Senescence in the Development and Response to Cancer with Immunotherapy: A Double-Edged Sword

International Journal of Molecular Sciences Review Senescence in the Development and Response to Cancer with Immunotherapy: A Double-Edged Sword Anthony M. Battram 1, Mireia Bachiller 1and Beatriz Martín-Antonio 1,2,* 1 Department of Hematology, Hospital Clinic, IDIBAPS, 08036 Barcelona, Spain; [email protected] (A.M.B.); [email protected] (M.B.) 2Department of Hematology, Hospital Clinic, IDIBAPS/Josep Carreras Leukaemia Research Institute, Carrer Rosselló149-153, 08036 Barcelona, Spain *Correspondence: [email protected]; Tel.: +34-93-227-45-28; Fax: +34-93-312-94-07 Received: 31 May 2020; Accepted: 13 June 2020; Published: 18 June 2020   Abstract: Cellular senescence was first described as a physiological tumor cell suppressor mechanism that leads to cell growth arrest with production of the senescence-associated secretory phenotype known as SASP. The main role of SASP in physiological conditions is to attract immune cells to clear senescent cells avoiding tumor development. However, senescence can be damage-associated and, depending on the nature of these stimuli, additional types of senescence have been described. In the context of cancer, damage-associated senescence has been described as a consequence of chemotherapy treatments that were initially thought of as a tumor suppressor mechanism. However, in certain contexts, senescence after chemotherapy can promote cancer progression, especially when immune cells become senescent and cannot clear senescent tumor cells. Moreover, aging itself leads to continuous inflammaging and immunosenescence which are responsible for rewiring immune cells to become defective in their functionality. Here, we define different types of senescence, pathways that activate them, and functions of SASP in these events. Additionally, we describe the role of senescence in cancer and its treatments, including how aging and chemotherapy contribute to senescence in tumor cells, before focusing on immune cell senescence and its role in cancer. Finally, we discuss potential therapeutic interventions to reverse cell senescence. Keywords: senescence; SASP; senescence surveillance; inflammaging; immunotherapy 1. Defining Senescence 1.1. From Old to New and Different Concepts of Senescence Cellular senescence is a cell fate that has the defining feature of stable growth arrest that is refractory to mitogenic stimulation. As such, senescent cells differ from those that are quiescent and able to reenter the cell cycle in favorable growth conditions. Senescence is distinct to apoptosis as well, and in fact, resistance to apoptosis is a feature of senescence [ 1 ]. Importantly, senescence does not correspond with a complete cellular shutdown, as evidenced by active metabolic reprogramming, changes to cell morphology and the large-scale production and secretion of proinflammatory molecules known as senescence-associated secretory phenotypes (SASP) [2]. Historically, senescence was first identified in vitro by Hayflick and Moorhead in 1961 when they observed during serial passage of human diploid fibroblasts that these cultures had limited replicative potential, becoming irreversibly arrested [ 3 ]. This impaired proliferation seemed to be associated with a progressive shortage of telomeres, which induced a persistent DNA damage response (DDR) and activated signals including p53 and p16 [ 4 ]. These events were hypothesized to be a physiological mechanism to prevent cancer initiation by avoiding proliferation of damaged cells [ 5 ] as Int. J. Mol. Sci. 2020,21, 4346; doi:10.3390/ijms21124346 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020,21, 4346 2 of 31 loss of telomerase activity appeared to show inhibition of tumor progression in various mice models [ 6 ]. Since these discoveries, a high number of studies have established that, with aging, progressive accumulation of DNA damage in the cell originate a cell senescent state initiated primarily to repair this DNA damage being termed “replicative senescence” or “DNA damage-associated senescence”. Replicative senescence occurs following excessive proliferation when a cell has reached its proliferative capacity and is no longer able to undergo cell division, a point known as the Hayflick limit. Initially, damage-associated senescence causes an arrest of the cell cycle progression to potentially allow the suppression of dysfunctional, transformed, or aged cells [ 2 , 7 ]. Thus, senescence can be perceived initially as a tumor-protective mechanism that prevents uncontrolled replication of those precancerous cells that contain some oncogene activation or the loss of tumor suppressor genes [ 8 ]. Furthermore, it has been found that the overexpression of p53 in malignant tumors has led to induction of cell senescence and tumor regression, supporting the idea of senescence as a neoplastic brake [ 9 ]. Moreover, cellular senescence can occur prematurely after exposure to a stress signal, being termed “premature senescence”, “stress-induced senescence”, or “oncogene-induced senescence” (OIS). The extracellular or internal triggers that cause premature senescence are wide-ranging and include a high-fat diet, cytokines, radiotherapy and chemotherapy drugs (known as “therapy-induced senescence”, (TIS), reactive oxygen species (ROS), mitochondria dysfunction, and oncogene activation [ 10 ]. These stress signals can cause other cell fates, primarily apoptosis, and therefore, the balance of many different signals is critical in the cellular decision to begin a program of senescence induction (Figure 1). Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 30 [5] as loss of telomerase activity appeared to show inhibition of tumor progression in various mice models [6]. Since these discoveries, a high number of studies have established that, with aging, progressive accumulation of DNA damage in the cell originate a cell senescent state initiated primarily to repair this DNA damage being termed “replicative senescence” or “DNA damage-associated senescence”. Replicative senescence occurs following excessive proliferation when a cell has reached its proliferative capacity and is no longer able to undergo cell division, a point known as the Hayflick limit. Initially, damage-associated senescence causes an arrest of the cell cycle progression to potentially allow the suppression of dysfunctional, transformed, or aged cells [2,7]. Thus, senescence can be perceived initially as a tumor-protective mechanism that prevents uncontrolled replication of those precancerous cells that contain some oncogene activation or the loss of tumor suppressor genes [8]. Furthermore, it has been found that the overexpression of p53 in malignant tumors has led to induction of cell senescence and tumor regression, supporting the idea of senescence as a neoplastic brake [9]. Moreover, cellular senescence can occur prematurely after exposure to a stress signal, being termed “premature senescence”, “stress-induced senescence”, or “oncogene-induced senescence” (OIS). The extracellular or internal triggers that cause premature senescence are wide-ranging and include a high-fat diet, cytokines, radiotherapy and chemotherapy drugs (known as “therapy-induced senescence”, (TIS), reactive oxygen species (ROS), mitochondria dysfunction, and oncogene activation [10]. These stress signals can cause other cell fates, primarily apoptosis, and therefore, the balance of many different signals is critical in the cellular decision to begin a program of senescence induction (Figure 1). Figure 1. Stress-mediated inducers of cellular senescence in cancer. Damage-associated senescence associated with cancer development can occur in cells of the immune system due to SASP released by tumor cells, after accumulation of DNA damage, after inflammatory insults such as ROS or mitochondrial dysfunction which also impacts ROS production, after radiotherapy damage and chemotherapy treatments in tumor cells and also as a result of oncogenic mutations. SASP, senescence-associated secretory phenotype; ROS, reactive oxygen species. Figure 1. Stress-mediated inducers of cellular senescence in cancer. Damage-associated senescence associated with cancer development can occur in cells of the immune system due to SASP released by tumor cells, after accumulation of DNA damage, after inflammatory insults such as ROS or mitochondrial dysfunction which also impacts ROS production, after radiotherapy damage and chemotherapy treatments in tumor cells and also as a result of oncogenic mutations. SASP, senescence-associated secretory phenotype; ROS, reactive oxygen species. On the other hand, there is a physiological and non-damage-associated senescence termed “developmentally-programmed senescence” that occurs during mammalian embryonic development for efficient tissue remodeling. Developmentally-programmed senescence is strictly dependent on p21, Int. J. Mol. Sci. 2020,21, 4346 3 of 31 followed by macrophage infiltration for clearance of senescent cells and, unlike age-related senescence, is independent of DNA damage. This mechanism has been proposed to be the origin of damage-associated senescence. Physiological cellular senescence occurring both during normal embryonic development and upon tissue damage follows a cycle of “senescence–clearance–regeneration” to trigger tissue remodeling and removal of senescent cells maintaining tissue homeostasis. This cycle is achieved with the help of the immune system that performs clearance of senescent cells. However, during aging, which is a relevant risk factor for cancer, a decline in the immune response termed “immunosenescence” causes these stages to not complete, compromising the clearance of senescent cells and exacerbating inflammation with detrimental effects [7,11]. 1.2. Senescence Biomarkers and the SASP Cellular senescence is complex and dynamic, with large variations in the observed phenotype depending on cell type, environment, and the time following senescence induction. Identification of senescent cells is dependent on multiple markers and functional properties, and no single marker has yet been identified to classify cells as being senescent. Other than the gold standard, detecting senescence-associated β -galactosidase (SAβ -gal) activity, some of the most popular senescence biomarkers are related to proliferation and the cell cycle, such as loss of Ki67 protein expression and increased levels of the cell cycle inhibitor p16. Lipofuscin, an aggregate of oxidized proteins, lipids, and metals that accumulates progressively mostly in aged postmitotic cells, colocalizes with SAβ -gal being also used as a senescence marker [ 12 ]. Other features of senescent cells include resistance to apoptosis by downregulation of caspase-3 and the proapoptotic BAX, and upregulation of antiapoptotic BCL-2, BCL-W and BCL-XL, and of cyclin-dependent kinase inhibitors p16 and p21 [ 13 – 15 ]. Genetic and epigenetic alterations can also serve as useful markers of senescence, in particular telomere length, trimethylation of histone H3 on lysine 9 (H3K9me3), phosphorylation of the histone H2AX ( γ H2AX), and loss of lamin B1 [16–18]. Secretion of SASP by senescent cells mediates many of their patho-physiological effects contributing to age-associated pathologies such as cancer [ 2 , 7 , 19 ]. The SASP contains a cocktail of factors that include proinflammatory cytokines, chemokines, growth factors, ROS, angiogenic factors, and proteases that are released by senescent cells [ 2 , 20 ] which can create a favorable cytokine microenvironment for tumorigenesis [ 21 ]. Although these proteins are secreted by many cell types in nonsenescent states, especially by tumor cells and immune cells, what makes the SASP unique is that it is induced by senescence. The exact components of the SASP are influenced by a variety of factors such as the senescence induction mechanism, cell type, and tissue of origin. Moreover, the SASP is dynamic and can change over time following the initiation of senescence [ 22 ]. The SASP is controlled by NFκ B, mammalian target of rapamycin (mTOR) [ 23 ], p38 mitogen-activated protein kinase (p38MAPK) signaling [ 24 ], and IL-1 signaling through the NLRP3 inflammasome [ 19 ]. In addition, damage-associated molecular patterns (DAMPs) can also activate the inflammasome triggering SASP activation [ 25 ]. SASP factors act in an autocrine way to create positive and negative feedback loops, and in a paracrine manner to modulate the activity of neighboring cells. Critically, the SASP can promote the propagation of senescence as it contains many prosenescent factors released in extracellular vesicles [ 26 ] which spread the SASP [ 19 ]. In the context of cancer, the SASP can have a major influence on tumor progression—it can either promote or hinder tumorigenesis depending on the exact components of the SASP [ 20 ]. In this regard, the SASP can be a double-edged sword in cancer treatment, as it is required by immune cells to mediate antitumor responses [ 27 – 29 ] promoting “senescence surveillance” and preventing tumor initiation [ 30 , 31 ], and at chronic levels and pathological conditions such as established tumors, SASP components, such as vascular endothelial growth factor (VEGF), CCL5, and IL-6, can induce cancer, drug resistance, cancer progression, and associated side effects such as cachexia [ 20 , 32 – 41 ]. Excellent reviews have addressed the different molecules and their functions present in the SASP [ 20 ]. Here, in Tables 1and 2we have summarized some of the common molecules found in the SASP released by senescent and/or immune cells and their impact in cancer progression. Int. J. Mol. Sci. 2020,21, 4346 4 of 31 Other relevant features of senescent cells include decreased mitophagy, which results in increased mitochondria levels and defective mitochondrial networks that may contribute to the SASP and metabolic dysfunction with aging [ 42 , 43 ]. It is also important to highlight that senescence can be transmissible from senescent to nonsenescent cells through cytoplasmic bridges [2]. 1.3. Epigenetics in Senescence The progression of senescence is frequently associated with extensive epigenetic remodeling and large-scale chromatin reorganization [ 18 ]. Epigenetic alterations are vital for the senescence-associated DDR and p16 expression. However, epigenetic mechanisms that control senescence go much further [ 44 ]. For instance, the H3K9me3 histone modification is fundamental, and changes in the activity or levels of the enzymes associated with the deposition or removal of the methylation (methyltransferases or demethylases, respectively) can induce or reverse senescence [ 45 – 47 ]. Similarly, redistribution of histone variants is a senescence mechanism to control gene expression [ 44 ]. In particular, incorporation of histone variant H3.3 drives cell cycle arrest by downregulating proliferation-promoting genes [ 48 ], whereas, H2A.J and macroH2A1 promote the SASP [ 49 , 50 ]. Other important epigenetic regulators of senescence are the DNA-distorting proteins HMGB1 and HMGB2, which are diminished in the nuclei of senescent cells [ 51 , 52 ]. Nuclear HMGB1 inhibits SASP gene expression and HMGB2 loss is associated with the genomic reorganization that occurs early in senescence development [ 51 , 52 ]. Interestingly, secreted HMGB1 promotes the SASP in an autocrine/paracrine manner, in complete contrast to its role in the nucleus [52]. In addition, a common feature of OIS, but not replicative senescence, is the formation of nuclear structures known as senescence-associated heterochromatin foci (SAHFs) [ 16 ]. SAHFs occur due to reorganization of the nuclear architecture, in which areas of heterochromatin containing repressive marks, such as H3K9me3, are brought together into clusters. Formation of OIS SAHFs is dependent on the nuclear lamina component lamin B1 and DNMT1-mediated upregulation of HMGA2 [ 17 , 18 ]. The abundance of repressive marks and loss of modifications associated with gene expression meant that SAHFs were originally thought of as areas of gene silencing, although recent studies of OIS showing that heterochromatin decondenses as it forms SAHFs suggests that this may not be universally true [ 16 , 17 ]. In fact, the 3D realignment of chromatin that occurs in the formation of SAHFs actually enhances expression of senescence-associated genes in regions adjacent to SAHFs [17]. 1.4. Signaling Pathways that Contribute to Senescence Independent of the method of senescence induction, the DDR is often critical, but not the only cause, to translating the senescence trigger into cell cycle arrest; a key feature of senescence. In replicative senescence, telomere loss leads to the exposure of chromosome ends that the DDR detects as it would a double-strand break in DNA [ 53 ]. In OIS, hyperproliferation causes faults in DNA replication, resulting in a DDR [ 10 ]. Radiation and chemotherapeutic drugs can initiate a DDR by generating a number of different DNA aberrations, depending on the nature of the damaging agent. Telomere attrition or irreparable/sustained DNA damage activate the kinases ataxia-telangiectasia mutated (ATM) and/or ATM and Rad3-related (ATR) to generate DDR foci (detected as γ H2AX). When ATM/ATR activity at DDR foci exceeds a certain threshold, a signaling cascade is triggered in which CHK2 and CHK1, phosphorylated by ATM and ATR, respectively, translocate away from chromatin to phosphorylate a number of substrates involved in cell cycle control and protein expression [ 31 ]. As a result of this signaling, p53 is activated and stabilized, allowing p53 to drive the transcription of the cell cycle inhibitor p21, which ultimately causes cell cycle arrest at the G1/S checkpoint. Although p21 is important for senescence initiation, it is thought that sustained cell G1 phase arrest requires a second cell cycle inhibitory protein, p16 [ 54 ]. The function of p16 is critical because it removes the inhibition of retinoblastoma protein, allowing it to repress E2F-mediated expression of DNA replication genes. Expression of p16 from the INK4A/ARF locus, which also encodes the tumor Int. J. Mol. Sci. 2020,21, 4346 5 of 31 suppressor proteins p14 and p15, is tightly controlled by chromatin modifiers, cofactor proteins and RNA molecules [ 55 ]. Although many details of this regulation are still unknown, it is well-established that polycomb repressive complexes restrain p16 transcription by adding chromatin-compacting modifications to the INK4A/ARF locus, especially H3K27 trimethylation (H3K27me3). Stress signals can contribute to senescence by suppressing the polycomb repressive complexes or by activating demethylases such as JMJD3 that removes the H3K27me3 mark, both of which abolish gene silencing at the INK4A/ARF locus and facilitate the transcription of p16 [56,57]. A number of signaling pathways cooperate to induce the development of the SASP. The DDR and signal transduction pathways mediated by oncogene activation, p38 MAPK, cGAS/STING, and