Elimination of Senescent Cancer-Associated Fibroblasts by Senolysis: A Potential Strategy for Cancer Therapy

Aging represents a fundamental risk factor for a wide spectrum of human diseases, including fibrotic disorders, neurodegeneration, and malignancies. Among the many hallmarks of aging, chronic low-grade inflammation has emerged as a central pathological feature, contributing significantly to the functional deterioration of tissues and organs and to the onset and progression of numerous age-associated conditions [1]. A growing body of research implicates senescent cells as key drivers of this chronic inflammatory state. Cellular senescence is defined by a stable and irreversible arrest of cell proliferation, commonly triggered by extrinsic insults such as ultraviolet radiation and chemical exposure, as well as intrinsic stressors including telomere attrition, oxidative damage, and genotoxic stress [2]. This state is orchestrated by the activation of tumor suppressor pathways, notably those involving p16^Ink4a, p21^Cip1 and p53. Senescent cells are characterized not only by growth arrest but also by a suite of distinctive phenotypic changes, including the robust secretion of pro-inflammatory and tissue-remodeling factors collectively termed the Senescence-Associated Secretory Phenotype (SASP), resistance to apoptosis, and metabolic reprogramming [3]. 

The role of senescent cells in cancer is multifaceted and highly context-dependent. While senescence serves as a potent tumor-suppressive mechanism by halting the proliferation of damaged cells and facilitating immune-mediated clearance, persistent senescent cells can paradoxically foster a tumor-promoting microenvironment via sustained SASP production [4]. Therefore, a comprehensive understanding of the multifaceted roles of senescent cells is essential for the development of effective cancer therapies. Cancer is increasingly understood as a disease not only of malignant cells but also of their surrounding microenvironment. The Tumor Microenvironment (TME), composed of non-neoplastic stromal components such as immune cells, fibroblasts, and endothelial cells, plays a pivotal role in shaping tumor behavior and progression [5]. Age-related alterations in the TME, including the accumulation of senescent stromal cells, may create permissive conditions for neoplastic transformation and exacerbate malignancy. Despite mounting evidence, the specific mechanisms and signaling cascades by which aging-associated microenvironmental changes drive tumorigenesis remain incompletely elucidated. 

Bladder cancer is notoriously resistant to chemotherapy and remains one of the malignancies associated with poor clinical outcomes [6]. Despite increasing recognition of age as a major risk factor, the molecular pathways by which aging contributes to the initiation and progression of bladder cancer remain insufficiently understood. Our recent study demonstrated that the accumulation of senescent cells in the aging bladder significantly influences the initiation and progression of bladder cancer [7]. Utilizing p16-CreERT2-tdTomato reporter mice, which enable precise identification and lineage tracing of p16^Ink4a-expressing cells at the single-cell level [8], we examined age-associated alterations in the senescent cell landscape of the bladder, revealing a progressive accumulation of p16^Ink4a-expressing cells in the bladder with advancing age. 

The majority of these p16^Ink4a-expressing cells were identified as Inflammatory Cancer-Associated Fibroblasts (iCAFs), characterized by robust expression of C-X-C motif chemokine ligand 12 (CXCL12). Furthermore, we observed that p16^Ink4a-expressing fibroblasts acquire the typical senescent characteristics and were abundantly present within the tumor stroma of bladder cancer. Genetic ablation of these p16^Ink4a-expressing cells in mice resulted in a marked reduction in tumor burden, underscoring their functional contribution to cancer progression.

Comparison of p16^Ink4a-expressing cell abundance in tumors before and after orthotopic transplantation of bladder cancer cells revealed no significant increase, suggesting that the tumor-promoting p16^Ink4a-expressing cells were not newly induced but rather pre-existing within aged bladder tissues. While previous studies have proposed that stromal senescence may be secondarily induced by tumor-derived signals [9], our findings support an alternative model wherein resident senescent fibroblasts in the aging bladder actively promote malignancy. This observation may help explain the increased incidence of bladder cancer with age. 

