## 13.2. *Immune Boosting Strategies to Improve Elimination of Senescent Cells*

As the immune system becomes less efficient with age, it is likely that a therapeutic boosting of immune responses can be useful for improving senescent cell elimination.

Senescent cells are naturally immunogenic and are subject to context-dependent immune surveillance mechanisms. Senescence induction by p53 remodeling stimuli an innate immune reaction typically consists of invading leukocytes, neutrophils, macrophages, and NK cells in a model of hepatocellular carcinoma [33] and age-associated hepatic stellate cells enhance their own NK cell-mediated eradication in cases of liver fibrosis [26]. Senescence induction by NRASG12V expression in hepatocytes leads to a dual release of both innate and adoptive immunological response, as CD4+ T cells collaborate with monocytes/macrophages to remove senescent hepatocytes [35]. This procedure is mediated by stimulating natural killer cell receptor NKG2D, which detects ligands on the external membrane of stressed, infected, or injured cells. Senescent cells increase the expression of the immunological recognition protein NKG2D, which is normally absent from the membrane of healthy cells [228]. Additionally, NKG2D was needed for natural killer cell-induced senescent cell elimination to protect from fibrotic liver formation [228]. Therefore, the expression of NKG2D may be useful in immunotherapy strategies.

Boosting the immune clearance of senescent cells can also be achieved by adapting cancer vaccine strategies. Even though no universal biomarker of cellular senescence is known, the contact between immune and senescent cells might induce an immunological response to undetected antigens.

Senescence vaccines would consist of isolating senescence-specific antigens and exposing them to antigen presenting cells and dendritic cells. Therefore, the dendritic cells would express these antigens on their cellular surface, which would flag them to T cells. The interaction response between T cells and these antigens provokes the activation and differentiation of T cells and eventually kills targetted senescent cells [231].

A useful strategy is the production of cells able to detect particular components of the senescent secretome. This was proved in creating cells harboring a chimeric IL-6 receptor (IL6Rchi), which produces a Ca²⁺ signal when in contact with IL-6, an important component of the senescent secretome [234].

## 13.3. *Antibody-Dependent Cell-Mediated Cytotoxicity Eliminates Senescent Cells*

Adding to the immune enhancing methods, adapting immunotherapy strategies normally dedicated to cancer treatments might reveal important outcomes for ageing disease interventions. Blocking the immune checkpoint Programmed Death 1 (PD-1) is an example of a potent anticancer therapy, which can help alleviate clinical symptoms in an Alzheimer's disease mouse model [235].

A great example of an anti-cancer therapy is illustrated by using engineered immune cells, such as chimeric antigen receptor T cells targeting individual molecules on tumor cells. Similarly, senescent cells can be targeted via engineered T cells expressing NKG2D chimeric antigen receptor, which detects NKG2D ligands on target cell surfaces [236,237]. The lack of selective senescence markers is a hurdle in immune-targeting techniques.

Senescent cells are mainly secured from NK cell-mediated cytotoxicity due to increased expression of the decoy receptor 2, DcR2 [238]. DcR2 prevents TNF-related apoptosis-inducing ligand (TRAIL) from activating death receptors 4 and 5 (DR4/DR5), limiting the killing process to perforin- and granzyme-mediated pathways. Targeting strategies to accelerate this process, such as by inhibiting DcR2, may render senescent cells more susceptible to immune defense and immunotherapy-based targeting.

As mentioned earlier, natural killer cells detect and destroy senescent cells through NKG2D ligand membrane expression [228]. These NKG2D ligands are also expressed by cancer cells, which make them an important target for cancer immunotherapeutic strategies [239] and consequently for senescent cell clearance.

DPP4 was discovered to be more abundant on the surface of fibroblasts that had undergone RS than on normal cells [122]. Treating senescent cells with an anti-DPP4
13.2. *Immune Boosting Strategies to Improve Elimination of Senescent Cells*

As the immune system becomes less efficient with age, it is likely that a therapeutic
boosting of immune responses can be useful for improving senescent cell elimination.

Senescent cells are naturally immunogenic and are subject to context-dependent im-
mune surveillance mechanisms. Senescence induction by p53 remodeling stimuli an innate
immune reaction typically consists of invading leukocytes, neutrophils, macrophages, and
NK cells in a model of hepatocellular carcinoma [33] and age-associated hepatic stellate
cells enhance their own NK cell-mediated eradication in cases of liver fibrosis [26]. Senes-
cence induction by NRASG12V expression in hepatocytes leads to a dual release of both
innate and adoptive immunological response, as CD4+ T cells collaborate with mono-
cytes/macrophages to remove senescent hepatocytes [35]. This procedure is mediated by
stimulating natural killer cell receptor NKG2D, which detects ligands on the external mem-
brane of stressed, infected, or injured cells. Senescent cells increase the expression of the
immunological recognition protein NKG2D, which is normally absent from the membrane
of healthy cells [228]. Additionally, NKG2D was needed for natural killer cell-induced
senescent cell elimination to protect from fibrotic liver formation [228]. Therefore, the
expression of NKG2D may be useful in immunotherapy strategies.

Boosting the immune clearance of senescent cells can also be achieved by adapting
cancer vaccine strategies. Even though no universal biomarker of cellular senescence is
known, the contact between immune and senescent cells might induce an immunological
response to undetected antigens.

Senescence vaccines would consist of isolating senescence-specific antigens and ex-
posing them to antigen presenting cells and dendritic cells. Therefore, the dendritic cells
would express these antigens on their cellular surface, which would flag them to T cells.
The interaction response between T cells and these antigens provokes the activation and
differentiation of T cells and eventually kills targetted senescent cells [231].

A useful strategy is the production of cells able to detect particular components of the
senescent secretome. This was proved in creating cells harboring a chimeric IL-6 receptor
(IL6Rchi), which produces a Ca²⁺ signal when in contact with IL-6, an important component
of the senescent secretome [234].

13.3. *Antibody-Dependent Cell-Mediated Cytotoxicity Eliminates Senescent Cells*

Adding to the immune enhancing methods, adapting immunotherapy strategies normally dedicated to cancer treatments might reveal important outcomes for ageing disease interventions. Blocking the immune checkpoint Programmed Death 1 (PD-1) is an example of a potent anticancer therapy. which can help alleviate clinical symptoms in an Alzheimer's disease mouse model [235].

A great example of an anti-cancer therapy is illustrated by using engineered immune
cells, such as chimeric antigen receptor T cells targeting individual molecules on tumor
cells. Similarly, senescent cells can be targeted via engineered T cells expressing NKG2D
chimeric antigen receptor, which detects NKG2D ligands on target cell surfaces [236,237].
The lack of selective senescence markers is a hurdle in immune-targeting techniques.

Senescent cells are mainly secured from NK cell-mediated cytotoxicity due to increased expression of the decoy receptor 2, DcR2 [238]. DcR2 prevents TNF-related apoptosis-inducing ligand (TRAIL) from activating death receptors 4 and 5 (DR4/DR5), limiting the killing process to perforin- and granzyme-mediated pathways. Targeting strategies to accelerate this process, such as by inhibiting DcR2, may render senescent cells more susceptible to immune defense and immunotherapy-based targeting.

As mentioned earlier, natural killer cells detect and destroy senescent cells through
NKG2D ligand membrane expression [228]. These NKG2D ligands are also expressed
by cancer cells, which make them an important target for cancer immunotherapeutic
strategies [239] and consequently for senescent cell clearance.

DPP4 was discovered to be more abundant on the surface of fibroblasts that had
undergone RS than on normal cells [122]. Treating senescent cells with an anti-DPP4
antibody induces an NK cell favorable killing in an antibody-dependent cell-mediated cytotoxicity assay [122,240].

Besides, recent studies are working on identifying particular epitopes and membrane receptors related to senescent cells in order to develop antibody-based therapies or screening factors that are more selective and have fewer negative effects. Both DEP1 and B2MG [241], in addition to DCR2 [242], are examples of overly expressed identified markers in senescent cells, but these markers also appeared among other cells and damaged tissues.

Recently, the use of CD9 receptors associated with increased SA-βGal activity is being tested in an in vitro study as a dual nanoparticle targeting better drug delivery to senescent cells [184]. Similarly, a senescent lung fibroblasts marker was discovered, a membrane-bound vimentin oxidized type [121]. In senescent fibroblasts, humoral innate immunity may recognize and attack the oxidized precursor of vimentin. Altogether, these findings therefore suggested a possible immunotherapeutic approach targeting cellular senescence. However, further in vivo studies are required to prove the clinical applicability of these experimental approaches.

One can believe that this approach might ultimately be used in healthy people to postpone aging. We should re-evaluate the best approaches to translate beneficial pre-clinical findings into well developed and effective therapeutic processes. Several approaches targeting senescent cells currently face to drug development challenges, including cost and time, to bring a potent and widely used anti-aging treatment to the pharmaceutical market, while being effective, safe, and tolerable, which is taken into consideration in the initial phases of development.

The major unanswered question in this discipline is the efficacy of various senotherapies in diverse human tissues. The majority of current data on such therapies come from in vitro cell culture research, which does not represent the setting of a pathology and may slightly replicate the condition in vivo. Pharmacological in vivo efficacy was frequently investigated in mice models, many of which are partially related to aging and age-associated diseases. As a result, prior to serotherapies' implementation in clinical trials, their significance to particular human diseases must be thoroughly explored. Furthermore, existing techniques target a broad population of senescent cells, despite the fact that senescent cells are very diverse. The varying sensitivity of cell subpopulations to a particular therapy may result from differences in the cells' origin, the stimulus of senescence, or the disease environment. Therefore, it is reasonable to assume that certain therapies will be more appropriate than others for a specific tissue or age-associated condition. Thus, the idea of considering senotherapies as a pan-antisenescence therapy is rather confusing and should be reconsidered. This will require evaluation of the impact of each therapy on various cell populations while employing the most appropriate model for every disease condition to which the drug can be used.

Because of the physiological importance of senescent cells and the adverse effects of current senolytics, safety concerns may arise when adopting senescence-targeted therapies. Upon systemic administration, many senolytics, particularly BCL-2 family inhibitors, are toxic to non-senescent cells [203]. In addition, many senescence epitopes may be displayed to some extent in non-senescent cells, such as cellular stress or secreting cells, in the case of immunotherapy. There are several alternative solutions to this issue. Drugs, for example, could be delivered directly to the target zone. More importantly, synergistic techniques based on the delivery of senescent cell-specific senolytic agents could have a synchronous effect, allowing the delivery of smaller, less aggressive doses with increased efficacy and safety. Another avenue for improving specific targeting is to modify the drug so that it becomes active only after exposure to an enzyme produced by senescent cells. In addition to the negative impact of the targeted component, decreased safety may result from a likely interaction with the positive activities of senescence. Although the senescence process is implicated in embryogenesis, tissue repair, rejuvenation, and tumor suppression, the impact of senescence targeting by senolytic therapy on such processes is yet unknown [42, 43, 243-246]. Current senolytics induce apoptosis of senescent cells during
antibody induces an NK cell favorable killing in an antibody-dependent cell-mediated cytotoxicity assay [122,240].

Besides, recent studies are working on identifying particular epitopes and membrane receptors related to senescent cells in order to develop antibody-based therapies or screening factors that are more selective and have fewer negative effects. Both DEP1 and B2MG [241], in addition to DCR2 [242], are examples of overly expressed identified markers in senescent cells, but these markers also appeared among other cells and damaged tissues.

Recently, the use of CD9 receptors associated with increased SA-βGal activity is being tested in an in vitro study as a dual nanoparticle targeting better drug delivery to senescent cells [184]. Similarly, a senescent lung fibroblasts marker was discovered, a membrane-bound vimentin oxidized type [121]. In senescent fibroblasts, humoral innate immunity may recognize and attack the oxidized precursor of vimentin. Altogether, these findings therefore suggested a possible immunotherapeutic approach targeting cellular senescence. However, further in vivo studies are required to prove the clinical applicability of these experimental approaches.

