX as a DNA damage marker was detected by DNA Damage Detection Kit - γH2AX-Red. As a result, total ROS and γH2AX were increased in SA-βGal-positive senescent cells, and the increase in oxidative stress-related markers associated with cellular senescence could be detected by multiple staining.


  Flow cytometry：SONY SA3800
　 SPiDER Blue: PacificBlue 　
    ROS Assay Kit: FITC
    γH2AX - Red: Cy3

＜Experimental Procedure>
 *Cellular senescence was induced in A549 cells by DOX (0.2 μM DOX for 3 days → normal medium for 3 days)
 The detail procedure for this experiment, please refer to the product page: SPiDER Blue.

 

 

 

Assessing Cellular Senescence

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What is Cellular Senescence?

 Cellular senescence was reported by Hayflick in 1981. It was discovered when pulmonary fibroblasts slowed down their proliferation and eventually ended in cell death after cell passaging had been performed for more than 8 months. Subsequent studies have revealed that cellular senescence is caused not only by telomere length reduction, but also by external factors such as oncogene activation, oxidative stress, and DNA damage.
The induction and control mechanisms of cellular senescence – in which genetic and external factors are intricately involved – have yet to be fully elucidated. However, it has been suggested that the process is closely related to cancer and various age-related diseases, inspiring large amounts of active research into the topic. The development of drugs that eliminate senescent cells in the body (senolytic drugs) is also attracting the attention of researchers as a possible strategy to extend healthy life expectancy.





 
 
 
 
 
 
 
 Cellular senescence is controlled by various factors such as cell type and physiological conditions, such as oxidative stress. None of the individual biomarkers that have been identified so far have been deemed to be specific to senescent cells. Therefore, it is desirable to determine and confirm cellular senescence using multiple indicators.
 Common detection indicators for assessing cellular senescence include features related to cell cycle progression (DNA synthesis, p16/p21 expression, etc.), features related to morphology (of the cell, nucleus, nucleolus, etc.), SA-ß-Gal activity, DNA damage, oxidative stress (ROS), telomere length, inflammatory cytokines (senescence-associated secretory phenotype (SASP)), and more.

 

< Video Seminar >
“Recent Findings of Cellular Senescence Studies and Analysis Method”

Chapters:
0:00 What is Cellular Senescence ?
5:00 Senescence Studies and Drug discovery
12:30 Methods of Senescence Detection and Analysis

 

 

 

Indicators Related to Cellular Senescence

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Correlation map of related indicators

 Research into apoptosis, necrosis, autophagy, and cellular senescence is very important for understanding the intracellular functions that control cell survival and death.
Recently, various fields have given particular attention to cellular senescence due to the recent discoveries of SASP (a known cancer-causing factor) and senescence-related phenomena in stem cell research.

 This correlation map shows the relationship between various intracellular indicators, resulting from cellular senescence. This information is based on currently available information. Please refer to the table with cited references below as reference for your experiments. The table lists the cell type, the method of senescence induction used, the senescence markers measured, and the variables affected by senescence in each reference for the map.

  Cell Senescence induction Senescence marker (s) Responding variable (s) Reference
① IMR90
(Human pulmonary fibroblasts)		 Several passages in culture SA-ß-Gal, p16, p21, Nucleosome hypertrophy Expression of SETD8↓,  H4K20me1↓, oxidative phosphorylation↑, ribosome synthesis↑ H. Tanaka, S. Takebayashi, A. Sakamoto, N. Saitoh, S. Hino and M. Nakao, “The SETD8/PR-Set7 Methyltransferase Functions as a Barrier to Prevent Senescence-Associated Metabolic Remodeling.”, Cell Reports, 2017, 18(9), 2148.
Inhibition of SETD8
(Methyltransferase)		 Oxidative phosphorylation↑,  ribosome synthesis↑
② Senescent mouse satellite cell
eletal muscle progenitor cells)		 – SA-ß-Gal, p16 Autophagy activity↓, ROS↑, mitochondrial membrane potential↓ L. Garcia-Prat, M. Martinez-Vicente and P. Munoz-Canoves, “Autophagy: a decisive process for stemness”, Oncotarget, 2016, 7(11), 12286.
Atg7 knockout mouse
(Satellite cells)		 Autophagy inhibition SA-ß-Gal, P15, p16, p21, γ-H2AX ROS↑, mitochondrial membrane potential↓
③ Rat fibroblast model of type 2 diabetes – SA-ß-Gal, p21, p53, γ-H2AX NADP+/ NADPH↓(resistance to oxidative stress↓), NADPH oxidase↑(ROS↑) M. Bitar, S. Abdel-Halim and F. Al-Mulla, “Caveolin-1/PTRF upregulation constitutes a mechanism for mediating p53-induced cellular senescence: implications for evidence-based therapy of delayed wound healing in diabetes”, Am J Physiol Endocrinol Metab., 2013, 305(8), E951.
④ IMR90
(Human pulmonary fibroblasts)		 Ethidium bromide (inhibition of mtDNA) + pyruvate deficiency SA-ß-Gal NAD+/NADH C. Wiley, M. Velarde, P. Lecot, A. Gerencser, E. Verdin, J. Campisi, et. al., “Mitochondrial Dysfunction Induces Senescence with a Distinct Secretory Phenotype”, Cell Metab., 2016, 23(2), 303.
⑤ MDA-MB-231
(Human breast cancer cells)		 X-ray irradiation + inhibition of cell cycle-related factor (securin) expression SA-ß-Gal Lactate↑, LDH activity↑, (glycolysis↑) E. Liao, Y. Hsu, Q. Chuah, Y. Lee, J. Hu, T. Huang, P-M Yang & S-J Chiu, “Radiation induces senescence and a bystander effect through metabolic alterations.”, Cell Death Dis., 2014, 5, e1255.
⑥ MEF
(Mouse Embryonic Fibroblast)		 Overexpression of oncogenes,several passages in culture, transcription factor overexpression(E2F1) SA-ß-Gal, p16, p21, Nucleosome hypertrophy Ribosome RNA↑, p53↑ K. Nishimura, T. Kumazawa, T. Kuroda, A. Murayama, J. Yanagisawa and K. Kimura, “Perturbation of Ribosome Biogenesis Drives Cells into Senescence through 5S RNP-Mediated p53 Activation”, Cell Rep. 2015, 10(8), 1310.
⑦ Mouse tail fibroblast 2 months old, 22 months old, p16 knockout (22 months old) SA-ß-Gal, p14, p16 NAD+↓, SIRT3↓ M. J. Son, Y. Kwon, T. Son and Y. S. Cho, “Restoration of Mitochondrial NAD+ Levels Delays Stem Cell Senescence and Facilitates Reprogramming of Aged Somatic Cells”, Stem Cells. 2016, 34(12), 2840.

