Cell Senescence & Analysis

Cell senescence is defined as permanent cell growth arrest, where cells are unable to progress through growth cycle. Biologically, most natural cell senescence occurs as eventual exposure of the uncapped free dsDNA will trigger permanent DNA damage, also known as progressive telomere shortening, and morphological characterization of senescent cells appears with a distinct vacuolated morphology.

Senescence may also be a response to various intrinsic stimuli, extrinsic stressors, or possibly even developmental signals. Some potential causative factors of senescence include structural changes in the telomere or chromatin, mitogenic signals, oncogenic activation, oxidative and genotoxicity, epigenetic changes, mitochondrial dysfunction, inflammation, tissue damage, radiation or chemotherapeutic agents, or nutrient deprivation. These senescence pathways are often unique, depending on the causative factor.

Senescence, then, can be categorized into two types; as telomere dependent where the impacting factor is due to repeated cellular replications, or non-telomeric where growth arrest is acute and premature.

Senescence plays a role in embryogenesis, short term wound healing, and in immune clearance by preventing dangerous cells, like tumor cells, from multiplying. Cell senescence has also been associated with negative downstream effects, particularly through senescence-associated secretory phenotype (SASP). SASP has been noted to run senescent fibroblasts into proinflammatory cells, and can impact neighboring cells and structural components. SASP may be a reason senescent cells contribute to tissue homeostasis as well as dysfunction, due to its roles in immune signaling, cell-cell crosstalk, and in the formation of pro-tumorigenic environments. Because of this, SASP has been linked to chronic inflammation, fibrosis, and induction of senescence in healthy cells and vulnerable tissues.

Methods and Visualization

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Because of the dynamic nature of cell senescence, various experiments that target differing characteristics of senescence, performed together, will provide a more well-rounded understanding of this phenomenon. Some experiments, including Western blot (WB), Immunohistochemistry (IHC), and immunofluorescence (IF) target cell cycle arrest using known protein markers from senescence-associated pathways. The most common pathways include the p53/ p21^WAF1/CIP1 pathway, p16^Ink4a /phosphorylated Retinoblastoma protein (RB) pathway, and DREAM complex pathway. These techniques are, however, only semi-quantitative and may need to be coupled with a reporter assay.

Often, techniques to target the extent of cellular replication in a sample may be used. To analyze the growth curve of a cell population over time, live/dead automated cell imaging, a cell counting software, or spectrophotometry at different time-points, where data is collected over time are a few techniques. Also, immunostaining for proliferation markers, like protein Ki-67, has been documented. This method is particularly useful in that most of these associated proteins are active during all phases of the cell cycle, however immunostaining for proliferation markers is still not a direct measurement of active proliferation like other methods that incorporate EdU or BrdU. Instead, assays that utilize EdU or BrdU are geared towards assessing DNA replication, which is done by determining levels of DNA synthesis through the incorporation of these modified nucleotides into the DNA of replicating cells.


Detection for all these methods can be performed by common laboratory technology including fluorescence imaging or flow analysis. Additionally, RT-qPCR may be used to quantify senescence-related mRNAs, although mRNA level does not always correlate with expressed proteins levels and primers as well as housekeeping genes used must be carefully considered.

Colorimetric assays through enzymatic staining are another common technique of analyzing senescence. Senescence-associated beta-galactosidase (SA- β -Gal), a hydrolase enzyme, catalyzes the hydrolysis of β-galactosides into monosaccharides. After experimental hydrolysis, the substrate will dimerize and form a blue colored precipitate, whose OD levels are linearly correlated to senescent activity within the sample. As SA- β -Gal is a lysosomal enzyme, this assay can monitor the increased expression and activity thereof, as well as provide information on the increase or decrease of lysosomal mass. Colorimetry may also target SA-α-fucosidase, though this technique is less used.

In colorimetry, incubation typically falls between 6 - 12 hours, but should be optimized for the choice of cell line as excessive incubation may result in unspecific positive staining. Non-senescent cells should also be probed in parallel to avoid these false positive readings.



Other methods have been adapted that manipulate SA-ꞵ-Gals cleavable biochemistry, including near infrared technology (NIR), two-photon fluorescence, probes adapted to positron emission tomography (PET), and light microscopy. Some fluorescent probes have even been modified to incorporate galactose, which function as OFF-ON fluorophores that activate when a sugar moiety is cleaved in the presence of SA- β -Gal. These fluorophores are self-quenching, and offer easy visualization and analysis through fluorescent microscopy and/or flow cytometry.

In senescence, chromatin often attempts to silence proliferation-promoting genes and form specialized dense structures known as senescence-associated heterochromatin foci (SAHF). Detection of SAHF is therefore a useful tool to assess senescence, and can be performed through DAPI staining or immunostaining of heterochromatin forming proteins like HP1, H3K9me3, and macroH2A.

Analysis is usually performed through fluorescent or confocal imaging. Instead of SAHF, SASP may be a preferred target due to its association with senescence. Detection of this secondary senescent marker can be performed through a number of techniques including qPCR, ELISA, WB, SASP-responsive alkaline phosphatase assay, or proteomic techniques like mass spectrometry (MS) or LC-MS.

Assessment of senescence may also be the analysis of telomeres, where telomere length may be measured by qPCR and quantitation can be performed by qFISH. Telomere dysfunction-induced focis (TIFs) are telomeric regions associated with DNA damage, and can be useful markers in immunoblotting techniques.



Another characteristic of senescent cells is nuclear membrane damage, which may be analyzed through the loss of laminin B1, a major component of the nuclear lamina, by qPCR, IF or WB.

Additionally, an increase in reactive oxidative species (ROS) may be caused by structural changes or alterations in the mitochondria, both causal activators of senescence. ROS can be detected through chemiluminescence analysis, fluorimetry, or flow cytometry where useful fluorophores measure levels of superoxides in various parts of the cell.



Commonly morphological assessment of the sample may be necessary to help determine structural size and internal cellular changes due to senescence. Senescent cells are abnormally large, flat, and samples may appear multinucleated. Samples can also appear with increased granularity due the increased size and number of lysosomes, and analysis may return with an increased cytoplasm to DNA ratio.

Considerations

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Due to the heterogeneous and diverse nature of senescence, there is currently no single totally reliable biomarker to measure total senescence in a sample. While senescence is naturally higher in aged tissues, the magnitude and extent of senescence varies remarkably depending upon tissue type, section, and target used for experimentation. Numerous diseases are associated with senescence, which make finding a therapeutic resolution of high necessity.

Senescence may contribute to pathophysiological conditions like fibrosis, diabetes, cancer, Alzheimer's disease, and aging in general. Senescence can accelerate cell deterioration, hyperplasia, tumorigenesis in cancer, and kidney disease. The senescence of stem cells and immunosenescence, a process of immune dysfunction characterized by the remodeling of lymphoid, are particularly common in old age.

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