One can believe that this approach might ultimately be used in healthy people to postpone aging. We should re-evaluate the best approaches to translate beneficial pre-clinical findings into well developed and effective therapeutic processes. Several approaches targeting senescent cells currently face to drug development challenges, including cost and time, to bring a potent and widely used anti-aging treatment to the pharmaceutical market, while being effective, safe, and tolerable, which is taken into consideration in the initial phases of development.

The major unanswered question in this discipline is the efficacy of various senotherapies in diverse human tissues. The majority of current data on such therapies come from in vitro cell culture research, which does not represent the setting of a pathology and may slightly replicate the condition in vivo. Pharmacological in vivo efficacy was frequently investigated in mice models, many of which are partially related to aging and age-associated diseases. As a result, prior to serotherapies' implementation in clinical trials, their significance to particular human diseases must be thoroughly explored. Furthermore, existing techniques target a broad population of senescent cells, despite the fact that senescent cells are very diverse. The varying sensitivity of cell subpopulations to a particular therapy may result from differences in the cells' origin, the stimulus of senescence, or the disease environment. Therefore, it is reasonable to assume that certain therapies will be more appropriate than others for a specific tissue or age-associated condition. Thus, the idea of considering senotherapies as a pan-antisenescence therapy is rather confusing and should be reconsidered. This will require evaluation of the impact of each therapy on various cell populations while employing the most appropriate model for every disease condition to which the drug can be used.

Because of the physiological importance of senescent cells and the adverse effects of current senolytics, safety concerns may arise when adopting senescence-targeted therapies. Upon systemic administration, many senolytics, particularly BCL-2 family inhibitors, are toxic to non-senescent cells [203]. In addition, many senescence epitopes may be displayed to some extent in non-senescent cells, such as cellular stress or secreting cells, in the case of immunotherapy. There are several alternative solutions to this issue. Drugs, for example, could be delivered directly to the target zone. More importantly, synergistic techniques based on the delivery of senescent cell-specific senolytic agents could have a synchronous effect, allowing the delivery of smaller, less aggressive doses with increased efficacy and safety. Another avenue for improving specific targeting is to modify the drug so that it becomes active only after exposure to an enzyme produced by senescent cells. In addition to the negative impact of the targeted component, decreased safety may result from a likely interaction with the positive activities of senescence. Although the senescence process is implicated in embryogenesis, tissue repair, rejuvenation, and tumor suppression, the impact of senescence targeting by senolytic therapy on such processes is yet unknown [42, 43, 243-246]. Current senolytics induce apoptosis of senescent cells during
limited drug exposures, which can be applied for a short period of time. New therapies must take this into account and be administered in a spatially and temporally regulated manner, in addition to well designed studies to avoid failure. First-wave senotherapies must be carefully selected for indications that may include an unusual age-related condition for which standard treatment is only symptomatic and not therapeutic. The function of cellular senescence in the pathophysiology of each condition or disorder must be established, and the underlying mechanisms must be well understood. The existence of appropriate preclinical models will enable the assessment of the risks and benefits of each therapy. Specific biomarkers of senescence or disease indicators to be used for prognosis and prediction are also critical to the success of clinical investigations. The lack of good senescence markers continues to limit fundamental research, where quantitative measurements are restricted to ex vivo cellular examination. Novel senescence biomarkers are needed to study senescence levels non-invasively in tissues so that participants can be selected based on their senescence status. Many factors can contribute to the success of experimental trials, including scientific evidence, experimental parameters, and individualized patient conditions. Altogether, cellular senescence is a complex process involving both advantageous and disadvantageous outcomes. Senotherapies have the potential to inhibit adverse outcomes and cure a wide range of age-related diseases, but proper administration is necessary to ensure both efficiency and safety. The importance of understanding the processes of potential adverse reactions when determining the method of administration and concentration will accelerate the clinical development of senolytic drugs and provide new opportunities for improving human lifespan and health.

## 14. Clinical Targeting of Senescence in Age-Associated Disorders

Age-related diseases and their corresponding social burden are increasing. Age-associated immune senescent modifications might lead to a decrease in the immune system, chronic inflammation, in addition to weakness, chronic disease, and loss of function among old people [247]. Translating approaches that target senescent cells into clinical practice could have a major impact on curing a range of diseases that occur with age and perhaps change our perspective of ageing.

Preclinical studies showed that the clearance of aged cells can improve and reverse multiple age-associated diseases [41,248,249], giving hope and promises to develop new therapeutics for these critical disorders. For this reason, clinical trials targeting senescent cells are under development.

Due to impressive preliminary results, many trials are focusing on D+Q as a potential bi-therapy to some age-associated diseases and mainly chronic kidney disease (NCT02848131), idiopathic pulmonary fibrosis (NCT02874989), and in cases of hematopoietic stem cell transplant, presenting a high risk of premature ageing (NCT02652052) (Table 1).

Persons diagnosed with activated PI3K Delta syndrome have a dominantly mutated PI3K catalytic subunit p110δ, causing senescence of T cells and immunodeficiency [250]. Leniolisib, a new effective and selective oral PI3Kδ (CDZ173) inhibitor, was able to reduce senescent T cells and to lower the inflammatory markers in clinical trials formed of six patients (NCT02435173); also, an extension study is progressing (NCT02859727). Further procedures are being developed, which include more trials to detect the effect of plasma transfer from healthy young male donors to patients over 40, assessing the ability to reverse epigenetic and other senescent markers (NCT03353597). Many factors can be evaluated from blood and skin biopsies, such as the DNA methylation level and other epigenetic alterations, the length of telomeres, as well as renal, pulmonary, cognitive, and muscle strength variations, in addition to IGF-1 and p16 expression.
limited drug exposures, which can be applied for a short period of time. New therapies must take this into account and be administered in a spatially and temporally regulated manner, in addition to well designed studies to avoid failure. First-wave senotherapies must be carefully selected for indications that may include an unusual age-related condition for which standard treatment is only symptomatic and not therapeutic. The function of cellular senescence in the pathophysiology of each condition or disorder must be established, and the underlying mechanisms must be well understood. The existence of appropriate preclinical models will enable the assessment of the risks and benefits of each therapy. Specific biomarkers of senescence or disease indicators to be used for prognosis and prediction are also critical to the success of clinical investigations. The lack of good senescence markers continues to limit fundamental research, where quantitative measurements are restricted to ex vivo cellular examination. Novel senescence biomarkers are needed to study senescence levels non-invasively in tissues so that participants can be selected based on their senescence status. Many factors can contribute to the success of experimental trials, including scientific evidence, experimental parameters, and individualized patient conditions. Altogether, cellular senescence is a complex process involving both advantageous and disadvantageous outcomes. Senotherapies have the potential to inhibit adverse outcomes and cure a wide range of age-related diseases, but proper administration is necessary to ensure both efficiency and safety. The importance of understanding the processes of potential adverse reactions when determining the method of administration and concentration will accelerate the clinical development of senolytic drugs and provide new opportunities for improving human lifespan and health.

## 14. Clinical Targeting of Senescence in Age-Associated Disorders

Age-related diseases and their corresponding social burden are increasing. Age-associated immune senescent modifications might lead to a decrease in the immune system, chronic inflammation, in addition to weakness, chronic disease, and loss of function among old people [247]. Translating approaches that target senescent cells into clinical practice could have a major impact on curing a range of diseases that occur with age and perhaps change our perspective of ageing.

Preclinical studies showed that the clearance of aged cells can improve and reverse multiple age-associated diseases [41,248,249], giving hope and promises to develop new therapeutics for these critical disorders. For this reason, clinical trials targeting senescent cells are under development.

Due to impressive preliminary results, many trials are focusing on D+Q as a potential bi-therapy to some age-associated diseases and mainly chronic kidney disease (NCT02848131), idiopathic pulmonary fibrosis (NCT02874989), and in cases of hematopoietic stem cell transplant, presenting a high risk of premature ageing (NCT02652052) (Table 1).

Persons diagnosed with activated PI3K Delta syndrome have a dominantly mutated PI3K catalytic subunit p110δ, causing senescence of T cells and immunodeficiency [250]. Leniolisib, a new effective and selective oral PI3Kδ (CDZ173) inhibitor, was able to reduce senescent T cells and to lower the inflammatory markers in clinical trials formed of six patients (NCT02435173); also, an extension study is progressing (NCT02859727). Further procedures are being developed, which include more trials to detect the effect of plasma transfer from healthy young male donors to patients over 40, assessing the ability to reverse epigenetic and other senescent markers (NCT03353597). Many factors can be evaluated from blood and skin biopsies, such as the DNA methylation level and other epigenetic alterations, the length of telomeres, as well as renal, pulmonary, cognitive, and muscle strength variations, in addition to IGF-1 and p16 expression.
<table><caption>**Table 1.** Ongoing and planned clinical trials using senolytic drug interventions.</caption><thead><tr><th>Intervention/Treatment</th><th>Disease/Condition</th><th>Outcome Measures</th><th>Eligible Age</th><th>Phase</th><th>Study ID (https://clinicaltrials.gov/, accessed on 24 June 2022)</th></tr></thead><tbody><tr><td rowspan="7">Dasatinib + Quercetin</td><td>Idiopathic pulmonary fibrosis patients</td><td>Improvements in physical function</td><td>≥50</td><td>Phase I</td><td>NCT02874989</td></tr><tr><td>Diabetes and kidney dysfunction</td><td>Decrease in senescence markers</td><td>50-80</td><td>Phase I</td><td>NCT02848131</td></tr><tr><td>Alzheimer</td><td>Change in cellular senescence blood marker</td><td>≥65</td><td>Phase II</td><td>NCT04063124</td></tr><tr><td>Skeletal disease</td><td>Skeletal health improvement</td><td>≥70</td><td>Phase II</td><td>NCT04313634</td></tr><tr><td>Chronic kidney disease</td><td>Senescent cells proportional change</td><td>40-80</td><td>Phase II</td><td>NCT02848131</td></tr><tr><td>Stem cell transplant</td><td>Frailty level</td><td>≥18</td><td>Recruiting</td><td>NCT02652052</td></tr><tr><td>Frailty, inflammation</td><td>Decrease in blood inflammation markers</td><td>≥70</td><td>Phase II</td><td>NCT03430037</td></tr><tr><td rowspan="2">Fisetin</td><td>Osteoarthritis, knee</td><td>Change in levels of proinflammatory markers associated with senescence</td><td>40-80</td><td>Phase I</td><td>NCT04210986</td></tr><tr><td>COVID-19</td><td>Change in COVID-19 severity</td><td>≥65</td><td>Phase II</td><td>NCT04771611</td></tr><tr><td>Dasatinib + Quercetin; Fisetin</td><td>Childhood cancer and Frailty</td><td>Senescence reduction and frailty improvement</td><td>≥18</td><td>Phase II</td><td>NCT04733534</td></tr><tr><td>UBX0101</td><td>Osteoarthritis, knee</td><td>Safety and efficacy level</td><td>40-85</td><td>Phase II</td><td>NCT04129944</td></tr><tr><td>UBX1325</td><td>Diabetic Macular Edema Neovascular age-related macular degeneration</td><td>Gain in visual acuity</td><td>≥50</td><td>Phase I</td><td>NCT04537884</td></tr><tr><td>UBX1967<br>UBX 2050</td><td>Macular degeneration</td><td>-</td><td>-</td><td>-</td><td>Planned</td></tr><tr><td>CDZ173</td><td>APDS/PASLI</td><td>Senescent T cells reduction</td><td>12-75</td><td>Phase II and III</td><td>NCT02435173 (Extension study NCT02859727)</td></tr><tr><td rowspan="2">Tamoxifen (MitoTam)</td><td rowspan="2">Solid metastatic tumours</td><td>Maximum tolerated dose identification</td><td rowspan="2">18-75</td><td rowspan="2">Phase I/Ib</td><td rowspan="2">EudraCT 2017-004441-25</td></tr><tr><td>Safety level<br>High efficacy against renal cell carcinoma</td></tr></tbody></table>

The early findings of a phase two experiment examining twelve weeks of senolytic therapy of knee osteoarthritis showed failure to meet the expected outcome, with the complete data still to be reported (UNITY, 2021). The phase 2 clinical study, UBX0101, in patients with painful osteoarthritis of the knee, was conducted by Unity Biotechnology. Many explanations could be given as to why the experiment failed: the strategy is not practical for this condition, maybe the drug dosage was wrong, or the inclusion criteria should have been narrower or broader. All of this aside, clinical trials of senescence-reducing therapies are in their earliest stages and need to be further explored. However, recently, a red-hot anti-aging approach almost passed its first test after 14 volunteers took drugs meant to kill off old, toxic cells present in their bodies. They suffered from a fatal, hard-to-treat lung condition called idiopathic pulmonary fibrosis. Idiopathic pulmonary fibrosis is a lethal condition characterized by age-related markers, such as DNA damage, inflammation, telomere attrition, oxidative stress, and, most significantly, cellular senescence [251]. Studies in mice with idiopathic pulmonary fibrosis show that the senolytic agents D+Q have a beneficial impact on pulmonary functioning [7]. At the same time, a study among patients with idiopathic pulmonary fibrosis, which primarily investigated the overall safety of this treatment, indicates that D+Q also has beneficial implications for physical function in humans [252]. For three weeks, fourteen patients suffering from idiopathic pulmonary fibrosis received D+Q treatment, and the results showed not only significant, but also clinically meaningful gains, in physical function. This analysis showed no effect on lung capacity, clinical chemistry, or frailty. As for the effects on SASP and proinflammatory markers, it was not entirely conclusive, but it showed some correlated changes [252].

