Cellular Senescence of Patient-derived Fibroblasts Reveals the Mid-old Stage as a Critical Window for Transcriptomic Signatures Linked to Alzheimer’s Disease Biomarkers and Classification

Cellular senescence prevents the proliferation of cells at risk for neoplastic transformation. Nonetheless, the senescence response is thought to be antagonistically pleiotropic and thus contribute to aging phenotypes, including, ironically, late life cancers. The cancer-promoting activity of senescent cells is likely due to secreted molecules, the identity of which remains largely unknown. Here, we have shown that senescent fibroblasts, much more than presenescent fibroblasts, stimulate tumor vascularization in mice. Weakly malignant epithelial cells co-injected with senescent fibroblasts had larger and greater numbers of blood vessels compared with controls. Accordingly, increased vascular endothelial growth factor (VEGF) expression was a frequent characteristic of senescent human and mouse fibroblasts in culture. Importantly, conditioned medium from senescent fibroblasts, more than medium from presenescent cells, stimulates cultured human umbilical vein endothelial cells to invade a basement membrane, a hallmark of angiogenesis. Increased VEGF expression was specific to the senescent phenotype and increased whether senescence was induced by replicative exhaustion, overexpression of p16(Ink4a), or overexpression of oncogenic RAS. The senescence-dependent increase in VEGF production was accompanied by very little increase in hypoxic-inducible (transcription) factor 1 alpha protein levels, and hypoxia further induced VEGF in senescent cells. This result suggests the rise in VEGF expression at senescence is not a hypoxic response. Our findings may in part explain why senescent cells stimulate tumorigenesis in vivo and support the idea that senescent cells may facilitate age-associated cancer development by secreting factors that promote malignant progression.

To identify plant extracts capable of inhibiting cellular senescence, the effects of plant extracts on adriamycin-induced cellular senescence in human fibroblasts, human umbilical vein endothelial cells (HUVECs), and vascular smooth muscle cells were investigated. After adriamcyin treatment, the effects of plant extracts on cellular senescence were evaluated by measuring senescenceassociated β-galactosidase activity of the cells. Among 799 plant extracts, those from Rhei Rhizoma, Cirsii Radix, and Plantagnis Semen repressed adriamycin-induced cellular senescence in fibroblasts. Extracts of Cinnamomi Cortex and Cinnamomi Cortex Spissus were effective in endothelial cells. Extracts of Euonymi Lignum Suberalatum, Salicis Radicis Cortex, Polygoni aviculari Herba, and Chaenomelis langenariae Radix reduced senescence in vascular smooth muscle cells. These results suggested the effectiveness of plant extracts in reducing cellular senescence of human cells. These plant extracts could be used to develop dietary supplements or cosmetics for modulating tissue aging or aging-associated diseases.

Although cellular senescence may be a protective mechanism in modulating proliferative capacity, fibroblast senescence is now recognized as a key pathogenic mechanism in idiopathic pulmonary fibrosis (IPF). In aged mice, abundance and persistence of apoptosis-resistant senescent fibroblasts play a central role in nonresolving lung fibrosis after bleomycin challenge. Therefore, we investigated whether quercetin can restore the susceptibility of senescent IPF fibroblasts to proapoptotic stimuli and mitigate bleomycin-induced pulmonary fibrosis in aged mice. Unlike senescent normal lung fibroblasts, IPF lung fibroblasts from patients with stable and rapidly progressing disease were highly resistant to Fas ligand (FasL)-induced and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Senescent IPF fibroblasts exhibited decreased expression of FasL and TRAIL receptors and caveolin-1, as well as increased AKT activation, compared with senescent normal lung fibroblasts. Although quercetin alone was not proapoptotic, it abolished the resistance to FasL- or TRAIL-induced apoptosis in IPF fibroblasts. Mechanistically, quercetin upregulated FasL receptor and caveolin-1 expression and modulated AKT activation. In vivo quercetin reversed bleomycin-induced pulmonary fibrosis and attenuated lethality, weight loss, and the expression of pulmonary senescence markers p21 and p19-ARF and senescence-associated secretory phenotype in aged mice. Collectively, these data indicate that quercetin reverses the resistance to death ligand-induced apoptosis by promoting FasL receptor and caveolin-1 expression and inhibiting AKT activation, thus mitigating the progression of established pulmonary fibrosis in aged mice. Therefore, quercetin may be a viable therapeutic option for IPF and other age-related diseases that progress with the accumulation of senescent fibroblasts.

