Acknowledgments
The authors thank Andrew Young for critical reading. This work was supported by the University of Cambridge, Cancer Research UK, Hutchison Whampoa.
References
1. Pérez-Mancera PA, Young ARJ, Narita M (2014) Inside and out: the activities of senes- cence in cancer. Nat Rev Cancer 14:547–558.
doi: 10.1038/nrc3773
2. Hoare M, Young ARJ, Narita M (2011) Autophagy in cancer: having your cake and eat- ing it. Semin Cancer Biol 21:397–404.
doi: 10.1016/j.semcancer.2011.09.004 3. Salama R, Sadaie M, Hoare M, Narita M
(2014) Cellular senescence and its effector pro- grams. Genes Dev 28:99–114. doi: 10.1101/
gad.235184.113
4. Coppé J-P, Desprez P-Y, Krtolica A, Campisi J (2010) The senescence-associated secretory phenotype: the dark side of tumor suppression.
Annu Rev Pathol Mech Dis 5:99–118.
doi: 10.1146/annurev-pathol-121808-102144 5. Kuilman T, Peeper DS (2009) Senescence-
messaging secretome: SMS-ing cellular stress.
Nat Rev Cancer 9:81–94. doi: 10.1038/nrc2560 6. Lamb CA, Yoshimori T, Tooze SA (2013) The autophagosome: origins unknown, biogenesis
complex. Nat Rev Mol Cell Biol 14:759–774.
doi: 10.1038/nrm3696
7. Klionsky DJ, Abdelmohsen K, Abe A et al (2016) Guidelines for the use and interpreta- tion of assays for monitoring autophagy (3rd edition). Autophagy 12(1):1–222. doi: 10.108 0/15548627.2015.1100356
8. Tooze SA, Dooley HC, Jefferies HBJ et al (2015) Assessing mammalian autophagy.
Methods Mol Biol 1270:155–165.
doi: 10.1007/978-1-4939-2309-0_12 9. Tabata K, Hayashi-Nishino M, Noda T et al
(2013) Morphological analysis of autophagy.
Methods Mol Biol 931:449–466.
doi: 10.1007/978-1-62703-056-4_23 10. Young A, Tooze S (2008) Protein traffi cking into
autophagosomes. Methods Mol Biol 445:147–
157. doi: 10.1007/978-1-59745-157-4_10 11. Mizushima N, Levine B, Cuervo AM, Klionsky
DJ (2008) Autophagy fi ghts disease through cellular self-digestion. Nature 451:1069–1075.
doi: 10.1038/nature06639
12. Johansen T, Lamark T (2011) Selective autophagy mediated by autophagic adapter proteins. Autophagy 7:279–296. doi: 10.4161/
auto.7.3.14487
13. Katsuragi Y, Ichimura Y, Komatsu M (2015) p62/SQSTM1 functions as a signaling hub and an autophagy adaptor. FEBS J. doi: 10.1111/
febs.13540
14. Young ARJ, Narita M, Ferreira M et al (2009) Autophagy mediates the mitotic senescence transition. Genes Dev 23:798–803.
doi: 10.1101/gad.519709
15. Narita M, Young ARJ, Arakawa S et al (2011) Spatial coupling of mTOR and autophagy aug- ments secretory phenotypes. Science 332:
966–970. doi: 10.1126/science.1205407 16. Kang C, Xu Q, Martin TD et al (2015) The
DNA damage response induces infl ammation and senescence by inhibiting autophagy of GATA4. Science 349:aaa5612. doi: 10.1126/
science.aaa5612
17. Laberge R-M, Sun Y, Orjalo AV et al (2015) MTOR regulates the pro-tumorigenic senescence- associated secretory phenotype by promoting IL1A translation. Nat Cell Biol 17:1049–1061. doi: 10.1038/ncb3195
18. Herranz N, Gallage S, Mellone M et al (2015) mTOR regulates MAPKAPK2 translation to control the senescence-associated secretory phenotype. Nat Cell Biol 17:1205–1217.