Diabetes and renal disease are associated with an increased senescent cell load, particularly in adipose tissue. A clinical experiment examined the effects of D+Q on senescent human adipocytes [253], in which 11 patients with diabetes and kidney disease received D+Q for three days. Results showed a substantial decrease in markers of senescent cells, as well as significantly lower numbers of senescent cells in adipose tissue. Ultimately, the
<table><caption>**Table 1.** Ongoing and planned clinical trials using senolytic drug interventions.</caption><thead><tr><th>Intervention/Treatment</th><th>Disease/Condition</th><th>Outcome Measures</th><th>Eligible Age</th><th>Phase</th><th>Study ID (https://clinicaltrials.gov/, accessed on 24 June 2022)</th></tr></thead><tbody><tr><td rowspan="6">Dasatinib + Quercetin</td><td>Idiopathic pulmonary fibrosis patients</td><td>Improvements in physical function</td><td>≥50</td><td>Phase I</td><td>NCT02874989</td></tr><tr><td>Diabetes and kidney dysfunction</td><td>Decrease in senescence markers</td><td>50-80</td><td>Phase I</td><td>NCT02848131</td></tr><tr><td>Alzheimer</td><td>Change in cellular senescence blood marker</td><td>≥65</td><td>Phase II</td><td>NCT04063124</td></tr><tr><td>Skeletal disease</td><td>Skeletal health improvement</td><td>≥70</td><td>Phase II</td><td>NCT04313634</td></tr><tr><td>Chronic kidney disease</td><td>Senescent cells proportional change</td><td>40-80</td><td>Phase II</td><td>NCT02848131</td></tr><tr><td>Stem cell transplant</td><td>Frailty level</td><td>≥18</td><td>Recruiting</td><td>NCT02652052</td></tr><tr><td rowspan="3">Fisetin</td><td>Frailty, inflammation</td><td>Decrease in blood inflammation markers</td><td>≥70</td><td>Phase II</td><td>NCT03430037</td></tr><tr><td>Osteoarthritis, knee</td><td>Change in levels of proinflammatory markers associated with senescence</td><td>40-80</td><td>Phase I</td><td>NCT04210986</td></tr><tr><td>COVID-19</td><td>Change in COVID-19 severity</td><td>≥65</td><td>Phase II</td><td>NCT04771611</td></tr><tr><td>Dasatinib + Quercetin; Fisetin</td><td>Childhood cancer and Frailty</td><td>Senescence reduction and frailty improvement</td><td>≥18</td><td>Phase II</td><td>NCT04733534</td></tr><tr><td>UBX0101</td><td>Osteoarthritis, knee</td><td>Safety and efficacy level</td><td>40-85</td><td>Phase II</td><td>NCT04129944</td></tr><tr><td>UBX1325</td><td>Diabetic Macular Edema Neovascular age-related macular degeneration</td><td>Gain in visual acuity</td><td>≥50</td><td>Phase I</td><td>NCT04537884</td></tr><tr><td>UBX1967<br>UBX 2050</td><td>Macular degeneration</td><td>-</td><td>-</td><td>-</td><td>Planned</td></tr><tr><td>CDZ173</td><td>APDS/PASLI</td><td>Senescent T cells reduction</td><td>12-75</td><td>Phase II and III</td><td>NCT02435173 (Extension study NCT02859727)</td></tr><tr><td rowspan="2">Tamoxifen (MitoTam)</td><td rowspan="2">Solid metastatic tumours</td><td>Maximum tolerated dose identification</td><td rowspan="2">18-75</td><td rowspan="2">Phase I/lb</td><td rowspan="2">EudraCT 2017-004441-25</td></tr><tr><td>Safety level<br>High efficacy against renal cell carcinoma</td></tr></tbody></table>

The early findings of a phase two experiment examining twelve weeks of senolytic therapy of knee osteoarthritis showed failure to meet the expected outcome, with the complete data still to be reported (UNITY, 2021). The phase 2 clinical study, UBX0101, in patients with painful osteoarthritis of the knee, was conducted by Unity Biotechnology. Many explanations could be given as to why the experiment failed: the strategy is not practical for this condition, maybe the drug dosage was wrong, or the inclusion criteria should have been narrower or broader. All of this aside, clinical trials of senescence-reducing therapies are in their earliest stages and need to be further explored. However, recently, a red-hot anti-aging approach almost passed its first test after 14 volunteers took drugs meant to kill off old, toxic cells present in their bodies. They suffered from a fatal, hard-to-treat lung condition called idiopathic pulmonary fibrosis. Idiopathic pulmonary fibrosis is a lethal condition characterized by age-related markers, such as DNA damage, inflammation, telomere attrition, oxidative stress, and, most significantly, cellular senescence [251]. Studies in mice with idiopathic pulmonary fibrosis show that the senolytic agents D+Q have a beneficial impact on pulmonary functioning [7]. At the same time, a study among patients with idiopathic pulmonary fibrosis, which primarily investigated the overall safety of this treatment, indicates that D+Q also has beneficial implications for physical function in humans [252]. For three weeks, fourteen patients suffering from idiopathic pulmonary fibrosis received D+Q treatment, and the results showed not only significant, but also clinically meaningful gains, in physical function. This analysis showed no effect on lung capacity, clinical chemistry, or frailty. As for the effects on SASP and proinflammatory markers, it was not entirely conclusive, but it showed some correlated changes [252].

Diabetes and renal disease are associated with an increased senescent cell load, particularly in adipose tissue. A clinical experiment examined the effects of D+Q on senescent human adipocytes [253], in which 11 patients with diabetes and kidney disease received D+Q for three days. Results showed a substantial decrease in markers of senescent cells, as well as significantly lower numbers of senescent cells in adipose tissue. Ultimately, the
study confirmed the postulated hypothesis by showing that D+Q significantly decreased circulating SASP components in the patients.

Moreover, numerous studies have been conducted on MitoTam, a mitochondria-targeted tamoxifen that has been shown to be a novel senolytic that selectively activates senescent cell death by decreasing mitochondrial membrane potential and preventing mitochondrial oxidative phosphorylation (OXPHOS), thereby improving mitochondrial stability [254]. Further than this, MitoTam has been shown to effectively destroy a variety of renal cancer cells and inhibit renal carcinomas in a rodent model via involvement of mitochondria, resulting in reduced tumorigenesis (EudraCT 2017-004441-25) and providing hope for future phase 2 trials [255]. Finally, MitoTam also appears to have the potential to be an anti-diabetic agent, helping to tackle one of the most prevalent pandemics, obesity and type 2 diabetes mellitus [256].

At present, 20 clinical trials on senolytics are ongoing. Two of the studies targeting frailty and aging (AFFIRM studies), comparing fisetin to placebo as a control group, are almost halfway complete. One of these studies is in elderly women with severe frailty who have a walking speed of less than 0.6 m per second, which is a significant indicator of a 2-year survival rate of about 50%. A pilot study in Alzheimer's disease (ALSENLITE) has been initiated, along with a multicenter, double-blinded, placebo-controlled study in Alzheimer's patients (SToMP-AD) and a study in renal disease, along with a nearly completed study in bone marrow transplant patients (HTSS). Patients requiring bone marrow transplantation receive high doses of chemotherapy and radiation to knock out their immune system prior to transplantation, which unfortunately can cause aging. A number of bone marrow transplant recipients have been found to develop accelerating signs of aging after three to five years. Therefore, this trial will test if elimination of senescent cells could mitigate this accelerated aging condition in which individuals develop many pathologies including diabetes, cognitive decline, secondary non-related cancers, and atherosclerosis with myocardial infarctions and strokes.

All trials are in preparation for a bigger double-blinded, placebo-controlled study. The NIH-funded study of age-related osteoporosis is nearly completed, with over half the participants recruited. Another trial by the Office of Naval Research supported a clinical trial on osteoarthritis, a condition associated with the accumulation of senescent cells in the joints of the knees. Additionally, a number of studies on coronaviruses and their consequences are still currently active or have been initiated (COVID-FIS, COVID-FISETIN, COVFIS-HOME).

**15. Future Perspectives**

The abnormal and permanent accumulation of various senescent cells causes aging and the emergence of chronic diseases. Cellular senescence regulation is crucial for healthy aging, as it also has short-term benefits. There are multiple clinical molecules and senolytics to treat aging and chronic diseases related to cellular senescence.

Even though immunological elimination of specific senescent cells is a novel and challenging technique for anti-aging and treatment of several chronic conditions, it is not costless and has negative effects. For clinical translation of this new technology, certain inquiries need to be conducted, including: (1) both preclinical and clinical research, identification of appropriate biomarkers or molecular mechanisms for personalized aging; (2) discovery of distinct senescent cell antigens or ligands for immune monitoring; and (3) incorporation of both sexes into the study due to the gender variations in aging and chronic disorders. Senotherapy can lead to adverse reactions and harm tissue homeostasis and function. This may be avoided by applying senotherapy in a temporary and discontinuous manner if it preserves the therapeutic effects of delaying aging.

In this review, we present certain important approaches to control cellular senescence, some of which were investigated and confirmed on animal models with aging-related diseases. A subset is now in clinical trials. Our main focus here is on senolytic drugs and manipulating the immune surveillance of senescent cells. Importantly, several clinical
study confirmed the postulated hypothesis by showing that D+Q significantly decreased circulating SASP components in the patients.

Moreover, numerous studies have been conducted on MitoTam, a mitochondria-targeted tamoxifen that has been shown to be a novel senolytic that selectively activates senescent cell death by decreasing mitochondrial membrane potential and preventing mitochondrial oxidative phosphorylation (OXPHOS), thereby improving mitochondrial stability [254]. Further than this, MitoTam has been shown to effectively destroy a variety of renal cancer cells and inhibit renal carcinomas in a rodent model via involvement of mitochondria, resulting in reduced tumorigenesis (EudraCT 2017-004441-25) and providing hope for future phase 2 trials [255]. Finally, MitoTam also appears to have the potential to be an anti-diabetic agent, helping to tackle one of the most prevalent pandemics, obesity and type 2 diabetes mellitus [256].