All human tissues experience aging that eventually causes organ dysfunction and disease. Cellular senescence was discovered in fibroblasts cultured in vitro. In adults, it is a primary defense mechanism against cancer, but also a major contributor to lifespan limits and disorders associated with aging. To assess how human blood vessels change in an aged environment, we developed an elementary tissue model-on-a-chip that comprises an in vitro three-dimensional model of a blood vessel embedded in a collagen gel with young or senescent skin fibroblasts. We found that senescent fibroblasts mechanically altered the surrounding extracellular matrix by exerting excessive traction stress. We then found that senescent fibroblasts induced sprouting angiogenesis of a microvessel via their senescence-associated secretory phenotype (SASP). Finally, we gathered evidence that the mechanical changes of the microenvironment play a role in sustaining SASP-induced angiogenesis. The model proved useful in monitoring morphological changes in blood vessels induced by senescent fibroblasts while controlling the proportion of senescent cells, and enabled the study of SASP inhibitors, a class of drugs useful in aging and cancer research.

What seems like a good idea can sometimes backfire later. A mechanism that foils cancer in young animals might induce it in older ones, according to a new study. Most mammalian cells, whether in animals or culture dishes, don't divide forever; instead, they eventually undergo cellular senescence, which permanently arrests cell division (see "More Than a Sum of Our Cells" ). In animals, that process might prevent cells with DNA damage or shortened telomeres from growing into tumors (see "Dangerous Liaisons" ). Senescent cells, however, remain in the body--dormant but perhaps not benign. To investigate how senescent cells affect the tissue in which they reside, Krtolica and colleagues grew cultures that contained either presenescent or senescent fibroblasts--cells that compose connective tissue--and then layered test cultures of various epithelial cell types on top of the fibroblasts. The researchers measured proliferation of the epithelial cells by tagging their DNA with fluorescent labels. Senescent, but not presenescent, fibroblasts provoked duplication of cell lines that had previously acquired mutations known to predispose them to cancer, the team found. Neither senescent nor presenescent fibroblasts promoted the growth of normal human epithelial cells. Cell-to-cell contact isn't required for the stimulation: Material secreted by senescent cells induced precancerous cells to grow approximately three times faster than did material secreted by presenescent cells. Senescent cells also appear to promote tumor growth in vivo: Tumors were more prevalent and grew larger in mice injected with precancerous epithelial cells and senescent fibroblasts than in those injected with precancerous cells and presenescent fibroblasts. Senescent cells apparently create a rich environment for tumor growth. This phenomenon, combined with the increased incidence of cancer-causing mutations as time passes, could partially explain the exponential increase in cancer rates as we age. Identifying exactly how senescent cells create fertile ground for cancer might well shed light on how a good deal in youth turns sour with age. --R. John Davenport; suggested by Nick Bishop A. Krtolica, S. Parrinello, S. Lockett, P.-Y. Desprez, J. Campisi, Senescent fibroblasts promote epithelial cell growth and tumorigenesis: A link between cancer and aging. Proc. Natl. Acad. Sci. U.S.A. 98 , 12072-12077 (2001). [Abstract] [Full Text]

Cellular senescence is a physiological process that serves as a powerful barrier for tumorigenesis. However, senescent cells can be deleterious for the tissue microenvironment. Such is the case of senescent fibroblasts that release several pro-tumorigenic factors that promote malignant transformation in the nearby epithelial cells. Occupational exposure to hexavalent chromium [Cr(VI)] compounds is a cause of respiratory cancers. Although Cr(VI) is known to induce senescence in human foreskin fibroblasts, the role of senescent fibroblasts in the Cr(VI)-induced malignant transformation of human bronchial epithelial cells was never assessed. Thus, to study the evolutionary dynamics generated by the interaction between human bronchial epithelial cells and senescent bronchial fibroblasts, the non-tumorigenic human bronchial epithelial BEAS-2B cells were co-cultured with Cr(VI)-induced senescent human bronchial fibroblasts for 4 weeks. Under the pressure of 0.5 µM Cr(VI), senescent fibroblasts promoted the acquisition of mesenchymal features on BEAS-2B cells, e.g. the fusiform shape and increased Vimentin expression, consistent with the occurrence of an epithelial-mesenchymal transition-like process. Features of transformed cells including larger nuclei, as well as nuclei with heterogeneous size, were also observed. Altogether the results obtained demonstrate that besides acting over the epithelium, Cr(VI) also affects bronchial fibroblasts driving them senescent. As a consequence, a paracrine communication loop is established with the above-placed epithelium prompting the epithelial cells for malignant transformation and thus facilitating the initial steps of tumorigenesis.