JAK/STAT ultimately converge to control the activity of NFκ B and/or C/EBP β transcription factors. In turn, NFκ B and C/EBP β promote the expression of SASP factors, such as IL-6, IL-8, and IL-1 β , which act in an autocrine and paracrine manner to generate a positive feedback loop and increase SASP production. Moreover, SASP-derived IL-1 β and TGF β promote senescence in surrounding cells by promoting a ROS-dependent DDR [ 58 ]. mTOR signaling is key to the regulation of the SASP as well. mTOR controls the translation of key proteins involved in the SASP, such as IL-1 α and MAPKAPK2 [ 59 ]. There are signaling pathways that control the flavor of the SASP as well, such as those downstream of NOTCH1 which inhibit a C/EBP β -mediated proinflammatory SASP in favor of a TGFβ-rich secretome [60]. Cellular senescence is also elicited independently of the DDR. Thus, metabolic rewiring is another important contributor to the senescent phenotype, particularly in cell cycle arrest and SASP production. Senescent cells often exhibit a glycolytic state, albeit with a reduced energy profile and dysfunction in other metabolic pathways, such as the malate–aspartate shuttle [ 61 – 63 ]. Reduced malate–aspartate shuttle activity causes a decrease in the cytosolic NAD+/NADH ratio, which is critical for replicative senescence and mitochondrial dysfunction-associated senescence (MiDAS) [ 61 , 64 ]. The associated increase in ADP/ATP and AMP/ATP ratios trigger AMP-activated protein kinase (AMPK) activation, which promotes p53-mediated cell cycle arrest [ 65 ]. In turn, p53 causes decreased expression of the ME1 and ME2 enzymes, which convert malate into pyruvate, to further increase p53 expression and enhance senescence [66]. The metabolite pyruvate is another important molecule for senescence induction, although the fate of pyruvate can differ depending on the senescence trigger. In replicative senescence and MiDAS, the increase in lactate dehydrogenase activity/expression causes more pyruvate to be converted into lactate, and thus taken away from potential use in the TCA cycle [ 61 , 62 ]. However, in models of OIS and TIS, both glycolytic flux and TCA cycle activity are heightened [ 63 ]. Increased activity of the enzyme pyruvate dehydrogenase directs pyruvate into the TCA cycle, and as such, mitochondrial energy production is increased [ 67 , 68 ]. Another major driver of heightened mitochondrial metabolism in OIS is the oxidation of fatty acids [ 69 ], which are generated more in OIS cells through the action of fatty acid synthase [ 70 ]. Interestingly, OIS is sensitive to perturbation of nucleotide metabolism as well—oncogenic Ras-driven repression of a critical dNTP synthesis enzyme results in a lack of dNTP production, stalled replication forks, and, as a result, DDR [71]. The mechanisms of many other aspects of senescence, including inhibition of autophagy, morphological changes, and resistance to apoptosis, have been studied to a varied degree, but the precise cellular signal transduction pathways that control them are as yet unclear or remain controversial. 1.5. Contribution of Inflammaging to Senescence Surveillance The clearance of senescent cells by immune cells known as “senescence surveillance” is a critical step for resolution of senescence. In a physiological context, the SASP promotes senescence surveillance, activating immune cells to drive the clearance of senescent cells and thus preventing tumor initiation [ 30 , 31 ]. With aging, the senescence of immune cells themselves, termed “immunosenescence”, avoids the elimination of senescent cells. Moreover, during aging, the SASP is responsible for the development of two different processes that lead to a chronic, sterile, highly self-reactive, systemic Int. J. Mol. Sci. 2020,21, 4346 6 of 31 inflammatory condition termed “inflammaging” which enhances immunosenescence. The first process is thymic involution related to aging that leads to a decline in immune function or “immunosenescence”, causing insufficient production of naïve T cells and amplified oligo-clonal expansion of memory T cells with reduced immune repertoire diversity [ 72 ]. This immunosenescence compromises the clearance of senescent cells and exacerbates inflammation through the SASP, causing inflammaging [ 73 ]. Second, thymic involution leads to a reduced capacity of regulatory T cells (Tregs) for autoimmune suppression and to an amplified release of autoreactive T cells leading to tissue damage with chronic inflammation that exacerbates inflammaging [73,74]. 2. Senescence in Cancer One important question that still remains in the oncology field is the extent to which senescence can be placed as one of the drivers of neoplastic malignancy, as opposed to a consequence of the pathology itself. While some studies support senescence as an aftereffect willing to stop tumor growth as a proliferation-suppressive mechanism [ 75 ], others support the idea of senescence as a mechanism to create a favorable microenvironment through SASP for the tumor cells to be protected from immunoclearance [ 4 ]. In addition, it is important to highlight the dual role of immune cells in the context of cancer, as multiple lines of evidence indicate that immune inflammatory cells attracted by chemokines present in the SASP can be actively tumor-promoting, capable of fostering angiogenesis, cancer cell proliferation, and invasiveness through the secretion of proinflammatory components [ 76 ]. Thus, there is a very-heterogeneous landscape of opinions about the role of senescence in cancer. Moreover, the positive and negative aspects of cellular senescence sometimes just depend on the time senescent cells remain in the organism and whether they can be cleared by the immune system. Context-dependent outcomes and possible different scenarios regarding the relationship between senescence and cancer are discussed here. 2.1. Before Cancer Treatment: Age-Related Senescence and Cancer Before cancer treatment the existence of senescence can have two different effects. On one hand, senescence can be beneficial when it is followed by immune clearance and tissue remodeling [ 7 ]. Moreover, senescence can prevent tumorigenesis by avoiding replication of precancerous cells [ 77 ]. In this context, the capacity of senescent cells entering cell cycle arrest was attributed to be a