At present, 20 clinical trials on senolytics are ongoing. Two of the studies targeting frailty and aging (AFFIRM studies), comparing fisetin to placebo as a control group, are almost halfway complete. One of these studies is in elderly women with severe frailty who have a walking speed of less than 0.6 m per second, which is a significant indicator of a 2-year survival rate of about 50%. A pilot study in Alzheimer's disease (ALSENLITE) has been initiated, along with a multicenter, double-blinded, placebo-controlled study in Alzheimer's patients (STOMP-AD) and a study in renal disease, along with a nearly completed study in bone marrow transplant patients (HTSS). Patients requiring bone marrow transplantation receive high doses of chemotherapy and radiation to knock out their immune system prior to transplantation, which unfortunately can cause aging. A number of bone marrow transplant recipients have been found to develop accelerating signs of aging after three to five years. Therefore, this trial will test if elimination of senescent cells could mitigate this accelerated aging condition in which individuals develop many pathologies including diabetes, cognitive decline, secondary non-related cancers, and atherosclerosis with myocardial infarctions and strokes.

All trials are in preparation for a bigger double-blinded, placebo-controlled study. The NIH-funded study of age-related osteoporosis is nearly completed, with over half the participants recruited. Another trial by the Office of Naval Research supported a clinical trial on osteoarthritis, a condition associated with the accumulation of senescent cells in the joints of the knees. Additionally, a number of studies on coronaviruses and their consequences are still currently active or have been initiated (COVID-FIS, COVID-FISETIN, COVFIS-HOME).

**15. Future Perspectives**

The abnormal and permanent accumulation of various senescent cells causes aging and the emergence of chronic diseases. Cellular senescence regulation is crucial for healthy aging, as it also has short-term benefits. There are multiple clinical molecules and senolytics to treat aging and chronic diseases related to cellular senescence.

Even though immunological elimination of specific senescent cells is a novel and challenging technique for anti-aging and treatment of several chronic conditions, it is not costless and has negative effects. For clinical translation of this new technology, certain inquiries need to be conducted, including: (1) both preclinical and clinical research, identification of appropriate biomarkers or molecular mechanisms for personalized aging; (2) discovery of distinct senescent cell antigens or ligands for immune monitoring; and (3) incorporation of both sexes into the study due to the gender variations in aging and chronic disorders. Senotherapy can lead to adverse reactions and harm tissue homeostasis and function. This may be avoided by applying senotherapy in a temporary and discontinuous manner if it preserves the therapeutic effects of delaying aging.

In this review, we present certain important approaches to control cellular senescence, some of which were investigated and confirmed on animal models with aging-related diseases. A subset is now in clinical trials. Our main focus here is on senolytic drugs and manipulating the immune surveillance of senescent cells. Importantly, several clinical
approaches targeting senescent cells might have a significant effect on treating some age-
related pathologies and on increasing lifespan through an aging delay in the damaged
tissues and organs. However, some critical risks should be taken into consideration for a
cautious clinical translation of senescence-targeted therapeutic methodologies.

Much of the data on senotherapies were from human cell cultures. The new approaches promote senescence through various stressors and controlling mechanisms and in various cell types. The induction of senescence in specific cell types from specific tissues through either replicative stress, oncogene activation, irradiation, carcinogens, damaging molecules, and many others helps determine the key elements and signaling pathways involved [257]. There are many different types of senescent cells, and consequently, the subtypes can have multiple vulnerabilities to senotherapies and senoprobes.

Thus, it is very crucial to clinically solve the correct questions as to how drug 1 affects drug 2 and in which conditions, by assessing different models accurately representing the human condition. Senescent cells are known to autonomously inhibit the spread of damaged and pre-malignant cells, and thus, they form an essential tumor-resistance barrier [179]. It was observed that the accumulation of mutations in the p53-p21 and the p16-Rb pathways are correlated with the majority of human cancer cells and among different senescence-related genes [258]. Additionally, escape from or reverting senescence contribute to carcinogenesis [259]; correspondingly, it was discovered that a family of therapy-induced senescent cancer cells can develop functional and phenotypic stemness characteristics, causing cell cycle entrance, self-renewal ability, and a higher tumor phenotypic aggressiveness [260]. Despite their many advantages, senescence-focussed approaches should be translated from relevant preclinical models with extreme caution due to the beneficial and harmful roles that senescence exerts, depending on the pathological situation [4].

Moreover, the secretome of senescent cells is importantly involved in recruiting T cells and macrophages, which facilitates immuno-surveillance in the case of liver precancerous lesions [231]. However, the senescent secretome might also intrinsically induce chronic inflammation and tumor progression [150]. In fact, secretion from senescent cells helps in repairing damaged and injured tissues. An example of this comes from senescent-activated stellate cells, which limit liver fibrosis by increasing immuno-surveillance and reducing secretion of extracellular matrix components, apart from extracellular matrix degrading enzymes [26].

The same kind of situation occurs when senescent fibroblasts assemble In healing cutaneous wounds of granulated tissues and activate anti-fibrotic genes [45]. Mouse studies revealed that senescence contributes to fibrotic pulmonary disease models, as fibrosis was reduced and lung function improved when these mice were treated with senolytics, anti-inflammatory molecules, and therapeutic nanoparticles [7,217,261].

The contradictory roles of senescent cells, as inhibitor and enhancer of fibrosis, in mouse models of human fibrotic diseases highlights the need for more comprehensive knowledge on the intrinsic mechanisms and pathways causing cell cycle arrest and the specific elements released by senescent cells prior to initiating clinical trials in humans.

**16. Conclusions**

As life expectancy has increased, so have the chances of contracting age-related diseases, such as cancer, so understanding the aging process is key to improving life. During aging in mice, as well as in humans, senescent cells accumulate, leading to tissue dysfunction and to different age-related pathologies. Preclinical studies in mice proved that eliminating senescent cells can improve and even reverse the pathological appearance of several disorders; however, many obstacles must be overcome to guarantee a perfectly strategic translation to the clinic.

When cells are damaged and stressed, senescence or apoptosis cell fates are safeguard mechanisms with bright and dark side. In some circumstances, it might be more interesting for tissue fitness to maintain senescence and mitigate the deleterious effect of the SASP
approaches targeting senescent cells might have a significant effect on treating some age-
related pathologies and on increasing lifespan through an aging delay in the damaged
tissues and organs. However, some critical risks should be taken into consideration for a
cautious clinical translation of senescence-targeted therapeutic methodologies.

Much of the data on senotherapies were from human cell cultures. The new approaches promote senescence through various stressors and controlling mechanisms and in various cell types. The induction of senescence in specific cell types from specific tissues through either replicative stress, oncogene activation, irradiation, carcinogens, damaging molecules, and many others helps determine the key elements and signaling pathways involved [257]. There are many different types of senescent cells, and consequently, the subtypes can have multiple vulnerabilities to senotherapies and senoprobes.

Thus, it is very crucial to clinically solve the correct questions as to how drug 1 affects drug 2 and in which conditions, by assessing different models accurately representing the human condition. Senescent cells are known to autonomously inhibit the spread of damaged and pre-malignant cells, and thus, they form an essential tumor-resistance barrier [179]. It was observed that the accumulation of mutations in the p53-p21 and the p16-Rb pathways are correlated with the majority of human cancer cells and among different senescence-related genes [258]. Additionally, escape from or reverting senescence contribute to carcinogenesis [259]; correspondingly, it was discovered that a family of therapy-induced senescent cancer cells can develop functional and phenotypic stemness characteristics, causing cell cycle entrance, self-renewal ability, and a higher tumor phenotypic aggressiveness [260]. Despite their many advantages, senescence-focussed approaches should be translated from relevant preclinical models with extreme caution due to the beneficial and harmful roles that senescence exerts, depending on the pathological situation [4].

Moreover, the secretome of senescent cells is importantly involved in recruiting T cells and macrophages, which facilitates immuno-surveillance in the case of liver precancerous lesions [231]. However, the senescent secretome might also intrinsically induce chronic inflammation and tumor progression [150]. In fact, secretion from senescent cells helps in repairing damaged and injured tissues. An example of this comes from senescent-activated stellate cells, which limit liver fibrosis by increasing immuno-surveillance and reducing secretion of extracellular matrix components, apart from extracellular matrix degrading enzymes [26].

The same kind of situation occurs when senescent fibroblasts assemble In healing cutaneous wounds of granulated tissues and activate anti-fibrotic genes [45]. Mouse studies revealed that senescence contributes to fibrotic pulmonary disease models, as fibrosis was reduced and lung function improved when these mice were treated with senolytics, anti-inflammatory molecules, and therapeutic nanoparticles [7,217,261].

The contradictory roles of senescent cells, as inhibitor and enhancer of fibrosis, in mouse models of human fibrotic diseases highlights the need for more comprehensive knowledge on the intrinsic mechanisms and pathways causing cell cycle arrest and the specific elements released by senescent cells prior to initiating clinical trials in humans.

**16. Conclusions**

As life expectancy has increased, so have the chances of contracting age-related dis-
eases, such as cancer, so understanding the aging process is key to improving life. During
aging in mice, as well as in humans, senescent cells accumulate, leading to tissue dys-
function and to different age-related pathologies. Preclinical studies in mice proved that
eliminating senescent cells can improve and even reverse the pathological appearance of
several disorders; however, many obstacles must be overcome to guarantee a perfectly
strategic translation to the clinic.

When cells are damaged and stressed, senescence or apoptosis cell fates are safeguard
mechanisms with bright and dark side. In some circumstances, it might be more interesting
for tissue fitness to maintain senescence and mitigate the deleterious effect of the SASP
with senomorphic strategies and, in other situations, elimination of senescent cells using senolytic therapy could be more advantageous clinically. As there are various molecular mechanisms behind the resistance of senescent cells to apoptosis, for each age-related disease it will be important to identify their signaling pathways, specific secretomes, and senescent arrest contributors. Additionally, a more stringent selection of better optimized senotherapies is required to improve efficacy and reduce toxicity. Bearing in mind inter-patient individuality, and that the aging process with its related disorders, is multifactorial and complex, it is most likely that the next generation of anti-aging health interventions will be specialized to depend on specific biomarkers that inform a clear risk-benefit ratio.

Many senotherapeutic and immunological studies of age-associated pathologies are currently in clinical trial. It is likely that, ultimately in the long term, immune cell-mediated elimination of senescent cells will be key to reduce senescent cell burden. Expeditious clinical advancement will thus depend on a better understanding of senescent cells and their interaction with the immune system.

This critical era that we are going through will allow us to develop anti-senescent therapies, which might have a major effect on personalized 'precision' medicine. These advances in tissue repair mechanisms and regeneration will likely further increase human healthspan and longevity.

**Author Contributions:** R.K. and J.-M.L. wrote the original draft of the review and M.D.-A. edited the text. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of Interest.