In addition, genetic removal of p16^Ink4a-expressing cells led to a reduction in CXCL12 expression within the tumor microenvironment, along with diminished activation of the downstream AKT signaling pathway. Pharmacological inhibition of the CXCL12 receptor CXCR4 using AMD3100 significantly suppressed tumor growth, reinforcing the critical role of the CXCL12-CXCR4 axis in bladder cancer progression. Although CXCL12 is known to be upregulated under hypoxic conditions and implicated in tumor proliferation, adhesion, angiogenesis, and metastasis [10], the precise molecular mechanisms by which it modulates bladder cancer cell behavior remain to be fully elucidated. 

Recent studies have underscored the functional heterogeneity of cancer-associated fibroblast subtypes, including myofibroblastic CAFs (myCAFs) and iCAFs, with emerging evidence suggesting that specific subsets may exert distinct influences on tumor biology [10,11]. Thus, comprehensive profiling of CAF populations, particularly those exhibiting senescent phenotypes, will be critical for informing the development of novel multimodal therapeutic strategies. 

CAFs are increasingly recognized as key facilitators of tumor progression across diverse malignancies. One major mechanism by which CAFs exert their pro-tumorigenic influence is through the deposition and remodeling of the Extracellular Matrix (ECM), which not only enhances tissue stiffness but also generates structural pathways that promote tumor cell invasion and dissemination [9]. Recent advances in single-cell and spatial transcriptomic technologies have enabled the identification of a Senescent CAF-Related Signature (SCRS) across multiple tumor types. In neuroblastoma, for example, the presence of myCAFs is common, and patients can be stratified into distinct prognostic subgroups based on SCRS levels. High SCRS scores are associated with diminished immune cell infiltration and reduced responsiveness to therapeutic interventions, underscoring the immunosuppressive and therapy-resistant microenvironment conferred by specific CAF populations [11]. 

In Esophageal Squamous Cell Carcinoma (ESCC), a subset of Hypoxia-Induced Senescent Fibroblasts (hsCAFs) has been characterized. These fibroblasts accumulate under low-oxygen conditions and secrete high levels of Insulin-like Growth Factor 1 (IGF1), which in turn suppresses AMP-activated Protein Kinase (AMPK) signaling. The inhibition of AMPK activity enhances the self-renewal capacity and plasticity of tumor cells, thereby promoting cancer aggressiveness under hypoxic stress [12]. Similarly, in breast cancer, the ECM produced by senescent CAFs (sn-CAFs) has been shown to impede Natural Killer (NK) cell infiltration, contributing to immune evasion and facilitating tumor expansion. Clinical data have also revealed a correlation between elevated sn-CAF abundance and poor patient prognosis, further supporting their deleterious role in the tumor microenvironment [13]. 

Given the significant impact of sn-CAFs on tumor biology, their selective elimination through senolytic strategies has gained considerable interest. The Bcl-2 family inhibitor ABT-263 (navitoclax), initially evaluated in models of bladder cancer, has shown efficacy in suppressing tumor growth in both breast and lung cancers. Notably, ABT-263 treatment results in elevated levels of cleaved caspase-3, a hallmark of apoptosis induction. Mechanistically, ABT-263 disrupts the anti-apoptotic function of Bcl-xL by competitively binding to it, thereby liberating pro-apoptotic proteins BAX and BAK to trigger mitochondrial outer membrane permeabilization and subsequent cell death. Importantly, this compound exhibits selective cytotoxicity toward senescent cells while sparing proliferating populations. These findings highlight the therapeutic promise of senolytic agents like ABT-263 in mitigating the tumor-promoting activities of sn-CAFs [14]. 

In our study, we elucidated the role of senescent CAFs in the progression of bladder cancer using a pathophysiologically relevant disease model. The interplay between cellular senescence and tumor development is multifaceted, influenced by cancer type, disease stage, and therapeutic context. Among the key components of TME, CAFs have emerged as critical regulators of tumor behavior and therapeutic resistance. Their ability to shape the immune landscape, remodel the extracellular matrix, and secrete tumor-promoting factors positions them as attractive targets for combinatorial treatment strategies. One such approach is the “one-two punch” strategy, which involves the deliberate induction of senescence in tumor cells through genotoxic therapies, followed by the targeted elimination of these cells using senolytic agents. This sequential intervention holds potential to enhance anti-cancer efficacy while minimizing the pro-tumorigenic risks associated with persistent senescent cells.

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