Cellular senescence refers to a state of irreversible arrest of cell proliferation. It was first described by Hayflick and colleagues in the 1960s when they observed cessation of cell proliferation after a finite number of cell divisions in human fibroblasts attributed to telomeric shortening. Senescent cells were subsequently found to adopt a senescence-associated secretory phenotype (SASP) in which they continuously secrete pro-inflammatory factors like cytokines and chemokines.
Accumulation of senescent cells can occur due to diminished clearance ability of the immune system or persistent exposure to senescence-inducing stimuli that produce more cells than can be cleared in time. Such accumulation can lead to a chronic inflammatory state in the surrounding tissue microenvironment, also referred to as inflammaging, that further promotes senescence in neighboring healthy cells.
Due to the deleterious effects of cellular senescence, the senescent cell has been the target of active research to tackle age-related pathologies either through the approach of seno-rejuvenation or senolytics. In a pioneering study done in 2011, Baker and colleagues showed that by removing p16Ink4a-positive senescent cells, there was observable delayed tissue dysfunction and extended healthspan in a progeroid mouse model. Here, we describe recent progress in senolytics as a form of therapy and the existing challenges for this field.
Promise of senolytics
Senolytics refers to a class of pharmacological agents that eliminate senescent cells by inducing apoptosis. Compared to their healthy counterparts, senescent cells are highly resistant to apoptosis even in the presence of cellular stresses due to activation of pro-survival and anti-apoptotic pathways. One of the most common senolytics approaches is to inhibit pro-survival pathways such as those regulated by the BCL-2 protein family and PI3K/AKT pathway.
In a 2021 study, Novais et al. found that long-term administration of senolytic drugs Dasatinib, a tyrosine kinase inhibitor, and Quercetin, a naturally occurring flavonoid, mitigated age-dependent intervertebral disc degeneration in mice. Mice given the drug combination for close to two years experienced lower incidence of disc degeneration while exhibiting less senescent-related biomarkers, including p16Ink4a-positive senescent cells, SASP molecules like interleukin (IL)-6, and matrix metalloproteinase (MMP)-13. Importantly, there was a higher composition of collagen I and chondroitin sulfate in drug-treated mice which preserved the integrity of the extracellular matrix (ECM) at the spinal disc.
However, this therapeutic effect was only observed in mice that were 6- (young)–14- (middle-aged) months-old and not those which were 18 (elderly) months of age, demonstrating an age-dependent efficacy.
Beyond the use of senolytic drugs, there is also increasing interest in the use of engineered immune cells for senolytics purposes, stemming from the observation on the role of immune system to clear senescent cells. For instance, CD4+ T cells have been found to work with monocytes and macrophages to clear senescent hepatocytes. Natural killer cells were also shown to be involved in the clearance of senescent activated stellate cells from the liver, leading to fibrosis resolution.
However, age-related decline in innate and adaptive immune functions is thought to impair immune surveillance, reducing their ability in clearing senescent cells. This in turn leads to the accumulation of senescent cells in aged tissues over time.
In a recent study, Corina Amor, MD, PhD, an independent fellow at the Cold Spring Harbor Laboratory studying cellular senescence in cancer and colleagues developed chimeric antigen receptor (CAR) T cells as senolytic agents to target cells expressing urokinase-type plasminogen activator receptor (uPAR). The team first showed that uPAR is a cell-surface protein biomarker of senescence by validating against biomolecular markers like β-galactosidase and comparing untreated controls with samples induced with senescence either through drugs or multiple cell passaging. CAR-T cells were able to selectively eliminate cells expressing uPAR, leading to enhanced survival of mice with lung adenocarcinoma and restoration of tissue homeostasis in mice with liver fibrosis.
“The major advantage for using CAR-T cells as senolytics instead of small compounds is their high efficacy and persistence. We know that one very low dose of CAR-T cells can lead to significant (and prolonged) clinical benefit in mouse models of senescence-associated diseases like liver fibrosis. I think this is a major advantage over compounds that would need to be taken several times per day on a continuous basis for years. On the other hand, I think the major challenge for senolytic CAR-T cells are the high costs of the therapy and the need for individualized treatment,” said Amor.
“I think the biggest challenge is that there is a lot of heterogeneity among senescent cells. Their dependencies, the surface molecules they upregulate, and the soluble factors they secrete all vary significantly based on cell type and trigger of senescence. This makes it very difficult to find senolytic approaches that are useful in all scenarios. I think in the years to come the focus is going to change to developing senolytics to target specific senescent cells in given clinical scenarios,” added Amor.
Difficulty in biomarker identification
Despite the promise of senolytics to enhance longevity and reduce the impact of chronic diseases, there are challenges facing this novel therapy, one of which being identifying a biomarker that is expressed exclusively by senescent cells and not healthy cells, to limit off-target effects and enhance safety.
One of the most used and well-established biomarkers of senescence is senescence-associated β-galactosidase (SA-β-Gal). This is attributed to the increase in activity of β-Gal, a lysosomal hydrolase, in senescent cells. While it is considered the gold standard in detecting cellular senescence, evidence has indicated that activity of β-Gal can be increased by other conditions such as contact inhibition which are independent of senescence.