## References

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2. López-Otín, C.; Blasco, M.A.; Partridge, L.; Serrano, M.; Kroemer, G. The hallmarks of aging. *Cell* **2013**, *153*, 1194–1217. [CrossRef] [PubMed]

3. López-Otín, C.; Blasco, M.A.; Partridge, L.; Serrano, M.; Kroemer, G. Hallmarks of aging: An expanding universe. *Cell* **2023**, *186*, 243–278. [CrossRef] [PubMed]

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5. van Deursen, J.M. The role of senescent cells in ageing. *Nature* **2014**, *509*, 439–446. [CrossRef] [PubMed]

6. Childs, B.G.; Baker, D.J.; Wijshake, T.; Conover, C.A.; Campisi, J.; van Deursen, J.M. Senescent intimal foam cells are deleterious at all stages of atherosclerosis. *Science* **2016**, *354*, 472–477. [CrossRef]

7. Schafer, M.J.; White, T.A.; Iijima, K.; Haak, A.J.; Ligresti, G.; Atkinson, E.J.; Oberg, A.L.; Birch, J.; Salmonowicz, H.; Zhu, Y.; et al. Cellular senescence mediates fibrotic pulmonary disease. *Nat. Commun.* **2017**, *8*, 14532. [CrossRef]

8. Farr, J.N.; Xu, M.; Weivoda, M.M.; Monroe, D.G.; Fraser, D.G.; Onken, J.L.; Negley, B.A.; Sfeir, J.G.; Ogrodnik, M.B.; Hachfeld, C.M.; et al. Targeting cellular senescence prevents age-related bone loss in mice. *Nat. Med.* **2017**, *23*, 1072–1079. [CrossRef]

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10. Jeon, O.H.; Kim, C.; Laberge, R.M.; Demaria, M.; Rathod, S.; Vasserot, A.P.; Chung, J.W.; Kim, D.H.; Poon, Y.; David, N.; et al. Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment. *Nat. Med.* **2017**, *23*, 775–781. [CrossRef]

11. Roos, C.M.; Zhang, B.; Palmer, A.K.; Ogrodnik, M.B.; Pirtskhalava, T.; Thalji, N.M.; Hagler, M.; Jurk, D.; Smith, L.A.; Casaclang-Verzosa, G.; et al. Chronic senolytic treatment alleviates established vasomotor dysfunction in aged or atherosclerotic mice. *Aging Cell* **2016**, *15*, 973–977. [CrossRef]
with senomorphic strategies and, in other situations, elimination of senescent cells using senolytic therapy could be more advantageous clinically. As there are various molecular mechanisms behind the resistance of senescent cells to apoptosis, for each age-related disease it will be important to identify their signaling pathways, specific secretomes, and senescent arrest contributors. Additionally, a more stringent selection of better optimized senotherapies is required to improve efficacy and reduce toxicity. Bearing in mind inter-patient individuality, and that the aging process with its related disorders, is multifactorial and complex, it is most likely that the next generation of anti-aging health interventions will be specialized to depend on specific biomarkers that inform a clear risk-benefit ratio.

Many senotherapeutic and immunological studies of age-associated pathologies are currently in clinical trial. It is likely that, ultimately in the long term, immune cell-mediated elimination of senescent cells will be key to reduce senescent cell burden. Expeditious clinical advancement will thus depend on a better understanding of senescent cells and their interaction with the immune system.

This critical era that we are going through will allow us to develop anti-senescent therapies, which might have a major effect on personalized 'precision' medicine. These advances in tissue repair mechanisms and regeneration will likely further increase human healthspan and longevity.

**Author Contributions:** R.K. and J.-M.L. wrote the original draft of the review and M.D.-A. edited the text. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of Interest.

## References

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2. López-Otín, C.; Blasco, M.A.; Partridge, L.; Serrano, M.; Kroemer, G. The hallmarks of aging. *Cell* **2013**, *153*, 1194–1217. [CrossRef] [PubMed]

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6. Childs, B.G.; Baker, D.J.; Wijshake, T.; Conover, C.A.; Campisi, J.; van Deursen, J.M. Senescent intimal foam cells are deleterious at all stages of atherosclerosis. *Science* **2016**, *354*, 472–477. [CrossRef]

7. Schafer, M.J.; White, T.A.; Iijima, K.; Haak, A.J.; Ligresti, G.; Atkinson, E.J.; Oberg, A.L.; Birch, J.; Salmonowicz, H.; Zhu, Y.; et al. Cellular senescence mediates fibrotic pulmonary disease. *Nat. Commun.* **2017**, *8*, 14532. [CrossRef]

8. Farr, J.N.; Xu, M.; Weivoda, M.M.; Monroe, D.G.; Fraser, D.G.; Onken, J.L.; Negley, B.A.; Sfeir, J.G.; Ogrodnik, M.B.; Hachfeld, C.M.; et al. Targeting cellular senescence prevents age-related bone loss in mice. *Nat. Med.* **2017**, *23*, 1072–1079. [CrossRef]

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120. Hildebrand, D.G.; Lehle, S.; Borst, A.; Haferkamp, S.; Essmann, F.; Schulze-Osthoff, K. α-Fucosidase as a novel convenient biomarker for cellular senescence. *Cell Cycle* **2013**, *12*, 1922–1927. [CrossRef]

121. Frescas, D.; Roux, C.M.; Aygun-Sunar, S.; Gleiberman, A.S.; Krasnov, P.; Kurnasov, O.V.; Strom, E.; Virtuoso, L.P.; Wrobel, M.; Osterman, A.L.; et al. Senescent cells expose and secrete an oxidized form of membrane-bound vimentin as revealed by a natural polyreactive antibody. *Proc. Natl. Acad. Sci. USA* **2017**, *114*, E1668–E1677. [CrossRef]

122. Kim, K.M.; Noh, J.H.; Bodogai, M.; Martindale, J.L.; Yang, X.; Indig, F.E.; Basu, S.K.; Ohnuma, K.; Morimoto, C.; Johnson, P.F.; et al. Identification of senescent cell surface targetable protein DPP4. *Genes. Dev.* **2017**, *31*, 1529–1534. [CrossRef] [PubMed]

123. Mills, K.F.; Yoshida, S.; Stein, L.R.; Grozio, A.; Kubota, S.; Sasaki, Y.; Redpath, P.; Migaud, M.E.; Apte, R.S.; Uchida, K.; et al. Long-Term Administration of Nicotinamide Mononucleotide Mitigates Age-Associated Physiological Decline in Mice. *Cell Metab.* **2016**, *24*, 795–806. [CrossRef] [PubMed]

124. Zhang, H.; Ryu, D.; Wu, Y.; Gariani, K.; Wang, X.; Luan, P.; D'Amico, D.; Ropelle, E.R.; Lutolf, M.P.; Aebersold, R.; et al. NAD(+) repletion improves mitochondrial and stem cell function and enhances life span in mice. *Science* **2016**, *352*, 1436–1443. [CrossRef] [PubMed]

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126. Irie, J.; Inagaki, E.; Fujita, M.; Nakaya, H.; Mitsuishi, M.; Yamaguchi, S.; Yamashita, K.; Shigaki, S.; Ono, T.; Yukioka, H.; et al. Effect of oral administration of nicotinamide mononucleotide on clinical parameters and nicotinamide metabolite levels in healthy Japanese men. *Endocr. J.* **2020**, *67*, 153–160. [CrossRef]

127. Latorre, E.; Torregrossa, R.; Wood, M.E.; Whiteman, M.; Harries, L.W. Mitochondria-targeted hydrogen sulfide attenuates endothelial senescence by selective induction of splicing factors HNRNPD and SRSF2. *Aging (Albany NY)* **2018**, *10*, 1666–1681. [CrossRef]

128. Roberts, M.N.; Wallace, M.A.; Tomilov, A.A.; Zhou, Z.; Marcotte, G.R.; Tran, D.; Perez, G.; Gutierrez-Casado, E.; Koike, S.; Knotts, T.A.; et al. A Ketogenic Diet Extends Longevity and Healthspan in Adult Mice. *Cell Metab.* **2017**, *26*, 539–546.e5. [CrossRef]

129. Newman, J.C.; Covarrubias, A.J.; Zhao, M.; Yu, X.; Gut, P.; Ng, C.P.; Huang, Y.; Haldar, S.; Verdin, E. Ketogenic Diet Reduces Midlife Mortality and Improves Memory in Aging Mice. *Cell Metab.* **2017**, *26*, 547–557.e8. [CrossRef]

130. de Cabo, R.; Mattson, M.P. Effects of Intermittent Fasting on Health, Aging, and Disease. *N. Engl. J. Med.* **2019**, *381*, 2541–2551. [CrossRef]

131. Sleiman, S.F.; Henry, J.; Al-Haddad, R.; El Hayek, L.; Abou Haidar, E.; Stringer, T.; Ulja, D.; Karuppagounder, S.S.; Holson, E.B.; Ratan, R.R.; et al. Exercise promotes the expression of brain derived neurotrophic factor (BDNF) through the action of the ketone body beta-hydroxybutyrate. *Elife* **2016**, *5*, e15092. [CrossRef]

132. Han, Y.M.; Bedarida, T.; Ding, Y.; Somba, B.K.; Lu, Q.; Wang, Q.; Song, P.; Zou, M.H. beta-Hydroxybutyrate Prevents Vascular Senescence through hnRNP A1-Mediated Upregulation of Oct4. *Mol. Cell* **2018**, *71*, 1064–1078.e5. [CrossRef] [PubMed]

133. Youm, Y.H.; Nguyen, K.Y.; Grant, R.W.; Goldberg, E.L.; Bodogai, M.; Kim, D.; D'Agostino, D.; Planavsky, N.; Lupfer, C.; Kanneganti, T.D.; et al. The ketone metabolite beta-hydroxybutyrate blocks NLRP3 inflammasome-mediated inflammatory disease. *Nat. Med.* **2015**, *21*, 263–269. [CrossRef] [PubMed]
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135. Lapasset, L.; Milhavet, O.; Prieur, A.; Besnard, E.; Babled, A.; Ait-Hamou, N.; Leschik, J.; Pellestor, F.; Ramirez, J.M.; De Vos, J.; et al. Rejuvenating senescent and centenarian human cells by reprogramming through the pluripotent state. *Genes. Dev.* **2011**, *25*, 2248–2253. [CrossRef] [PubMed]

136. Jaskelioff, M.; Muller, F.L.; Paik, J.H.; Thomas, E.; Jiang, S.; Adams, A.C.; Sahin, E.; Kost-Alimova, M.; Protopopov, A.; Cadinanos, J.; et al. Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice. *Nature* **2011**, *469*, 102–106. [CrossRef]

137. Bernardes de Jesus, B.; Vera, E.; Schneeberger, K.; Tejera, A.M.; Ayuso, E.; Bosch, F.; Blasco, M.A. Telomerase gene therapy in adult and old mice delays aging and increases longevity without increasing cancer. *EMBO Mol. Med.* **2012**, *4*, 691–704. [CrossRef]

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139. Sarkar, T.J.; Quarta, M.; Mukherjee, S.; Colville, A.; Paine, P.; Doan, L.; Tran, C.M.; Chu, C.R.; Horvath, S.; Qi, L.S.; et al. Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells. *Nat. Commun.* **2020**, *11*, 1545. [CrossRef]

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142. Milhavet, O.; Lemaitre, J.M. Single short reprogramming early in life increases healthspan. *Aging (Albany NY)* **2022**, *14*, 9779–9781. [CrossRef] [PubMed]

143. Coppe, J.P.; Rodier, F.; Patil, C.K.; Freund, A.; Desprez, P.Y.; Campisi, J. Tumor suppressor and aging biomarker p16(INK4a) induces cellular senescence without the associated inflammatory secretory phenotype. *J. Biol. Chem.* **2011**, *286*, 36396–36403. [CrossRef] [PubMed]

144. Acosta, J.C.; O'Loghlen, A.; Banito, A.; Guijarro, M.V.; Augert, A.; Raguz, S.; Fumagalli, M.; Da Costa, M.; Brown, C.; Popov, N.; et al. Chemokine signaling via the CXCR2 receptor reinforces senescence. *Cell* **2008**, *133*, 1006–1018. [CrossRef]

145. Coppe, J.P.; Patil, C.K.; Rodier, F.; Krtolica, A.; Beausejour, C.M.; Parrinello, S.; Hodgson, J.G.; Chin, K.; Desprez, P.Y.; Campisi, J. A human-like senescence-associated secretory phenotype is conserved in mouse cells dependent on physiological oxygen. *PLoS ONE* **2010**, *5*, e9188. [CrossRef] [PubMed]

146. Kuilman, T.; Michaloglou, C.; Vredeveld, L.C.; Douma, S.; van Doorn, R.; Desmet, C.J.; Aarden, L.A.; Mooi, W.J.; Peeper, D.S. Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network. *Cell* **2008**, *133*, 1019–1031. [CrossRef] [PubMed]

147. Hong, D.S.; Angelo, L.S.; Kurzrock, R. Interleukin-6 and its receptor in cancer: Implications for translational therapeutics. *Cancer* **2007**, *110*, 1911–1928. [CrossRef]