tumor-suppressive mechanism [ 4 ] and the loss of telomerase activity appeared to inhibit tumor progression in various mice models [ 6 ]. Senescence seemed to prevent uncontrolled replication of those precancerous cells that contain some oncogene activation or loss of tumor suppressor genes [ 8 ]. These observations suggested that cellular senescence does not simply arise from the accumulation of cell divisions but can also be prompted by stress inducers, such as oncogenic activation [10]. On the other hand, immunosenescence is a hallmark of aging which concerns both the innate and adaptive immune system. This deterioration of the immune system contributes to the increased susceptibility to neoplastic transformation with age. Senescence can present a detrimental outcome during aging when emerging senescent cells are not cleared by the immune system leading to their accumulation. This accumulation promotes SASP and a proinflammatory state within the tissue that can cause or facilitate the appearance of neoplastic pathologies, especially in the elderly population [ 20 ]. The linkage between age, cancer, and cellular senescence remains one of the examples why senescence could end in detrimental outcomes for patients [ 78 ]. The elderly population faces different degenerative and neoplastic pathologies derived from the loss of proper cellular function. Age-related diseases, such as cancer, occur on a background of dysfunctional tissue and moreover, the appearance and accumulation of senescent cells with age could worsen the tissue status. Although the emergence of ‘primary’ senescent cells is a physiologic process of tissue maintenance for regeneration, when these cells are not efficiently cleared by the immune system due to impaired or aged immune cells, the senescent cells accumulate within the tissue [ 7 ]. Those are known as ‘secondary’ senescent cells Int. J. Mol. Sci. 2020,21, 4346 7 of 31 and are generated at sites of pathology after disease initiation and are thought to be responsible for disease amplification [79] (Figure 2). Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 30 Figure 2. The complex and contrasting roles of senescence in the immune response to cancer. (A) Healthy conditions: proliferative cells react to stresses, such as DNA damage or oncogene activation, by inducing senescence to avoid malignancy. Immune cells, recruited by immunosupportive elements of the SASP, prevent the harmful effects of senescent cell accumulation by mediating their clearance in a process known as immunosurveillance. As a further protective mechanism, malignant cells are detected and eliminated by effector T cells and NK cells. (B) During aging: immunosenescence leads to loss of immune cell function and thus a diminished ability to detect and clear senescent cells. The SASP released from persistent senescent cells has multiple roles in cancer progression, including promoting tumor cell expansion and the development of an immunosuppressive tumor microenvironment. Cancer cells reinforce T cell senescence through metabolite competition and other mechanisms. (C) Chemotherapy treatments: senescence-inducing chemotherapeutic agents force malignant cells into senescence. An undesirable effect of these chemotherapies is the induction of immune cell senescence, resulting in lack of senescent cell clearance and increased risk of cancer relapse. Nonetheless, when immune cell senescence is avoided, immune cells effectively remove senescent cancer cells and tumor progression is prevented. (D) Immunotherapy treatments: senescence in CAR-T cells causes cell cycle arrest and loss of function, and is therefore associated with decreased in vivo persistence and cancer relapse. This problem is likely to be more pronounced in elderly cancer patients due to immunosenescence of many immune cell types. Nondysfunctional chimeric antigen receptor modified T (CAR-T) cells expand in patients to become an effective antitumor therapy. Functional NK cells, macrophages and B cells also have important roles to play in tumor clearance, making these cell types exciting future immunotherapeutic options. 2.2. After Cancer Treatment with Chemotherapy Therapy-induced senescence (TIS) appears as a result of pharmacological intervention. TIS can end either in an advantageous outcome or an unwanted side effect [79]. The most dangerous side effect derived from TIS is cancer relapse. Cancer relapse comes from dormant cancer cells that survive therapy because they become apoptosis-resistant. Anticancer agents possess the ability to induce senescent-like phenotypes in some subset of tumor cells, creating a chemo-resistant niche. Although senescent cells remain in a durable and prolonged growth arrest, it may not be permanent. Figure 2. The complex and contrasting roles of senescence in the immune response to cancer. ( A ) Healthy conditions: proliferative cells react to stresses, such as DNA damage or oncogene activation, by inducing senescence to avoid malignancy. Immune cells, recruited by immunosupportive elements of the SASP, prevent the harmful effects of senescent cell accumulation by mediating their clearance in a process known as immunosurveillance. As a further protective mechanism, malignant cells are detected and eliminated by effector T cells and NK cells. ( B ) During aging: immunosenescence leads to loss of immune cell function and thus a diminished ability to detect and clear senescent cells. The SASP released from persistent senescent cells has multiple roles in cancer progression, including promoting tumor cell expansion and the development of an immunosuppressive tumor microenvironment. Cancer cells reinforce T cell senescence through metabolite competition and other mechanisms. ( C ) Chemotherapy treatments: senescence-inducing chemotherapeutic agents force malignant cells into senescence. An undesirable effect of these chemotherapies is the induction of immune cell senescence, resulting in lack of senescent cell clearance and increased risk of cancer relapse. Nonetheless, when immune cell senescence is avoided, immune cells effectively remove senescent cancer cells and tumor progression is prevented. ( D ) Immunotherapy treatments: senescence in CAR-T cells causes cell cycle arrest and loss of function, and is therefore associated with decreased in vivo persistence and cancer relapse. This problem is likely to be more pronounced in elderly cancer patients due to immunosenescence of many immune cell types. Nondysfunctional chimeric antigen receptor modified T (CAR-T) cells expand in patients to become an effective antitumor therapy. Functional NK cells, macrophages and B cells also have important roles to play in tumor clearance, making these cell types exciting future immunotherapeutic options. 