Cellular senescence and its associated secretory phenotype (SASP) can promote cancer progression in the tumor microenvironment (TME). The TME includes tumor cells, stromal cells, immune cells, endothelial cells, and extracellular matrix. Senescent cancer-associated fibroblasts (CAF) may contribute to tumor growth and therapy resistance. Targeting senescent CAF by means of removal, modulation of the SASP, or through cellular reprogramming might provide therapeutic avenues for treating cancer. We investigated the impact of chemotherapy-induced fibroblast senescence in the TME on tumor growth and response to cancer therapy. Expression of cytokines in chemo-induced senescent fibroblasts and cancer cells was assessed by bulk and single cell cytokine profiling. As expected, there were alterations in SASP factors with increased pro-tumorigenic immune factors and decreased anti-tumor cytokines during IMR90 etoposide-induced fibroblast senescence. We co-cultured luciferase-labeled HT29 cancer cells with senescent IMR90 and found that the senescent fibroblasts promoted HT29 cell growth in culture and accelerated xenograft tumor formation in mice. We next inhibited cellular senescence and its SASP with the senolytic drug ABT263 or the senostatic/senomorphic drug lamivudine (3TC). Both ABT263 and 3TC significantly reduced bioluminescence of HT29-Luc cells co-cultured with senescent IMR90 compared to non-treated IMR90 cells. Therapy-induced senescence confers 5-Fluorouracil (5-FU) resistance in colorectal cancer. We found that 5-FU treatment significantly reduced colony formation of HT29 cells in the presence of proliferating or senescent IMR90 cells, with a lesser reduction in the presence of the senescent IMR90 cells. This suggests that a microenvironment that includes senescent cells promotes tumor cell resistance to 5-FU. We hypothesize that SASP factors might confer cancer cell resistance to 5-FU treatment. Cytokine profiling showed that TRAIL expression is reduced in senescent cells. Treatment with the TRAIL-inducer ONC201 reduced colony formation and cell viability of HT29 cells co-cultured with senescent IMR90 fibroblasts. Single-cell cytokine profiling showed subpopulations of cancer cells with increased polyfunctionality strength index (PSI, secretion of more than 2 dominant types of cytokines per cell in a population). Combined treatment with ABT263 and ONC201 synergically reduced viability HT29 cells co-cultured with senescent IMR90, and this correlated with reduced PSI. Our results indicate that TME targeting by increasing antitumor cytokines in conjunction with senolytic therapies can inhibit tumor growth. We are continuing to unravel the cytokine landscape of chemotherapy-induced cell senescence to gain insights into therapeutic strategies targeting chemotherapy-induced TME-senescence and drug resistance. Citation Format: Shengliang Zhang, Kelsey E. Huntington, Bianca Kun, Lanlan Zhou, Jill Kreiling, John M. Sedivy, Wafik S. El-Deiry. ONC201 suppresses cancer cell growth in a reconstructed tumor microenvironment that includes chemotherapy-induced senescent fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5297.

Cellular senescence was first reported in human fibroblasts as a state of stable in vitro growth arrest following extended culture. Since that initial observation, a variety of other phenotypic characteristics have been shown to co-associate with irreversible cell cycle exit in senescent fibroblasts. These include (1) a pro-inflammatory secretory response, (2) the up-regulation of immune ligands, (3) altered responses to apoptotic stimuli and (4) promiscuous gene expression (stochastic activation of genes possibly as a result of chromatin remodeling). Many features associated with senescent fibroblasts appear to promote conversion to an immunogenic phenotype that facilitates self-elimination by the immune system. Pro-inflammatory cytokines can attract and activate immune cells, the presentation of membrane bound immune ligands allows for specific recognition and promiscuous gene expression may function to generate an array of tissue restricted proteins that could subsequently be processed into peptides for presentation via MHC molecules. However, the phenotypes of senescent cells from different tissues and species are often assumed to be broadly similar to those seen in senescent human fibroblasts, but the data show a more complex picture in which the growth arrest mechanism, tissue of origin and species can all radically modulate this basic pattern. Furthermore, well-established triggers of cell senescence are often associated with a DNA damage response (DDR), but this may not be a universal feature of senescent cells. As such, we discuss the role of DNA damage in regulating an immunogenic response in senescent cells, in addition to discussing less established "atypical" senescent states that may occur independent of DNA damage.