148. Anestakis, D.; Petanidis, S.; Kalyvas, S.; Nday, C.M.; Tsave, O.; Kioseoglou, E.; Salifoglou, A. Mechanisms and applications of interleukins in cancer immunotherapy. *Int. J. Mol. Sci.* **2015**, *16*, 1691–1710. [CrossRef]

149. Perez-Mancera, P.A.; Young, A.R.; Narita, M. Inside and out: The activities of senescence in cancer. *Nat. Rev. Cancer* **2014**, *14*, 547–558. [CrossRef]

150. Gonzalez-Meljem, J.M.; Apps, J.R.; Fraser, H.C.; Martinez-Barbera, J.P. Paracrine roles of cellular senescence in promoting tumourigenesis. *Br. J. Cancer* **2018**, *118*, 1283–1288. [CrossRef]

151. Lee, S.; Schmitt, C.A. The dynamic nature of senescence in cancer. *Nat. Cell Biol.* **2019**, *21*, 94–101. [CrossRef]

152. Faget, D.V.; Ren, Q.; Stewart, S.A. Unmasking senescence: Context-dependent effects of SASP in cancer. *Nat. Rev. Cancer* **2019**, *19*, 439–453. [CrossRef] [PubMed]

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155. Freund, A.; Orjalo, A.V.; Desprez, P.Y.; Campisi, J. Inflammatory networks during cellular senescence: Causes and consequences. *Trends Mol. Med.* **2010**, *16*, 238–246. [CrossRef]

156. Moiseeva, O.; Deschenes-Simard, X.; St-Germain, E.; Igelmann, S.; Huot, G.; Cadar, A.E.; Bourdeau, V.; Pollak, M.N.; Ferbeyre, G. Metformin inhibits the senescence-associated secretory phenotype by interfering with IKK/NF-kappaB activation. *Aging Cell* **2013**, *12*, 489–498. [CrossRef]

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135. Lapasset, L.; Milhavet, O.; Prieur, A.; Besnard, E.; Babled, A.; Ait-Hamou, N.; Leschik, J.; Pellestor, F.; Ramirez, J.M.; De Vos, J.; et al. Rejuvenating senescent and centenarian human cells by reprogramming through the pluripotent state. *Genes. Dev.* **2011**, *25*, 2248–2253. [CrossRef] [PubMed]

136. Jaskelioff, M.; Muller, F.L.; Paik, J.H.; Thomas, E.; Jiang, S.; Adams, A.C.; Sahin, E.; Kost-Alimova, M.; Protopopov, A.; Cadinanos, J.; et al. Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice. *Nature* **2011**, *469*, 102–106. [CrossRef]

137. Bernardes de Jesus, B.; Vera, E.; Schneeberger, K.; Tejera, A.M.; Ayuso, E.; Bosch, F.; Blasco, M.A. Telomerase gene therapy in adult and old mice delays aging and increases longevity without increasing cancer. *EMBO Mol. Med.* **2012**, *4*, 691–704. [CrossRef]

138. Jaijyan, D.K.; Selariu, A.; Cruz-Cosme, R.; Tong, M.; Yang, S.; Stefa, A.; Kekich, D.; Sadoshima, J.; Herbig, U.; Tang, Q.; et al. New intranasal and injectable gene therapy for healthy life extension. *Proc. Natl. Acad. Sci. USA* **2022**, *119*, e2121499119. [CrossRef]

139. Sarkar, T.J.; Quarta, M.; Mukherjee, S.; Colville, A.; Paine, P.; Doan, L.; Tran, C.M.; Chu, C.R.; Horvath, S.; Qi, L.S.; et al. Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells. *Nat. Commun.* **2020**, *11*, 1545. [CrossRef]

140. Ocampo, A.; Reddy, P.; Martinez-Redondo, P.; Platero-Luengo, A.; Hatanaka, F.; Hishida, T.; Li, M.; Lam, D.; Kurita, M.; Beyret, E.; et al. In Vivo Amelioration of Age-Associated Hallmarks by Partial Reprogramming. *Cell* **2016**, *167*, 1719–1733.e12. [CrossRef]

141. Alle, Q.; Le Borgne, E.; Bensadoun, P.; Lemey, C.; Béchir, N.; Gabanou, M.; Estermann, F.; Bertrand-Gaday, C.; Pessemesse, L.; Toupet, K.; et al. A single short reprogramming early in life initiates and propagates an epigenetically related mechanism improving fitness and promoting an increased healthy lifespan. *Aging Cell* **2022**, *21*, e13714. [CrossRef]

142. Milhavet, O.; Lemaitre, J.M. Single short reprogramming early in life increases healthspan. *Aging (Albany NY)* **2022**, *14*, 9779–9781. [CrossRef] [PubMed]

143. Coppe, J.P.; Rodier, F.; Patil, C.K.; Freund, A.; Desprez, P.Y.; Campisi, J. Tumor suppressor and aging biomarker p16(INK4a) induces cellular senescence without the associated inflammatory secretory phenotype. *J. Biol. Chem.* **2011**, *286*, 36396–36403. [CrossRef] [PubMed]

144. Acosta, J.C.; O'Loghlen, A.; Banito, A.; Guijarro, M.V.; Augert, A.; Raguz, S.; Fumagalli, M.; Da Costa, M.; Brown, C.; Popov, N.; et al. Chemokine signaling via the CXCR2 receptor reinforces senescence. *Cell* **2008**, *133*, 1006–1018. [CrossRef]

145. Coppe, J.P.; Patil, C.K.; Rodier, F.; Krtolica, A.; Beausejour, C.M.; Parrinello, S.; Hodgson, J.G.; Chin, K.; Desprez, P.Y.; Campisi, J. A human-like senescence-associated secretory phenotype is conserved in mouse cells dependent on physiological oxygen. *PLoS ONE* **2010**, *5*, e9188. [CrossRef] [PubMed]

146. Kuilman, T.; Michaloglou, C.; Vredeveld, L.C.; Douma, S.; van Doorn, R.; Desmet, C.J.; Aarden, L.A.; Mooi, W.J.; Peeper, D.S. Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network. *Cell* **2008**, *133*, 1019–1031. [CrossRef] [PubMed]

147. Hong, D.S.; Angelo, L.S.; Kurzrock, R. Interleukin-6 and its receptor in cancer: Implications for translational therapeutics. *Cancer* **2007**, *110*, 1911–1928. [CrossRef]

148. Anestakis, D.; Petanidis, S.; Kalyvas, S.; Nday, C.M.; Tsave, O.; Kioseoglou, E.; Salifoglou, A. Mechanisms and applications of interleukins in cancer immunotherapy. *Int. J. Mol. Sci.* **2015**, *16*, 1691–1710. [CrossRef]

149. Perez-Mancera, P.A.; Young, A.R.; Narita, M. Inside and out: The activities of senescence in cancer. *Nat. Rev. Cancer* **2014**, *14*, 547–558. [CrossRef]

150. Gonzalez-Meljem, J.M.; Apps, J.R.; Fraser, H.C.; Martinez-Barbera, J.P. Paracrine roles of cellular senescence in promoting tumourigenesis. *Br. J. Cancer* **2018**, *118*, 1283–1288. [CrossRef]

151. Lee, S.; Schmitt, C.A. The dynamic nature of senescence in cancer. *Nat. Cell Biol.* **2019**, *21*, 94–101. [CrossRef]

152. Faget, D.V.; Ren, Q.; Stewart, S.A. Unmasking senescence: Context-dependent effects of SASP in cancer. *Nat. Rev. Cancer* **2019**, *19*, 439–453. [CrossRef] [PubMed]

153. Salminen, A.; Kauppinen, A.; Kaarniranta, K. Emerging role of NF-kappaB signaling in the induction of senescence-associated secretory phenotype (SASP). *Cell Signal* **2012**, *24*, 835–845. [CrossRef] [PubMed]

154. Chien, Y.; Scuoppo, C.; Wang, X.; Fang, X.; Balgley, B.; Bolden, J.E.; Premiered, P.; Luo, W.; Chicas, A.; Lee, C.S.; et al. Control of the senescence-associated secretory phenotype by NF-kappaB promotes senescence and enhances chemosensitivity. *Genes. Dev.* **2011**, *25*, 2125–2136. [CrossRef] [PubMed]

155. Freund, A.; Orjalo, A.V.; Desprez, P.Y.; Campisi, J. Inflammatory networks during cellular senescence: Causes and consequences. *Trends Mol. Med.* **2010**, *16*, 238–246. [CrossRef]

156. Moiseeva, O.; Deschenes-Simard, X.; St-Germain, E.; Igelmann, S.; Huot, G.; Cadar, A.E.; Bourdeau, V.; Pollak, M.N.; Ferbeyre, G. Metformin inhibits the senescence-associated secretory phenotype by interfering with IKK/NF-kappaB activation. *Aging Cell* **2013**, *12*, 489–498. [CrossRef]

157. Martin-Montalvo, A.; Mercken, E.M.; Mitchell, S.J.; Palacios, H.H.; Mote, P.L.; Scheibye-Knudsen, M.; Gomes, A.P.; Ward, T.M.; Minor, R.K.; Blouin, M.J.; et al. Metformin improves healthspan and lifespan in mice. *Nat. Commun.* **2013**, *4*, 2192. [CrossRef]
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160. Lamming, D.W.; Ye, L.; Sabatini, D.M.; Baur, J.A. Rapalogs and mTOR inhibitors as anti-aging therapeutics. *J. Clin. Investig.* **2013**, *123*, 980–989. [CrossRef]

161. Leontieva, O.V.; Demidenko, Z.N.; Blagosklonny, M.V. Dual mTORC1/C2 inhibitors suppress cellular geroconversion (a senescence program). *Oncotarget* **2015**, *6*, 23238–23248. [CrossRef]

162. Herranz, N.; Gallage, S.; Mellone, M.; Wuestefeld, T.; Klotz, S.; Hanley, C.J.; Raguz, S.; Acosta, J.C.; Innes, A.J.; Banito, A.; et al. mTOR regulates MAPKAPK2 translation to control the senescence-associated secretory phenotype. *Nat. Cell Biol.* **2015**, *17*, 1205–1217. [CrossRef]

163. Imrali, A.; Mao, X.; Yeste-Velasco, M.; Shamash, J.; Lu, Y. Rapamycin inhibits prostate cancer cell growth through cyclin D1 and enhances the cytotoxic efficacy of cisplatin. *Am. J. Cancer Res.* **2016**, *6*, 1772–1784.

