Stepping Towards Better Assays for Cellular Senescence

Growth in the number of lingering senescent cells in all tissues is one of the root causes of aging. These cells generate signals that provoke chronic inflammation, destructively remodel nearby extracellular matrix structures, and alter the behavior of other cells for the worse. As their numbers grow, so does the negative impact on organ function and the acceleration of dysfunction that eventually becomes age-related disease. Cells become senescent when they reach the limit of on cell divisions that is imposed on most of the cells in the body, but also in response to genetic or other damage, or in the face of a toxic environment. Near all such cells destroy themselves, or are destroyed by the immune system. A tiny fraction evade this fate, however, and given enough time that tiny fraction would be enough to push us into frailty, disease, and death, even absent the other causes of degenerative aging.

Fortunately, targeted removal of senescent cells as a treatment for aging is becoming a reality. Numerous methods and drug candidates are under development, with varying degrees of evidence resulting from animal studies, ability to destroy senescent cells, and unwanted side-effects. A number of the drug candidates are chemotherapeutics, such as navitoclax, with significant and unpleasant side-effects to account for, but other approaches, such as the Oisin Biotechnologies gene therapy or the new FOXO4-p53 technique may well have next to no side-effects. Many people are presently in a position to order delivery of at least some of the tested compounds that already in the drug databases and give it a try, though most of the manufacturers are unwilling to sell to the public at large, for regulatory and liability reasons. There is at least a little more forethought that has to go into unofficial self-experimentation beyond just ordering the stuff and guessing a dosage, even for compounds for which the pharmacology is well-defined because they were previously tested in humans for one purpose or another.

That said, the big hurdle for self-experimentation is the lack of good assays. It is pointless to try this out unless you believe you have a good way to assess the results. Admittedly the results in mice seem pretty impressive, but it may nonetheless still be the case that unless you are very impacted by senescent cells, then just running bloodwork - or checking kidney function, or using CT scans to assess arterial calcification, or estimating your own joint pain, and so forth - will give you an ambiguous result. What is really needed is a way to see how many senescent cells are in your tissue, before and after a modest, limited dose. Today that really requires tissue sections and staining approaches, which is custom lab work, somewhat clunky, and there is some question as to how clearly a tissue biopsy from a human subject is going to show the desired information. Since senescent cells are involved in wound healing, the whole biopsy process might involve generating enough new senescent cells to confuse the data. To me it seems largely pointless to embark upon a personal test of the more easily obtained drug candidates without the ability to check before and after in a rigorous way.

This is essentially why I chose to support the work of CellAge, given their focus on clinically useful assays of cellular senescence. There are numerous groups working on improving the situation for assays of senescent cells, but the academic researchers are largely aiming at something other than improvements that are convenient for human self-experimentation or later clinical tests. There is probably more interest, as illustrated in this paper, in trying to better capture variations in senescent cell biochemistry, or better classify different types of senescence.

Quantitative identification of senescent cells in aging and disease

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-β-gal). However, SA-β-gal activity alone is not enough to allow us to conclude with confidence that cells are senescent, as positive staining can also occur in other biological contexts. Therefore, SA-β-gal staining is usually combined with staining for additional markers such as γH2AX-a marker for activation of DNA damage response. In addition, negative markers that should be absent in senescent cells can be used to exclude the cells that are not senescent. These markers indicate cell proliferation, like Ki67 or BrdU incorporation, or proteins ubiquitously present in the cell nuclei, but secreted from senescent cells and thus absent in their nucleus, like HGMB1.

The SA-β-gal and each of the markers are usually evaluated separately in consecutive sections. This procedure is not only laborious and expensive but also 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. Alternatively, SA-β-gal activity within cells can be quantified by flow cytometry using 5-dodecanoylaminofluorescein di-β-D-galactopyranoside as a substrate. However, this method can be performed only on intact cells and therefore does not allow identification of intracellular markers in the same cells. Altogether, 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.

Conventional SA-β-gal staining fails to distinguish between different cell types that can be a source of senescent cells within complex tissues, limiting our understanding of the underlying biological phenomena. In an attempt to overcome the limitations of current methods for identification of senescent cells, we utilized ImageStreamX, an advanced imaging flow cytometer capable of producing multiple high-resolution, fluorescent and bright-field (BF) images of every cell directly in flow. Our approach combines the quantitative power of flow cytometry with high-content image analysis. We modified the traditional SA-β-gal assay to meet the requirements of the ImageStreamX and performed the assay in a single-cell suspension. Using this method, we identified and quantified senescent cells in tumors, fibrotic tissues, and normal tissues of young and aged mice.

In this study, we evaluated several biomarkers of cellular senescence and found a significant correlation between SA-β-gal staining and the lack of nuclear HMGB1 staining in vitro. This combination might allow more reliable identification of senescent cells, compared to SA-β-gal assay alone. Therefore, it provides significant advantage over existing techniques, including the use of fluorescent β-gal substrate, which does not allow combination staining with any intracellular molecular markers. Accordingly, it seems possible to take advantage of this method to screen for new senescence biomarkers that correlate with SA-β-gal activity in vivo, and would consequently open the way to a deeper understanding of the senescent state in vivo. Furthermore, the use of senescence biomarkers will potentially yield greater biological insight by allowing protein localization and colocalization to be monitored and compared between senescent and nonsenescent cells.

Through its use of cell-type-specific biomarkers, our protocol can successfully determine which cell types undergo cellular senescence and which do not. Importantly, in the experiments with mice of different age, SA-β-gal staining was performed for 12 hours in all tissues to ensure consistency. We suggest that in future studies SA-β-gal staining time has to be calibrated for each tissue and in some circumstances even different cell population, to achieve the most accurate results. Moreover, staining of the cells for live-dead markers immediately following tissue dissociation will allow quantification of SA-β-gal-positive cells specifically from the live cell population. This is particularly pertinent since the dissociation of cells from tissues might result in a certain amount of cell death. We showed that about 96% of the cells are viable following tumor dissociation, but this percentage can diverse greatly depending on the tissue examined.


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