doi: 10.1038/ncb3225
19. Bar-Peled L, Sabatini DM (2014) Regulation of mTORC1 by amino acids. Trends Cell Biol 24:400–406. doi: 10.1016/j.tcb.2014.03.003 20. Yu L, Mcphee CK, Zheng L et al (2010)
Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature 465:942–946. doi: 10.1038/nature09076 21. Mizushima N, Yoshimori T, Levine B (2010)
Methods in mammalian autophagy research. Cell 140:313–326. doi: 10.1016/j.cell.2010.01.028 22. Mizushima N, Yoshimori T (2007) How to
interpret LC3 immunoblotting. Autophagy 3:542–545
23. Berthois Y, Katzenellenbogen JA, Katzenellen bogen BS (1986) Phenol red in tissue culture media is a weak estrogen: implications con- cerning the study of estrogen-responsive cells in culture. Proc Natl Acad Sci U S A 83:2496–2500
99
Mikhail A. Nikiforov (ed.), Oncogene-Induced Senescence: Methods and Protocols, Methods in Molecular Biology, vol. 1534, DOI 10.1007/978-1-4939-6670-7_9, © Springer Science+Business Media New York 2017
Chapter 9
Detecting the Senescence-Associated Secretory
Phenotype (SASP) by High Content Microscopy Analysis
Priya Hari and Juan Carlos Acosta
Abstract
The diverse arrays of proteins secreted by senescent cells have been described to infl uence aging and to have both pro-tumorigenic and anti-tumorigenic infl uences on the surrounding microenvironment.
Further characterization of these proteins, known as the senescence-associated secretory phenotype (SASP), and their regulators is required to understand and further manipulate such activities. The use of high-throughput technology allows us to obtain visual and quantitative data on a large number of samples quickly and easily. Not only is this an invaluable tool for conducting large-scale RNAi or compound screen- ings, but also allows rapid validation of candidates of interest. Here, we describe how we use the Widefi eld High-Content Analysis Systems to characterize the phenotypes of cells following modulation by potential regulators of Oncogene-Induced Senescence (OIS) by measuring numerous markers of senescence, includ- ing the SASP. This approach can be also used to screen for siRNA able to perturb the expression of SASP components during OIS.
Key words Senescence , OIS , SASP , SMS , Senescence secretome , High content analysis , IL-8 , IL-6 , IL-1α , IL-1β
1 Introduction
Cellular senescence, a state of irreversible cell cycle arrest , is a piv- otal cell state. Triggered largely by internal and environmental agents inducing DNA damage , oncogenic activation , or telomere attrition , senescence prevents the proliferation of cells with accu- mulated damage that could lead to tumor formation [ 1 ]. Despite the lack of proliferative capacity , senescent cells remain metaboli- cally active, infl uencing the surrounding environment through the secretion of an assorted array of proteins, known as the senescence- associated secretory phenotype (SASP). This mixture of growth factors, chemokines, cytokines , matrix metalloproteinases, and proteases, among others, can reinforce senescence in the cell, induce senescence in neighboring cells, activate immune-mediated clearance of the cells, and promote angiogenesis [ 1 ]. By studying the role and regulation of the SASP we aim to modulate the
function of senescent cells in different physiological and pathological conditions [ 2 , 3 ].
Over the last decade, signifi cant studies have unraveled not only some key components of the SASP, but also the mechanism through which they are regulated and their impact of the senes- cence phenotype. In 2008, IL- 6 and IL- 8 were found to be key players in the execution and reinforcement of Oncogene-Induced Senescence (OIS) [ 4 , 5 ]. Their expression is controlled under the response of NF-kB and C/EBPβ transcription factors and persis- tent DNA damage signalling is required for secretion of IL-6 [ 5 , 6 ] . It was later found that IL- 1α and IL- 1β are among the most robust inducers or IL- 6 and IL- 8 and that during OIS the activity of the infl ammasome is increased [ 7 ]. The infl ammasome is a multicom- ponent platform with caspase-1 activity, functioning to cleave pro-IL-1β to its mature form and thereby enabling the induction of other SASP factors.
Thus, by providing information on the DNA damage response , transcriptional regulation, and infl ammasome activation, the cel- lular levels of these four SASP factors, IL- 1α , IL-1β, IL-6, and IL-8, can provide huge insights into the regulation of the SASP from various triggers and inhibitors of senescence. By coupling immunofl uorescence staining with high content analysis, we are able to rapidly identify genes and conditions that are either mani- festing or perturbing the SASP, and thus OIS. The relative levels of key SASP factors can be used to identify and characterize novel regulators of OIS and the SASP .
OIS is induced through the stable expression of the oncogenic ER: RAS fusion protein, which is activated upon addition of 4-hydroxytamoxifen (4OHT). Cells can then be treated with drugs, RNAi, or other means of genetic modifi cation as per inter- est and seeded into 96-well plates in preparation for staining.
The use of the multiwell format allows numerous conditions to be tested simultaneously, while only requiring a small number of cells and reagents. Following a standard immunofl uorescent staining protocol with antibodies targeting the SASP factors, the plate is loaded into an automated high content analysis microscope that, after minimal setup, can automatically acquire images and perform quantitative analysis on the images [ 7 ] .
2 Materials