164. Wang, R.; Yu, Z.; Sunchu, B.; Shoaf, J.; Dang, I.; Zhao, S.; Caples, K.; Bradley, L.; Beaver, L.M.; Ho, E.; et al. Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2-independent mechanism. *Aging Cell* **2017**, *16*, 564–574. [CrossRef]

165. Freund, A.; Patil, C.K.; Campisi, J. p38MAPK is a novel DNA damage response-independent regulator of the senescence-associated secretory phenotype. *Embo J* **2011**, *30*, 1536–1548. [CrossRef]

166. Alimbetov, D.; Davis, T.; Brook, A.J.; Cox, L.S.; Faragher, R.G.; Nurgozhin, T.; Zhumadilov, Z.; Kipling, D. Suppression of the senescence-associated secretory phenotype (SASP) in human fibroblasts using small molecule inhibitors of p38 MAP kinase and MK2. *Biogerontology* **2016**, *17*, 305–315. [CrossRef]

167. Niehof, M.; Streetz, K.; Rakemann, T.; Bischoff, S.C.; Manns, M.P.; Horn, F.; Trautwein, C. Interleukin-6-induced tethering of STAT3 to the LAP/C/EBPbeta promoter suggests a new mechanism of transcriptional regulation by STAT3. *J. Biol. Chem.* **2001**, *276*, 9016–9027. [CrossRef]

168. Borghesan, M.; Fafian-Labora, J.; Eleftheriadou, O.; Carpintero-Fernandez, P.; Paez-Ribes, M.; Vizcay-Barrena, G.; Swisa, A.; Kolodkin-Gal, D.; Ximenez-Embun, P.; Lowe, R.; et al. Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3. *Cell Rep.* **2019**, *27*, 3956–3971.e6. [CrossRef]

169. Xu, D.; Tahara, H. The role of exosomes and microRNAs in senescence and aging. *Adv. Drug Deliv. Rev.* **2013**, *65*, 368–375. [CrossRef]

170. Urbanelli, L.; Buratta, S.; Sagini, K.; Tancini, B.; Emiliani, C. Extracellular Vesicles as New Players in Cellular Senescence. *Int. J. Mol. Sci.* **2016**, *17*, 1408. [CrossRef]

171. Takasugi, M.; Okada, R.; Takahashi, A.; Virya Chen, D.; Watanabe, S.; Hara, E. Small extracellular vesicles secreted from senescent cells promote cancer cell proliferation through EphA2. *Nat. Commun.* **2017**, *8*, 15729. [CrossRef]

172. Li, T.; Chen, Z.J. The cGAS-cGAMP-STING pathway connects DNA damage to inflammation, senescence, and cancer. *J. Exp. Med.* **2018**, *215*, 1287–1299. [CrossRef]

173. Zhang, Q.; Lenardo, M.J.; Baltimore, D. 30 Years of NF-kappaB: A Blossoming of Relevance to Human Pathobiology. *Cell* **2017**, *168*, 37–57. [CrossRef]

174. Karkera, J.; Steiner, H.; Li, W.; Skradski, V.; Moser, P.L.; Riethdorf, S.; Reddy, M.; Puchalski, T.; Safer, K.; Prabhakar, U.; et al. The anti-interleukin-6 antibody siltuximab down-regulates genes implicated in tumorigenesis in prostate cancer patients from a phase I study. *Prostate* **2011**, *71*, 1455–1465. [CrossRef]

175. van Rhee, F.; Wong, R.S.; Munshi, N.; Rossi, J.F.; Ke, X.Y.; Fossa, A.; Simpson, D.; Capra, M.; Liu, T.; Hsieh, R.K.; et al. Siltuximab for multicentric Castleman's disease: A randomised, double-blind, placebo-controlled trial. *Lancet Oncol.* **2014**, *15*, 966–974. [CrossRef]

176. Emery, P.; Keystone, E.; Tony, H.P.; Cantagrel, A.; van Vollenhoven, R.; Sanchez, A.; Alecock, E.; Lee, J.; Kremer, J. IL-6 receptor inhibition with tocilizumab improves treatment outcomes in patients with rheumatoid arthritis refractory to anti-tumour necrosis factor biologicals: Results from a 24-week multicentre randomised placebo-controlled trial. *Ann. Rheum. Dis.* **2008**, *67*, 1516–1523. [CrossRef]

177. Khan, S.S.; Shah, S.J.; Klyachko, E.; Baldridge, A.S.; Eren, M.; Place, A.T.; Aviv, A.; Puterman, E.; Lloyd-Jones, D.M.; Heiman, M.; et al. A null mutation in SERPINE1 protects against biological aging in humans. *Sci. Adv.* **2017**, *3*, eaao1617. [CrossRef]

178. Eren, M.; Boe, A.E.; Murphy, S.B.; Place, A.T.; Nagpal, V.; Morales-Nebreda, L.; Urich, D.; Quaggin, S.E.; Budinger, G.R.; Mutlu, G.M.; et al. PAI-1-regulated extracellular proteolysis governs senescence and survival in Klotho mice. *Proc. Natl. Acad. Sci. USA* **2014**, *111*, 7090–7095. [CrossRef]

179. Collado, M.; Serrano, M. Senescence in tumours: Evidence from mice and humans. *Nat. Rev. Cancer* **2010**, *10*, 51–57. [CrossRef]

180. Sharpless, N.E.; Sherr, C.J. Forging a signature of in vivo senescence. *Nat. Rev. Cancer* **2015**, *15*, 397–408. [CrossRef]

181. Agostini, A.; Mondragon, L.; Bernardos, A.; Martinez-Manez, R.; Marcos, M.D.; Sancenon, F.; Soto, J.; Costero, A.; Manguan-Garcia, C.; Perona, R.; et al. Targeted cargo delivery in senescent cells using capped mesoporous silica nanoparticles. *Angew. Chem. Int. Ed. Engl.* **2012**, *51*, 10556–10560. [CrossRef]
158. Campbell, J.M.; Bellman, S.M.; Stephenson, M.D.; Lisy, K. Metformin reduces all-cause mortality and diseases of ageing independent of its effect on diabetes control: A systematic review and meta-analysis. *Ageing Res. Rev.* **2017**, *40*, 31–44. [CrossRef]

159. Barzilai, N.; Crandall, J.P.; Kritchevsky, S.B.; Espeland, M.A. Metformin as a Tool to Target Aging. *Cell Metab.* **2016**, *23*, 1060–1065. [CrossRef]

160. Lamming, D.W.; Ye, L.; Sabatini, D.M.; Baur, J.A. Rapalogs and mTOR inhibitors as anti-aging therapeutics. *J. Clin. Investig.* **2013**, *123*, 980–989. [CrossRef]

161. Leontieva, O.V.; Demidenko, Z.N.; Blagosklonny, M.V. Dual mTORC1/C2 inhibitors suppress cellular geroconversion (a senescence program). *Oncotarget* **2015**, *6*, 23238–23248. [CrossRef]

162. Herranz, N.; Gallage, S.; Mellone, M.; Wuestefeld, T.; Klotz, S.; Hanley, C.J.; Raguz, S.; Acosta, J.C.; Innes, A.J.; Banito, A.; et al. mTOR regulates MAPKAPK2 translation to control the senescence-associated secretory phenotype. *Nat. Cell Biol.* **2015**, *17*, 1205–1217. [CrossRef]

163. Imrali, A.; Mao, X.; Yeste-Velasco, M.; Shamash, J.; Lu, Y. Rapamycin inhibits prostate cancer cell growth through cyclin D1 and enhances the cytotoxic efficacy of cisplatin. *Am. J. Cancer Res.* **2016**, *6*, 1772–1784.

164. Wang, R.; Yu, Z.; Sunchu, B.; Shoaf, J.; Dang, I.; Zhao, S.; Caples, K.; Bradley, L.; Beaver, L.M.; Ho, E.; et al. Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2-independent mechanism. *Aging Cell* **2017**, *16*, 564–574. [CrossRef]

165. Freund, A.; Patil, C.K.; Campisi, J. p38MAPK is a novel DNA damage response-independent regulator of the senescence-associated secretory phenotype. *Embo J* **2011**, *30*, 1536–1548. [CrossRef]

166. Alimbetov, D.; Davis, T.; Brook, A.J.; Cox, L.S.; Faragher, R.G.; Nurgozhin, T.; Zhumadilov, Z.; Kipling, D. Suppression of the senescence-associated secretory phenotype (SASP) in human fibroblasts using small molecule inhibitors of p38 MAP kinase and MK2. *Biogerontology* **2016**, *17*, 305–315. [CrossRef]

167. Niehof, M.; Streetz, K.; Rakemann, T.; Bischoff, S.C.; Manns, M.P.; Horn, F.; Trautwein, C. Interleukin-6-induced tethering of STAT3 to the LAP/C/EBPbeta promoter suggests a new mechanism of transcriptional regulation by STAT3. *J. Biol. Chem.* **2001**, *276*, 9016–9027. [CrossRef]

168. Borghesan, M.; Fafian-Labora, J.; Eleftheriadou, O.; Carpintero-Fernandez, P.; Paez-Ribes, M.; Vizcay-Barrena, G.; Swisa, A.; Kolodkin-Gal, D.; Ximenez-Embun, P.; Lowe, R.; et al. Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3. *Cell Rep.* **2019**, *27*, 3956–3971.e6. [CrossRef]

169. Xu, D.; Tahara, H. The role of exosomes and microRNAs in senescence and aging. *Adv. Drug Deliv. Rev.* **2013**, *65*, 368–375. [CrossRef]

170. Urbanelli, L.; Buratta, S.; Sagini, K.; Tancini, B.; Emiliani, C. Extracellular Vesicles as New Players in Cellular Senescence. *Int. J. Mol. Sci.* **2016**, *17*, 1408. [CrossRef]

171. Takasugi, M.; Okada, R.; Takahashi, A.; Virya Chen, D.; Watanabe, S.; Hara, E. Small extracellular vesicles secreted from senescent cells promote cancer cell proliferation through EphA2. *Nat. Commun.* **2017**, *8*, 15729. [CrossRef]

172. Li, T.; Chen, Z.J. The cGAS-cGAMP-STING pathway connects DNA damage to inflammation, senescence, and cancer. *J. Exp. Med.* **2018**, *215*, 1287–1299. [CrossRef]

173. Zhang, Q.; Lenardo, M.J.; Baltimore, D. 30 Years of NF-kappaB: A Blossoming of Relevance to Human Pathobiology. *Cell* **2017**, *168*, 37–57. [CrossRef]

174. Karkera, J.; Steiner, H.; Li, W.; Skradski, V.; Moser, P.L.; Riethdorf, S.; Reddy, M.; Puchalski, T.; Safer, K.; Prabhakar, U.; et al. The anti-interleukin-6 antibody siltuximab down-regulates genes implicated in tumorigenesis in prostate cancer patients from a phase I study. *Prostate* **2011**, *71*, 1455–1465. [CrossRef]

175. van Rhee, F.; Wong, R.S.; Munshi, N.; Rossi, J.F.; Ke, X.Y.; Fossa, A.; Simpson, D.; Capra, M.; Liu, T.; Hsieh, R.K.; et al. Siltuximab for multicentric Castleman's disease: A randomised, double-blind, placebo-controlled trial. *Lancet Oncol.* **2014**, *15*, 966–974. [CrossRef]

176. Emery, P.; Keystone, E.; Tony, H.P.; Cantagrel, A.; van Vollenhoven, R.; Sanchez, A.; Alecock, E.; Lee, J.; Kremer, J. IL-6 receptor inhibition with tocilizumab improves treatment outcomes in patients with rheumatoid arthritis refractory to anti-tumour necrosis factor biologicals: Results from a 24-week multicentre randomised placebo-controlled trial. *Ann. Rheum. Dis.* **2008**, *67*, 1516–1523. [CrossRef]

177. Khan, S.S.; Shah, S.J.; Klyachko, E.; Baldridge, A.S.; Eren, M.; Place, A.T.; Aviv, A.; Puterman, E.; Lloyd-Jones, D.M.; Heiman, M.; et al. A null mutation in SERPINE1 protects against biological aging in humans. *Sci. Adv.* **2017**, *3*, eaao1617. [CrossRef]

178. Eren, M.; Boe, A.E.; Murphy, S.B.; Place, A.T.; Nagpal, V.; Morales-Nebreda, L.; Urich, D.; Quaggin, S.E.; Budinger, G.R.; Mutlu, G.M.; et al. PAI-1-regulated extracellular proteolysis governs senescence and survival in Klotho mice. *Proc. Natl. Acad. Sci. USA* **2014**, *111*, 7090–7095. [CrossRef]

179. Collado, M.; Serrano, M. Senescence in tumours: Evidence from mice and humans. *Nat. Rev. Cancer* **2010**, *10*, 51–57. [CrossRef]

180. Sharpless, N.E.; Sherr, C.J. Forging a signature of in vivo senescence. *Nat. Rev. Cancer* **2015**, *15*, 397–408. [CrossRef]

181. Agostini, A.; Mondragon, L.; Bernardos, A.; Martinez-Manez, R.; Marcos, M.D.; Sancenon, F.; Soto, J.; Costero, A.; Manguan-Garcia, C.; Perona, R.; et al. Targeted cargo delivery in senescent cells using capped mesoporous silica nanoparticles. *Angew. Chem. Int. Ed. Engl.* **2012**, *51*, 10556–10560. [CrossRef]
182. Hall, B.M.; Balan, V.; Gleiberman, A.S.; Strom, E.; Krasnov, P.; Virtuoso, L.P.; Rydkina, E.; Vujcic, S.; Balan, K.; Gitlin, I.; et al. Aging of mice is associated with p16(Ink4a)- and beta-galactosidase-positive macrophage accumulation that can be induced in young mice by senescent cells. *Aging (Albany NY)* **2016**, *8*, 1294–1315. [CrossRef]