 

 

 

Reagent Selection Guide

Dojindo offers four types of kits and reagents that can be selected according to the evaluation method and purpose of cell senescence.

Product Cellular Senescence Detection Kit – SPiDER-ßGal, SPiDER Blue Cellular Senescence Plate Assay Kit – SPiDER-ßGal Cell Cycle Assay Solution Deep Red / Blue Nucleolus Bright Green / Red
Detection Fluorescence Fluorescence Fluorescence Fluorescence
Wavelength
(Ex/Em)		 [SPiDER-ßGal]
Ex. 500–540 nm/Em. 530–570 nm
[SPiDER Blue]
Ex. 350-450 nm/Em. 400-500 nm		 Ex. 535 nm / Em. 580 nm Deep Red: Ex. 633-647 nm /
Em. 780/60 nm
Blue: Ex. 405-407 nm /
Em. 450/50 nm		 Green: Ex. 513 nm /
Em. 538 nm
Red: Ex. 537 nm /
Em. 605 nm
Target SA-ß-gal activity SA-ß-gal activity Nucleus Changes in the nucleolus
Detection
Method		 Imaging, Flow cytometry
Substrate: SPiDER-ßGal, SPiDER Blue		 Plate assay
Substrate: SPiDER-ßGal		 Flow cytometry Imaging Detection of the nucleolus by RNA-staining reagent
Instrument Fluorescence microscope, FCM Fluorescence microplate reader FCM Fluorescence microscope
Sample SPiDER-ßGal: Live or fixed cells
SPiDER Blue: Fixed cells
(Tissue: some examples from published articles using SG02)		 Live cells
(lysis of live cells)		 Live cells, fixed cells Fixed cells
Best for Those who have difficulty quantifying data or performing multiple staining with X-gal Those who process multiple samples
Those who are evaluating senescent cells for the first time Small size package (20 tests) is available		 Those who wish to evaluate using indicators other than SA-ß-Gal Those who wish to evaluate using indicators other than SA-ß-Gal
Examples of reports using nucleolus as an indicator are available on the product page
data
Item# SPiDER-ßGal: SG04
SPiDER Blue: SG07		 SG05 Deep Red: C548
Blue: C549		 Green: N511
Red: N512

 

 

Lipid Accumulation (Lipotoxicity)

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 Lipotoxicity, which is caused by excessive lipid accumulation in non-adipose tissue cells, is thought to be involved in cancer, diabetes, heart failure, and obesity. It has been shown that lipid accumulation in cells causes cellular senescence and mitochondrial dysfunction. The figure below shows the changes in various indices caused by excessive lipid accumulation.

For more information click here or image below

 

 

 

Cell Cycle Arrest

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 Irreversible cell cycle arrest is one of the phenomena that characterize cellular senescence. p16, p21, p53, and pRB (phosphorylated retinoblastoma protein) are known as representative protein markers. The activation/upregulation of these proteins are used as indicators of cellular senescence. These marker proteins are known to be tumor suppressors and regulate the cell cycle mainly through two pathways (p16Ink4a-RB and p53-p21CIP1).

Doxorubicin (DOX) is known as an anticancer drug that acts in the G2/M phase of the cell cycle to arrest cell proliferation and induce cellular senescence (see the figure below in center). Below are the results of an experiment in which DOX was added to A549 cells. As a result, changes in SA-ß-Gal expression, cell cycle progression, and mitochondrial membrane potential were observed.

Changes in Intracellular Metabolism

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 It is well known that senescent cells maintain high metabolic activity despite their reduced proliferative capacity. In general, senescent cells show a decrease in NAD+, an increase in lactate efflux, and a decrease in AMP/ATP ratio. This is due to conversion from oxidative phosphorylation to aerobic glycolysis and mitochondrial dysfunction, in addition to activation of the glycolytic system.
Changes in intracellular metabolism are thus closely related to cellular senescence. Therefore, these changes in intracellular metabolism are very important – not only as indicators of cellular senescence, but also in clinical and basic research targeting cellular senescence.
Our webpage on intracellular metabolism provides maps focusing on senescence-associated changes in intracellular metabolism, such as SIRT1-related changes in NAD+ levels, and cells that have become senescent due to DNA damage.
(Please click on the "Senescence" tab in the link)
 
 
 

 

 

 

Related Scientific Information

Autophagy

 

 

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