2.2. After Cancer Treatment with Chemotherapy Therapy-induced senescence (TIS) appears as a result of pharmacological intervention. TIS can end either in an advantageous outcome or an unwanted side effect [ 79 ]. The most dangerous side effect derived from TIS is cancer relapse. Cancer relapse comes from dormant cancer cells that survive therapy Int. J. Mol. Sci. 2020,21, 4346 8 of 31 because they become apoptosis-resistant. Anticancer agents possess the ability to induce senescent-like phenotypes in some subset of tumor cells, creating a chemo-resistant niche. Although senescent cells remain in a durable and prolonged growth arrest, it may not be permanent. In fact, studies show that while replicative senescence maintains the irreversibility of senescence [ 80 ], those caused by premature stress such as TIS or OIS can reactivate the cell cycle and bring on cancer daughter cells more transformed than the original population, suggesting that the arrested state is not indefinite. TIS cells could be contemplated as the minimal residual disease and be the origin of relapse [ 81 ]. The main hypothesis is that some senescent cancer cells generated by therapeutic intervention can eventually escape from the arrest and acquire self-renewing properties. Saleh et al. [ 82 ] showed high expression of senescence markers in cancer cell lines after they escaped TIS induced by etoposide and doxorubicin. Some detrimental features observed in those cells include aggressiveness and stemness, making them a dangerous source of therapeutic failure and cancer relapse [83] (Figure 2). 2.3. After Cancer Treatment with Immunotherapy Senescence occurrence after TIS is an explored pathway after several decades of using chemotherapeutic agents for cancer treatment. Nevertheless, more and more approaches are appearing to address anticancer treatment, such as immunotherapy [ 84 ]. Thus, the appearance of senescence after immunotherapy has not been explored as deeply as TIS. Recently, it has been identified that IFN γ and TNF α , two cytokines highly secreted after cell immunotherapy [ 85 ], induce senescence in different murine and human neoplastic diseases [ 86 ]. Moreover, we observed that natural killer (NK) cells, which are used as a source for immunotherapy [ 87 ], secrete a high variety of proinflammatory molecules, including matrix metalloproteases, heat shock proteins [ 88 ] and others present in the SASP. 3. Senescence in the Immune Response to Fight Cancer The removal of senescent tumor and harmful nontumor cells by immune cells requires the inter-collaboration of different immune cell populations [ 9 , 30 , 89 , 90 ]. Therefore, restricting the proliferation of immune cells is likely to promote tumor growth and progression. This is particularly true for T and B lymphocytes whose function declines with aging as immunosenescence and further diminishes the natural anticancer response in older cancer patients [ 72 ]. The central hallmarks of immunosenescence include a poor immune response to novel antigens due to a decrease in naïve cells, an increase in memory cells, and also the chronic, low level of inflammation or ‘inflammaging’ with subsequent development of age-related diseases, such as cancer [ 73 , 91 , 92 ]. The cellular senescence that occurs in individual cells of the immune system is detailed below. 3.1. T Cells T cell dysfunction occurs through the development of exhaustion, anergy, and/or senescence [ 93 ]. T cell senescence occurs naturally with aging as a result of multiple rounds of proliferation, loss of activity of the telomere-extending enzyme complex telomerase, and shortening of telomeres. As a result, this form of T cell senescence is common in older individuals but can also be found in younger people whose T cells have undergone excessive proliferation, such as in X-linked lymphoproliferative syndrome patients [ 94 ]. With aging, a thymic atrophy results in a decrease of functional naïve CD4+and CD8 T cells [ 95 ]. This decline is more pronounced in males than in females [ 96 ] and is more noticeable for CD8 T cells with a peripheral oligo-clonal expansion of memory T cells, which in general provides a contracted T cell antigen receptor (TCR)-repertoire diversity inducing immunosenescence. In addition, aging causes CD4+T cells to become longer-lived but functionally impaired with reduced proliferation and IL-2 production. These cells express reduced levels of the proapoptotic BCL-2 interacting mediator of cell death (Bim), which is associated with development of age-associated dysfunctions [ 97 , 98 ]. This T cell dysfunctionality is a common phenomenon occurring in cancer patients that leads to deficient antitumor immune responses. Int. J. Mol. Sci. 2020,21, 4346 9 of 31 Moreover, this replicative senescence is exacerbated by certain diseases, including cancer and chronic viral infections (especially cytomegalovirus (CMV)), whereby long-term exposure of antigen causes repeated T cell stimulation [ 99 ]. An alternative method of senescence induction in T cells occurs that is independent of telomere length. This premature senescence is likely to be triggered by mechanisms that provoke the DNA damage response, such as excessive ROS [ 100 , 101 ]. Correspondingly, metabolic competition by Tregs can promote DNA damage in effector T cells and consequently induce senescence [ 102 – 104 ]. It appears that both types of T cell senescence are immunosuppressive in the tumor microenvironment [105,106]. Senescent T cells are found within CD4+and CD8+T cell compartments that have lost expression of CD27 and CD28. Absence of the costimulatory molecules CD27 and CD28 in T cells corresponds with short telomeres and an upregulation of CD57 and KLRG1 [ 100 , 107 – 110 ]. Further classifying CD27-CD28T cells with CD45RA expression identifies a CD45RA+or T EMRA population that has multiple characteristics of senescence, including decreased proliferation, an inability to upregulate