A Cost-Effective Method of Senescent Cell Visualization in Living Tissue
Researchers here report on the development of a method to enable real-time visualization of the current degree of cellular senescence present in living tissues, using an improved version of existing florescence techniques. If accurate enough, this could replace the current standard approach of biopsy and staining of the sample for analysis. Note that the paper isn't open access; you'll have to obtain a copy from the usual underground sources. A practical method of assessing cellular senescence burden that works in live animals will be a big step forward for the field, as it should reduce the cost of many of the activities involved in the production of senolytic therapies, treatments capable of destroying senescent cells and thus reversing their contribution to the aging process. Consider the ability to accurately track the presence of senescent cells in the same animal from moment to moment across a lifetime and through varied senolytic dosages and treatments, for example, information that at present is challenging and costly to obtain.
The main purpose of cellular senescence is to prevent the proliferation of damaged or stressed cells and to trigger tissue repair. However, upon persistent damage or during aging, the dynamic process of tissue repair becomes inefficient and senescent cells tend to accumulate. This accumulation in tissues is believed to impair tissue functions and accelerate aging. It has been demonstrated that genetic ablation of senescent cells ameliorates a variety of aging-associated diseases, reverts long-term degenerative processes, and extends longevity. Inspired by these findings, strategies to prevent, replace, or remove senescent cells have become of interest. For instance, there is an increasing interest in the development of senolytic molecules able to induce apoptosis preferentially in senescent cells.
A related key issue in this field is the design of probes to accurately detect senescent cells in aged or damaged tissues. However, one of the major obstacles limiting progress in this research area is the lack of real-time methods to selectively track senescence in in vivo systems. Detection of senescent cells usually relies on the detection of senescence-associated βGal (SAβGal), and several fluorescent or chromogenic probes have been reported for the visualization of this enzymatic activity. However, these first-generation probes are usually unsuitable for in vivo imaging as they rely on chromogenic changes or on the use of classical one-photon fluorescence excitation. As an alternative, recent stimulating studies developing two-photon fluorescent probes for the visualization of βGal activity have been described. However, some of the reported probes are synthesized by using tedious multistep protocols. Another common drawback is the fact that probes are tested in cultured cells or in animal models that were not directly related to senescence.
In view of the aspects mentioned above, we report herein a novel molecular probe for the two-photon fluorogenic in vivo detection of senescence. The probe (AHGa) is based on a naphthalimide fluorophore as a signaling unit containing an L-histidine methyl ester linker and an acetylated galactose attached to one of the aromatic nitrogen atoms of the L-histidine through a hydrolyzable N-glycosidic bond. Probe AHGa is transformed into AH in senescent cells resulting in an enhanced fluorescent emission intensity. Targeting of senescent cells in vitro with AHGa was validated with the SKMEL-103 cancer cell line treated with the chemotherapeutic palbociclib to induce senescence. A remarkable fluorescence emission enhancement (ca. 10-fold) in the presence of AHGa for palbociclib-treated SK-MEL-103 (senescent) cells was observed when compared with control SK-MEL-103 cells, due to the formation of AH.
The ability of tracking senescence of probe AHGa was also studied in vivo by employing mice bearing subcutaneous tumor xenografts generated with SK-MEL-103 melanoma cells and treated with palbociclib. Tumors in palbociclib-untreated mice showed negligible fluorescence emission both in the absence or in the presence of AHGa, whereas tumors in mice treated with palbociclib and intravenously injected with AHGa showed a clear fluorescent signal. A marked emission enhancement (ca. 15-fold) in tumors treated with palbociclib compared to nontreated tumors was observed. AH fluorescence was only found in senescent tumors but not in other organs. The combination of selectivity, sensitivity, and straightforward synthesis make AHGa an efficient OFF-ON two-photon probe for the in vivo signaling of senescence.
It's amazing how once the initial breakthrough was made research and interest in the area just starts snowballing. I hope this is repeated in the area of crosslinks and mitochondria.
Are there any features that make certain classes of the 7 types of SENS damage more or less susceptible to this snowballing of research and interest? The ND4 mitochondrial gene is now in stage 3 clinical trails, yet this doesn't seem to have led to any more research on any of the other mitochondrial genes. Maybe because allotopically expressing ND4 in a mouse hasn't led to lifespan extension?
@Jim: I suspect that the number of distinct targets has a lot to do with it. So senescence and glucosepane cross-links will go pretty rapidly once past the first demonstration, but allotopic expression is harder, and clearance of other metabolic garbage will be a long slog through scores of compounds.
Multiphoton techniques are great, you even have three-photon imaging or even multiphoton 3D printing for high specificity; but most dyes still questionable.
One man's senescent cell is anothers man's stem cell, the cell police have a tough job.
Still haven't a more succinct reality check for hard allotopic expression then the CORR theory, ie:
'Why chloroplasts and mitochondria retain their own genomes and genetic systems: Colocation for redox regulation of gene expression'
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4547249/
There is test procedure called Sodium MRI which uses a specialized MRI machine to identify general and localized pockets of high Sodium. I wonder if a comparable MRI could be used to quantify Senescent cells.
Does the rate of accumulation of senescent cells vary much between species? I'm conscious that most of the work done thus far has used short lived mice. Would a 50+ year old human have accumulated a relatively similar amount of senescent cells as a 2-3 year mouse? Or would a human have relatively many more senescent cells?
@TheRage: Answers to that question are pretty sketchy right now. Expect them to get much better very quickly in the next few years, as companies quantify the results of their human trials in ways other than just positive outcomes to disease states.
It seems like this staining method can be combined with in-vivo confocal microscopy, i.e. you'd get good images of sections through the skin or the cornea of the eye but getting much deeper is a challenge for light microscopy.