Vanadium pentoxide induced oxidative stress and cellular senescence in human lung fibroblasts

Oral submucous fibrosis (OSMF) is a pre-malignant condition that is strongly associated with the areca nut alkaloids, arecoline (ARC) and arecaidine (ARD). The condition is characterised by the presence of senescent fibroblasts in the subepithelial mesenchyme which have the potential to promote malignancy in the neighbouring epithelial cells. We tested the hypothesis that areca nut alkaloids induce senescence in oral fibroblasts and promote the secretion of invasion-promoting transforming growth factor β (TGF-β) and matrix metalloproteinase-2 (MMP-2). Two oral fibroblast lines were treated for 48h with ARC and ARD. Senescence-associated β-galactosidase (SA-βGal) activity, Ki67 (cycling cells), large 53BP1 foci (irreparable DNA strand breaks) and p16(INK) (4A) (late senescence) were used as markers of cellular senescence and were quantified using indirect immunofluorescence and the ImageJ program. TGF-β and MMP-2 levels were measured using ELISA. Statistical analyses were performed with the two-tailed unpaired t-test where n = 3 and the Wilcoxon-Mann-Whitney test where n = 6. ARC (100 and 300 μM) and ARD (30 and 100 μM) significantly (P < 0.05) induced fibroblast senescence, as determined by the increased expression of SA-βGal, 53BP1 staining and CDKN2A/p16(INK) (4A) ; there was also a non-significant reduction in Ki67 staining. Treated cells also showed a three- fivefold increase in TGF-β and a small non-significant increase in MMP-2. Areca nut alkaloids induce senescence in oral fibroblasts and promote increased secretion of TGF-β and perhaps MMP-2 that may create a tissue environment thought to be critical in the progression of OSMF to malignancy.

Normal cells in culture invariably undergo senescence, whereby they cease proliferation after a finite number of doublings. Irreversible changes in gene expression occurred in senescent human fetal lung fibroblasts: a non-cell cycle-regulated mRNA was partially repressed; an unusual polyadenylated histone mRNA was expressed; although serum induced c-H-ras, c-myc, and ornithine decarboxylase mRNA normally, ornithine decarboxylase activity was deficient; and serum did not induce mRNA for a replication-dependent histone and for the c-fos proto-oncogene. The loss of c-fos inducibility was the result of a specific, transcriptional block. The results suggest that senescent fibroblasts were unable to proliferate because of, at least in part, selective repression of c-fos; moreover, the multiple changes in gene expression support the view that cellular senescence is a process of terminal differentiation.

Metabolic stabilization of MAP kinase phosphatase-2 in senescence of human fibroblasts

Downregulation of DNA topoisomerase I in old versus young human diploid fibroblasts

Effect of Cellular Senescence and Retinoic Acid on the Expression of Cellular Retinoic Acid Binding Proteins in Skin Fibroblasts

As a gene with antiaging functions, sirtuin6 (SIRT6) belonging to the sirtuin family plays a vital role in DNA repair, telomerase function, and cellular senescence, as well as maintains epigenomic stability and promotes longevity. However, its role in cell senescence in large animals, such as buffaloes, remains unknown. Fibroblasts are commonly used for somatic reprogramming, and their physiological characteristics affect the efficiency of this process. We aimed to elucidate the role of SIRT6 in cellular senescence and proliferation and analyze its effect on the biological function of buffalo fibroblasts to help improve the efficiency of buffalo somatic cell reprogramming. The expression of SIRT6 and related DNA damage was measured in buffalo fibroblasts obtained at different developmental stages (in the fetus and at 3 and 10 years of age), and the effect of SIRT6 knockdown on the senescence of buffalo fetal fibroblast was investigated. An inverse relationship was observed between SIRT6 expression and senescence in buffalo fibroblasts obtained from animals of various ages. This was accompanied by decreased cell growth, viability, and increased DNA damage. Short hairpin RNA-mediated SIRT6 knockdown accelerated the senescence of buffalo fetal fibroblasts. It blocked the cell cycle during in vitro cell culture, which further enhanced DNA damage, particularly with respect to the telomeres. Collectively, our findings suggest that SIRT6 expression was closely associated with buffalo senescence in fibroblasts. These findings serve as a foundation to better understand the cellular functions of SIRT6 and also aid in selecting donor cells for buffalo somatic cell reprogramming.