telomerase activity, and higher levels of γ H2AX [ 100 , 109 , 111 ]. Interestingly, these EMRA T cells do not have shorter telomeres than CD27-CD45RAcells and the senescence phenotype is reversible, which suggests that their senescence is not purely telomere-dependent [ 109 , 112 , 113 ]. Furthermore, CD27-CD28T cells are not resistant to apoptosis, in contrast to senescent fibroblasts, and in fact, they are more prone to activation-induced apoptosis [ 112 , 114 ]. Long term T cell cultures are, however, resistant to apoptosis, possibly because they are in a later stage of senescence than freshly isolated senescent T cells, or because they have been selected during the in vitro T cell expansion [ 115 , 116 ]. Senescent T cells also persist in vivo due to the survival signals they receive, and senescent T cells from rheumatoid arthritis patients also appear to be apoptosis-resistant, which could have implications for overall T cell responses in elderly people with cancer. Senescent T cells lack proliferative ability and display features of cell cycle arrest at the G1/S phase transition [ 117 ]. In terms of effector function, CD27-CD45RA+senescent T cells are very much active, retaining the ability to release inflammatory cytokines (including IFN γ and TNF α ) and produce cytotoxic molecules (granzyme B and perforin) [ 100 , 110 , 111 , 114 ]. Interestingly, in CD4+T cells, IL-2 production is high in CD27-CD45RA+cells, but for CD8+T cells, the opposite is true [ 113 , 114 ]. Senescent CD8+T cells also release a cocktail of molecules that shows similarities with the SASP generated by other senescent cell types [ 118 ]. It is also important to remember that senescent T cells remain metabolically active, although senescence has a profound impact on cellular metabolism and vice versa. The ability to uptake nutrients and mitochondrial mass directly impacts on T cell senescence, as evidenced when comparing the more senescent/less proliferative CD8+T EMRA cells with less senescent/more proliferative CD8+TEM cell or CD4+TEMRA cells [100,119]. In many types of cancer, the presence of senescent-like T cells has been associated with malignancy and poor prognosis [ 120 – 122 ]. Indeed, tumor-infiltrating lymphocytes (TILs) have short telomeres and lack telomerase activity [ 123 ]. Although not well-researched, it has been shown that tumor cells themselves can induce a senescent-like phenotype in human T cells [ 120 , 124 , 125 ]. In a nonsuppressive environment, CD8+TILs react to tumor antigens by proliferating, differentiating, and producing effector molecules. However, senescence causes downregulation of costimulatory molecules and, at least in some cases, effector molecules [ 126 ]. Moreover, senescent T cells repress the activity of other immune cells in the tumor microenvironment, making it even more immunosuppressive [102,103]. Recently, it has been shown that senescent T cells are increased in different hematologic malignancies, including leukemias, lymphomas, and multiple myeloma (MM) [ 122 , 127 , 128 ]. In chronic lymphocytic leukemia (CLL), patients with a CD4+:CD8+T cell ratio below 1 had more senescent-like CD8+T cells and a poorer prognosis [ 122 ]. A comprehensive analysis of T cells from acute myeloblastic leukemia (AML) patients showed an increase in senescent CD8+T cells compared to healthy controls and a correlation between the expression of senescent markers and the patient response to chemotherapy [ 129 ]. Similarly, T cell senescence is associated with therapy response in MM. Specifically, when treated with autologous stem cell transplantation (ASCT), relapsed patients had Int. J. Mol. Sci. 2020,21, 4346 16 of 31 Clinical trials testing combination therapies containing the TLR8-specific agonist motolimod (VTX-2337) for the treatment of various tumor types have been published [ 215 – 220 ], and more are currently ongoing or planned (clinicaltrials.gov; NCT03906526, NCT02431559 and NCT04272333). Finally, chimeric antigen receptor modified T cells (CAR-T cells) have appeared in recent years as a successful immunotherapy for certain hematological malignancies [ 84 ]. However, some patients treated with CAR-T cells relapse, which is partially caused by a lack of functional persistence following treatment administration [ 221 ]. As the effectiveness of CAR-T cell therapy is dependent on the fitness of the patients’ T cells from which they are derived [ 222 ], the lack of sustained functional capacity of CAR-T cells could be due to the inflated levels of T cell senescence observed in cancer patients [ 107 ]. Furthermore, many cancer patients, including those with hematological malignancies, are elderly, meaning that they are likely to exhibit immunosenescence. In the case of anti-BCMA CAR-T cell clinical trials in MM, individuals receive CAR-T cell therapy after relapse, but at this stage of disease progression, the patient T cells are highly senescent and have a phenotype associated with a diminished response to anti-BCMA CAR-T cell treatment [130,222,223]. Therefore, therapeutic interventions that reverse CAR-T cell senescence, either in the process of their development or in vivo , would be of great benefit. 4.4. Potential Use of B Cells in Immunotherapy and Reversal of B Cell Senescence B cells clearly play an important role in the progression of many cancer types [ 141 ], and should therefore not be overlooked when discussing novel immunotherapies. Although some studies have shown that B cells are able to promote tumor progression and abrogate immunogenic chemotherapies [ 141 , 144 ], there is a clear correlation between increased TIBs and better patient outcome [ 141 ]. In fact, recent studies have shown a link between better response to immunotherapy and higher numbers of TIBs, and moreover, that this positive relationship is caused by the formation of tertiary lymphoid structures which promote T cell activation [ 224 – 226 ]. Interestingly, Helmink et al. found that there was a trend for fewer late memory CD27IgDB cells, which correspond to the cells that display senescent-like features, in the tumors of responders compared to nonresponders of immune checkpoint blockers [ 224 ], suggesting that fewer senescent B cells could promote immunotherapy response. A possible strategy to reverse B cell senescence could be to enhance autophagy. Reduced autophagy levels in old B lymphocytes corresponds with compromised B cell responses, but treatment with spermidine restores autophagy in old B cells so that they regain function [227]. 