183. Singh, M.; Piekorz, R.P. Senescence-associated lysosomal alpha-L-fucosidase (SA-alpha-Fuc): A sensitive and more robust biomarker for cellular senescence beyond SA-beta-Gal. *Cell Cycle* **2013**, *12*, 1996. [CrossRef]

184. Thapa, R.K.; Nguyen, H.T.; Jeong, J.H.; Kim, J.R.; Choi, H.G.; Yong, C.S.; Kim, J.O. Progressive slowdown/prevention of cellular senescence by CD9-targeted delivery of rapamycin using lactose-wrapped calcium carbonate nanoparticles. *Sci. Rep.* **2017**, *7*, 43299. [CrossRef]

185. Han, B.; Park, D.; Li, R.; Xie, M.; Owonikoko, T.K.; Zhang, G.; Sica, G.L.; Ding, C.; Zhou, J.; Magis, A.T.; et al. Small-Molecule Bcl2 BH4 Antagonist for Lung Cancer Therapy. *Cancer Cell* **2015**, *27*, 852–863. [CrossRef]

186. Fuhrmann-Stroissnigg, H.; Ling, Y.Y.; Zhao, J.; McGowan, S.J.; Zhu, Y.; Brooks, R.W.; Grassi, D.; Gregg, S.Q.; Stripay, J.L.; Dorronsoro, A.; et al. Identification of HSP90 inhibitors as a novel class of senolytics. *Nat. Commun.* **2017**, *8*, 422. [CrossRef]

187. Karkoulis, P.K.; Stravopodis, D.J.; Konstantakou, E.G.; Voutsinas, G.E. Targeted inhibition of heat shock protein 90 disrupts multiple oncogenic signaling pathways, thus inducing cell cycle arrest and programmed cell death in human urinary bladder cancer cell lines. *Cancer Cell Int.* **2013**, *13*, 11. [CrossRef]

188. Sato, S.; Fujita, N.; Tsuruo, T. Modulation of Akt kinase activity by binding to Hsp90. *Proc. Natl. Acad. Sci. USA* **2000**, *97*, 10832–10837. [CrossRef]

189. Astle, M.V.; Hannan, K.M.; Ng, P.Y.; Lee, R.S.; George, A.J.; Hsu, A.K.; Haupt, Y.; Hannan, R.D.; Pearson, R.B. AKT induces senescence in human cells via mTORC1 and p53 in the absence of DNA damage: Implications for targeting mTOR during malignancy. *Oncogene* **2012**, *31*, 1949–1962. [CrossRef]

190. Deschenes-Simard, X.; Gaumont-Leclerc, M.F.; Bourdeau, V.; Lessard, F.; Moiseeva, O.; Forest, V.; Igelmann, S.; Mallette, F.A.; Saba-El-Leil, M.K.; Meloche, S.; et al. Tumor suppressor activity of the ERK/MAPK pathway by promoting selective protein degradation. *Genes. Dev.* **2013**, *27*, 900–915. [CrossRef]

191. Trepel, J.; Mollapour, M.; Giaccone, G.; Neckers, L. Targeting the dynamic HSP90 complex in cancer. *Nat. Rev. Cancer* **2010**, *10*, 537–549. [CrossRef]

192. Dorr, J.R.; Yu, Y.; Milanovic, M.; Beuster, G.; Zasada, C.; Dabritz, J.H.; Lisec, J.; Lenze, D.; Gerhardt, A.; Schleicher, K.; et al. Synthetic lethal metabolic targeting of cellular senescence in cancer therapy. *Nature* **2013**, *501*, 421–425. [CrossRef] [PubMed]

193. Soto-Gamez, A.; Quax, W.J.; Demaria, M. Regulation of Survival Networks in Senescent Cells: From Mechanisms to Interventions. *J. Mol. Biol.* **2019**, *431*, 2629–2643. [CrossRef] [PubMed]

194. Kastenhuber, E.R.; Lowe, S.W. Putting p53 in Context. *Cell* **2017**, *170*, 1062–1078. [CrossRef] [PubMed]

195. Shay, J.W.; Pereira-Smith, O.M.; Wright, W.E. A role for both RB and p53 in the regulation of human cellular senescence. *Exp. Cell Res.* **1991**, *196*, 33–39. [CrossRef] [PubMed]

196. Di Micco, R.; Fumagalli, M.; Cicalese, A.; Piccinin, S.; Gasparini, P.; Luise, C.; Schurra, C.; Garre, M.; Nuciforo, P.G.; Bensimon, A.; et al. Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication. *Nature* **2006**, *444*, 638–642. [CrossRef] [PubMed]

197. Yosef, R.; Pilpel, N.; Papismadov, N.; Gal, H.; Ovadya, Y.; Vadai, E.; Miller, S.; Porat, Z.; Ben-Dor, S.; Krizhanovsky, V. p21 maintains senescent cell viability under persistent DNA damage response by restraining JNK and caspase signaling. *EMBO J.* **2017**, *36*, 2280–2295. [CrossRef]

198. Baar, M.P.; Brandt, R.M.C.; Putavet, D.A.; Klein, J.D.D.; Derks, K.W.J.; Bourgeois, B.R.M.; Stryeck, S.; Rijksen, Y.; van Willigenburg, H.; Feijtel, D.A.; et al. Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging. *Cell* **2017**, *169*, 132–147.e16. [CrossRef]

199. Wang, F.; Marshall, C.B.; Yamamoto, K.; Li, G.Y.; Plevin, M.J.; You, H.; Mak, T.W.; Ikura, M. Biochemical and structural characterization of an intramolecular interaction in FOXO3a and its binding with p53. *J. Mol. Biol.* **2008**, *384*, 590–603. [CrossRef]

200. Singh, R.; Letai, A.; Sarosiek, K. Regulation of apoptosis in health and disease: The balancing act of BCL-2 family proteins. *Nat. Rev. Mol. Cell Biol.* **2019**, *20*, 175–193. [CrossRef]

201. Zhu, Y.; Tchkonia, T.; Fuhrmann-Stroissnigg, H.; Dai, H.M.; Ling, Y.Y.; Stout, M.B.; Pirtskhalava, T.; Giorgadze, N.; Johnson, K.O.; Giles, C.B.; et al. Identification of a novel senolytic agent, navitoclax, targeting the Bcl-2 family of anti-apoptotic factors. *Aging Cell* **2016**, *15*, 428–435. [CrossRef]

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183. Singh, M.; Piekorz, R.P. Senescence-associated lysosomal alpha-L-fucosidase (SA-alpha-Fuc): A sensitive and more robust biomarker for cellular senescence beyond SA-beta-Gal. *Cell Cycle* **2013**, *12*, 1996. [CrossRef]

184. Thapa, R.K.; Nguyen, H.T.; Jeong, J.H.; Kim, J.R.; Choi, H.G.; Yong, C.S.; Kim, J.O. Progressive slowdown/prevention of cellular senescence by CD9-targeted delivery of rapamycin using lactose-wrapped calcium carbonate nanoparticles. *Sci. Rep.* **2017**, *7*, 43299. [CrossRef]

185. Han, B.; Park, D.; Li, R.; Xie, M.; Owonikoko, T.K.; Zhang, G.; Sica, G.L.; Ding, C.; Zhou, J.; Magis, A.T.; et al. Small-Molecule Bcl2 BH4 Antagonist for Lung Cancer Therapy. *Cancer Cell* **2015**, *27*, 852–863. [CrossRef]

186. Fuhrmann-Stroissnigg, H.; Ling, Y.Y.; Zhao, J.; McGowan, S.J.; Zhu, Y.; Brooks, R.W.; Grassi, D.; Gregg, S.Q.; Stripay, J.L.; Dorronsoro, A.; et al. Identification of HSP90 inhibitors as a novel class of senolytics. *Nat. Commun.* **2017**, *8*, 422. [CrossRef]

187. Karkoulis, P.K.; Stravopodis, D.J.; Konstantakou, E.G.; Voutsinas, G.E. Targeted inhibition of heat shock protein 90 disrupts multiple oncogenic signaling pathways, thus inducing cell cycle arrest and programmed cell death in human urinary bladder cancer cell lines. *Cancer Cell Int.* **2013**, *13*, 11. [CrossRef]

188. Sato, S.; Fujita, N.; Tsuruo, T. Modulation of Akt kinase activity by binding to Hsp90. *Proc. Natl. Acad. Sci. USA* **2000**, *97*, 10832–10837. [CrossRef]

189. Astle, M.V.; Hannan, K.M.; Ng, P.Y.; Lee, R.S.; George, A.J.; Hsu, A.K.; Haupt, Y.; Hannan, R.D.; Pearson, R.B. AKT induces senescence in human cells via mTORC1 and p53 in the absence of DNA damage: Implications for targeting mTOR during malignancy. *Oncogene* **2012**, *31*, 1949–1962. [CrossRef]

190. Deschenes-Simard, X.; Gaumont-Leclerc, M.F.; Bourdeau, V.; Lessard, F.; Moiseeva, O.; Forest, V.; Igelmann, S.; Mallette, F.A.; Saba-El-Leil, M.K.; Meloche, S.; et al. Tumor suppressor activity of the ERK/MAPK pathway by promoting selective protein degradation. *Genes. Dev.* **2013**, *27*, 900–915. [CrossRef]

191. Trepel, J.; Mollapour, M.; Giaccone, G.; Neckers, L. Targeting the dynamic HSP90 complex in cancer. *Nat. Rev. Cancer* **2010**, *10*, 537–549. [CrossRef]

192. Dorr, J.R.; Yu, Y.; Milanovic, M.; Beuster, G.; Zasada, C.; Dabritz, J.H.; Lisec, J.; Lenze, D.; Gerhardt, A.; Schleicher, K.; et al. Synthetic lethal metabolic targeting of cellular senescence in cancer therapy. *Nature* **2013**, *501*, 421–425. [CrossRef] [PubMed]

193. Soto-Gamez, A.; Quax, W.J.; Demaria, M. Regulation of Survival Networks in Senescent Cells: From Mechanisms to Interventions. *J. Mol. Biol.* **2019**, *431*, 2629–2643. [CrossRef] [PubMed]

194. Kastenhuber, E.R.; Lowe, S.W. Putting p53 in Context. *Cell* **2017**, *170*, 1062–1078. [CrossRef] [PubMed]

195. Shay, J.W.; Pereira-Smith, O.M.; Wright, W.E. A role for both RB and p53 in the regulation of human cellular senescence. *Exp. Cell Res.* **1991**, *196*, 33–39. [CrossRef] [PubMed]

196. Di Micco, R.; Fumagalli, M.; Cicalese, A.; Piccinin, S.; Gasparini, P.; Luise, C.; Schurra, C.; Garre, M.; Nuciforo, P.G.; Bensimon, A.; et al. Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication. *Nature* **2006**, *444*, 638–642. [CrossRef] [PubMed]

197. Yosef, R.; Pilpel, N.; Papismadov, N.; Gal, H.; Ovadya, Y.; Vadai, E.; Miller, S.; Porat, Z.; Ben-Dor, S.; Krizhanovsky, V. p21 maintains senescent cell viability under persistent DNA damage response by restraining JNK and caspase signaling. *EMBO J.* **2017**, *36*, 2280–2295. [CrossRef]

198. Baar, M.P.; Brandt, R.M.C.; Putavet, D.A.; Klein, J.D.D.; Derks, K.W.J.; Bourgeois, B.R.M.; Stryeck, S.; Rijksen, Y.; van Willigenburg, H.; Feijtel, D.A.; et al. Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging. *Cell* **2017**, *169*, 132–147.e16. [CrossRef]

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**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
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**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.