Telomere shortening is also a commonly used biomarker for replicative senescence. Numerous other biomolecular markers associated with intracellular alternations have also been described including DNA damage markers including histone protein γH2AX and secretomes of senescent cells. Likewise, these biomolecular markers of senescence can also be induced due to non-specific cellular stresses.
For robust identification of senescence, it has been proposed that at least three different senescence biomarkers representing different hallmarks of the senescent cell be used together for a multi-marker combinatorial approach.
Given that biophysical properties are representative of a whole host of molecular inputs, Phillip et al. demonstrated that cellular biophysical properties may more accurately reflect the cellular age as compared to biomolecular markers. Senescent cells are typically characterized by a flattened cellular morphology with an irregular shape and larger nuclei and cellular sizes. Senescent cells also exhibit changes in mechanical properties including increased stiffness. In an in vitro study on epithelial cells, it was observed that Young’s modulus indicative of cellular stiffness of older cells was two to four times higher than in younger cells.
There are also some established relationships both between biomolecular and biophysical features of senescent cells. For instance, cells with an enlarged and flattened morphology exhibited increased β-galactosidase expression. Studies had also shown the correlation between different biophysical markers like between cell motility, cell morphology, and cell size.
In our recent review, we also suggest a list of biophysical markers which we believe can be helpful when used in conjunction with biomolecular markers to better identify senescent cells to isolate them and learn about their biology.
Poor drug delivery specificity
Despite the promising results shown by senolytic drugs such as BCL-2 family inhibitors in eliminating senescent cells, clinical translation still remains limited, owing to safety concerns. One of the key reasons is that senolytics are typically drugs used for treating other disorders that have been repurposed to kill senescent cells, as Salvador Macip, MD, PhD, an associate professor at the University of Leicester studying aging and cancer, pointed out.
“[Senolytics] were used for other things, but it was found that senescent cells were very sensitive to them. The problem this has is that these drugs have many off-target effects,” explained Macip. “Thus, current senolytics are likely to have important side effects, which would greatly limit their clinical use.”
Navitoclax is a prime example of a senolytic compound exhibiting panolytic behavior, meaning that off-target apoptosis is induced in other cell types besides senescent cells. Navitoclax has been found to induce the apoptosis of non-senescent cells including neutrophils and platelets, resulting in neutropenia and thrombocytopenia during clinical trials. Attributing to its associated toxicities, it has not been approved for clinical use by the FDA.
To enhance the specificity of drug delivery, researchers are now looking into developing second-generation senolytics, which are more selective towards senescent cells and not healthy cells. To this end, researchers have designed materials that target senescence biomarkers. Agostini et al. designed mesoporous silica nanoparticles that encapsulated cargo and were capped with galacto-oligosaccharides, a substrate of SA-β-gal, which acts as a molecular gate. The cargo was found to be preferentially released in cells with high SA-β-gal activity, which mediates the enzymatic hydrolysis of the cap.
Further studies to improve this system were done by Muñoz-Espín et al. who showed that when coated with a homogenous coating of a 6-mer galacto-oligosaccharide, more specific cargo release into senescent cells was achieved.
Another direction that has been taken to increase the specificity in targeting is by leveraging surface markers found on senescent cells. For instance, in a recent study, Macip and colleagues designed antibody-drug conjugates (ADCs) which were shown to selectively kill senescent cells without toxicity to proliferating cells. Here, the antibody was designed to recognize and bind to β2 microglobulin (B2M), a membrane marker of senescence they had previously identified.
“We opted for copying an idea that has already been used successfully in cancer: train an antibody to recognize a mark on the surface of the target cell and load it with a drug that will then be released into the cell and kill it, without harming the neighbor cells,” explained Macip on the inspiration behind this design. “We compare it to an ‘intelligent bomb,’ because it finds and eventually only kills the target.”
In our recent review, we also propose that the altered biophysical phenotype of senescent cells can affect drug delivery and may be exploited to influence intracellular delivery. By tuning the parameters used in these techniques based on cellular biophysical properties, we propose that more targeted delivery towards senescent cells could be achieved.
Intracellular delivery techniques extend beyond biochemical means like nanoparticles to include other physical membrane-disruption techniques such as electroporation and high aspect-ratio nanostructures. Particularly for physical delivery approaches where targeting using biomolecular markers will be challenging, the use of biophysical markers may be more favorable to provide a physical basis for targeting, which can be tunable.
Intracellular delivery via nanoparticles can also benefit from the complementary use of both biophysical and biomolecular markers to further enhance their selectivity. For instance, senescent cells with a higher membrane stiffness may favor the uptake of stiffer nanoparticles which lower the energy barrier for endocytotic uptake.
The advent of an aging population has pushed the development of senolytics to become even more critical. It is encouraging to see rapid progression towards second-generation senolytics as a way to enhance their safety and efficacy for clinical use, albeit still in its infancy.
Deeper understanding of the behavior and characteristics of senescent cells will be imperative, which opportunely is in line with various emerging analytical strategies such as single-cell RNA sequencing and next-generation biophysical cytometry. It is hopeful that with this, more selective and targeted senolytics can be developed, impacting human health and longevity.
Andy Tay, PhD, is a freelance writer based in Singapore. Jessalyn Low is a PhD student at the National University of Singapore.