4.5. Retuning the Tumor Microenvironment to Promote Macrophage-Mediated Cancer Cell Clearance In the case of macrophages, the repolarization from M2 to M1 seems to be more important than senescence reversal, as M1 appears to be the phenotype capable of proinflammatory actions to initiate clearance of senescent cells [ 228 ]. The reversal of immune incompetence could be prevented by the restoration of the tumor microenvironment into one with a less immunosuppressive profile. There are studies that suggest a different role of molecules present in the SASP depending on the tumor stage [ 39 ]. Thus, in models of hepatocellular carcinoma, early stages of senescent precancerous hepatocytes secrete CCL2 via their SASP which acts as a tumor suppressive mechanism promoting the recruitment of macrophages to remove senescent cells. However, in developed tumors, senescent peritumoral tissue induces NK cell inhibition via CCL2-CCR2, promoting the growth of hepatocellular carcinoma. Moreover, CCL2 inhibits maturation of monocytes to macrophages causing more accumulation of senescent cells and contributing to tumor immune escape. This study demonstrates a dual context-dependent function of SASP components depending on the stage of the disease [39]. Int. J. Mol. Sci. 2020,21, 4346 17 of 31 Table 1. SASP factors released by immune cells. Senescent Immune Cell SASP Factors References CD4+T cells IL-6, IL-10, TNFα, IFNγ, TGF-β1 [102] CD8+T cells IL-6, IL-8, TNFα, IL-18, IFNγ, TGF-β1, CCL16, ADAM28 [102,111,117,118] B cells IL-6, IL-8, TNFα[229] NK cells MMPs, cathepsins [88] Macrophages IL-6, TNFα, PDGF-BB, TGFβReviewed in [230] IL, interleukin; TNF, tumor necrosis factor; CCL, chemokine ligand; ADAM, a disintegrin and metalloproteinase; NK, natural killer; MMP, matrix metalloproteinase; PDGF, platelet-derived growth factor; TGF, transforming growth factor. Table 2. SASP factors that influence immune cell function in cancer. SASP Factor Senescent Cells That Secrete Factor Protumorigenic Mechanisms Antitumorigenic Mechanisms Reference IL-6 CD8+T cells, B cells, macrophages Recruit MDSCs, impair DC differentiation, inhibit antitumor T cell responses Recruit macrophages and NKT cells [231–233] IL-8 Tumor cells in solid tumors and hematological malignancies Enhancement of angiogenesis, attraction of neutrophils and MDSCs Reviewed in [234] IL-1α Senescent fibroblasts. Breast cancer cells. Colon cancer cells Regulation of IL-6 and IL-8 protumorigenic effects. Induction of production of tumor survival factors. Oncogene Ras-induced cell senescence, doxorubicin-induced cancer cell senescence and replicative senescence. Macrophage immune surveillance [235–238] IL-1βFibroblasts Inflammaging, induction of ROS-mediated DDR [58] IL-10 Macrophages Immunosuppression [233] CXCL2/CXCR2 Prostate cancer cells T cell suppression through macrophage polarization to an anti-inflammatory phenotype. Recruit iMCs that hinder tumor cell senescence. [239,240] PGE2Hepatic stellate cells Inhibit antitumor responses through PTGER4 receptor in hepatocellular carcinoma [241] CCL2 Senescent hepatocytes Promotes accumulation of immunosuppressive iMCs promoting hepatocellular carcinoma through NK cell inhibition Recruits myeloid cells that differentiate into macrophages to clear senescent precancerous cells [39] CCL3 (MIP-1 α)Senescent hepatocytes Recruit immune NK cells for clearance of senescent cells in hepatocellular carcinoma [242] TGF-β1 Fibroblasts Inflammaging, induction of ROS-mediated DDR [58] TGF-β3 Senescent adipose-derived mesenchymal stem cells Decreased angiogenic potential [243] Int. J. Mol. Sci. 2020,21, 4346 18 of 31 Table 2. Cont. SASP Factor Senescent Cells That Secrete Factor Protumorigenic Mechanisms Antitumorigenic Mechanisms Reference CCL5 Melanoma cells Recruit TILs to eliminate cancer cells [244] TNFαInduce T cell senescence [109] IFNγBone marrow-derived macrophages Induce M1 macrophage differentiation [245] IFNα Induce CD8+T cell senescence, accelerates loss of CD27 and CD28 [135] MDSC, myeloid-derived suppressor cell; NKT, natural killer T; TIL, tumor-infiltrating lymphocyte; iMC, Gr1 + CD11b + immature myeloid cell; DC, dendritic cell; IL, interleukin; PGE 2 , prostaglandin E2; TNF, tumor necrosis factor; IFN, interferon; ROS, reactive oxygen species; DDR, DNA damage response; CXCL, CXC-chemokine ligand; CXCR, CXC chemokine receptor; CCL, chemokine ligand; MIP; macrophage inflammatory protein; TGF, transforming growth factor; NK, natural killer 5. Conclusions To summarize, in recent years, senescence is attracting increased attention in the field of cancer treatment as new studies are revealing its complex roles in cancer prevention, development, and progression. Whereas originally, in physiological conditions, senescence was described as a tumor suppressor mechanism, additional studies showed that damage-associated senescence after chemotherapy treatment can both hinder and promote cancer progression. The SASP has a crucial role in these seemingly contradictory outcomes and in the promotion of senescence in cells of the immune system that are crucial for the removal of tumor cells. Immunotherapy requires fit immune cells in order to succeed, and the development of novel cancer therapies should consider treatments that do not negatively affect immune cells, and moreover, that the achievement of senescence reversal in immune cells is likely to provide effective immunotherapy treatments. Author Contributions: A.M.B., M.B. and B.M.-A. wrote and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Instituto de Salud Carlos III, grant number PI17/01043, and the “la Caixa” Foundation, grant number P-CP042702. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. Wang, E. Senescent Human Fibroblasts Resist Programmed Cell Death, and Failure to Suppress bell Is Involved. Cancer Res. 1995,55, 2284–2292. [PubMed] 2. Gorgoulis, V.; Adams, P.D.; Alimonti, A.; Bennett, D.C.; Bischof, O.; Bishop, C.; Campisi, J.; Collado, M.; Evangelou, K.; Ferbeyre, G.; et al. Cellular Senescence: Defining a Path Forward. Cell 2019 ,179, 813–827. [CrossRef] 3. Hayflick, L.; Moorhead, P.S. 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