A human-like senescence-associated secretory phenotype is conserved in mouse cells dependent on physiological oxygen

Abstract

Cellular senescence irreversibly arrests cell proliferation in response to oncogenic stimuli. Human cells develop a senescence-associated secretory phenotype (SASP), which increases the secretion of cytokines and other factors that alter the behavior of neighboring cells. We show here that "senescent" mouse fibroblasts, which arrested growth after repeated passage under standard culture conditions (20% oxygen), do not express a human-like SASP, and differ from similarly cultured human cells in other respects. However, when cultured in physiological (3%) oxygen and induced to senesce by radiation, mouse cells more closely resemble human cells, including expression of a robust SASP. We describe two new aspects of the human and mouse SASPs. First, cells from both species upregulated the expression and secretion of several matrix metalloproteinases, which comprise a conserved genomic cluster. Second, for both species, the ability to promote the growth of premalignant epithelial cells was due primarily to the conserved SASP factor CXCL-1/KC/GRO-alpha. Further, mouse fibroblasts made senescent in 3%, but not 20%, oxygen promoted epithelial tumorigenesis in mouse xenographs. Our findings underscore critical mouse-human differences in oxygen sensitivity, identify conditions to use mouse cells to model human cellular senescence, and reveal novel conserved features of the SASP.

Figure 1. Secretory profiles of presenescent and senescent mouse fibroblasts.

A) Soluble factors secreted by the indicted cells were detected by antibody arrays and analyzed as described . For each cell strain, PRE and SEN signals were averaged and used as the baseline (average of PRE 3%, PRE 20%, SEN (XRA) 3%, SEN (OXI) 20%). Signals above baseline are shown in yellow; signals below baseline are in blue. The heat map key indicates log2-fold changes from baseline (changes greater than the scale show as saturated colors). The number of samples analyzed is shown below each lane (see also Dataset S1). B) Model of mouse cell proliferation in culture showing conditions under which SASPs do (+) and do not (−) develop. C–D) Correlation between the secretory profiles of mouse (C) or human (D) cells cultured and induced to senesce in 3% vs 20% O₂. Baselines for the senescent profiles are the corresponding PRE profiles of cells cultured in the same O₂ concentration. E) Comparisons of the number of secreted factors that change in human and mouse cells induced to senesce in 20% or 3% O₂. F–G) Unsupervised hierarchical clustering analysis of PRE and SEN mouse (F) or human (G) fibroblasts (see also Fig. S1).

Figure 2. Orthology analyses of human and mouse secretory profiles.

A) Correlation between human and mouse SEN cells cultured and induced to senesce in 3% O₂ (top panel) and 20% O₂ (lower panel). Baselines for each SEN profile are the secretory profiles of PRE cells cultured under the same O₂ concentration (see Datasets S2, S3). B) Unsupervised hierarchical clustering analysis of cells in A. C) Direct comparisons of the secretory profiles of human and mouse cells cultured in 3% O₂.

Figure 3. DNA damage, genomic instability and c-Fos response.

A–B) DNA damage foci in mouse and human PRE and SEN fibroblasts cultured at 3% or 20% O₂. Cells were immunostained for 53BP1 (red) and nuclei were stained with DAPI (blue). Bar graph shows the percentage of cells with 1 or more 53BP1 focus. C) Comparative genomic hybridization profiles of PRE and SEN mouse cells cultured at 3% or 20% O₂. D) c-Fos response in mouse cells. PRE, SEN(XRA) or SEN(OXI) – induced either by incubation with 400 uM H₂0₂ or passage in 20% O₂ – mouse cells were cultured at 3% or 20% O₂, incubated in 0.5% serum for 48 h, then stimulated (+ serum) or not (−) with 10% serum. Cell lysates were prepared and analyzed by western blotting for c-Fos and tubulin (control) protein.

Figure 4. mRNA and intracellular protein expression and identification of MMP, CXCL and CCL loci as SASP components.

A) IL-6 mRNA and intracellular protein in PRE and SEN mouse and human cells. IL-6 mRNA was quantified by RT-PCR (TaqMan). Intracellular protein was detected by immunofluorescence (green). Nuclei were stained with DAPI (blue). B) Intracellular IGFBP-6 protein was detected by immunofluorescence (green) of DAPI-stained (blue) cultures. C) mRNA levels of other senescence-associated genes (COX-2, TIMP-1, PAI-1 and VEGF) in mouse and human cells. mRNA was quantified by RT-PCR using TaqMan. D) Matrix metalloproteinases (MMP) are mouse and human SASP factors. Shown is the organization of the mouse (left) and human (right) MMP gene clusters. mRNA was isolated from mouse and human cells cultured as indicated: PRE cells in 3% (x) or 20% (gray dot) O₂ (black line); SEN(XRA) cultured in 3% O₂ (red line); SEN(OXI) mouse cells made senescent by passage in 20% O₂ (green line); SEN(REP) human cells made senescent by passage in 20% O₂ (green line). Abundance of the indicated MMP mRNAs was quantified by RT-PCR. PRE and SEN(XRA) mouse cells were immunostained for MMP-3 (bottom left). Conditioned media (CM) were assayed for MMP3 activity (zymography, bottom right) and protein level (western blotting, bottom right). E) Expression of Cxcl and Ccl gene clusters in PRE, SEN(XRA) and SEN(OXI) mouse cells. The genes are listed vertically in 5′ (top) →3′ order. Antibody arrays results are shown to the right. Bottom panels show immunostaining for intracellular Ccl2 and Ccl12.

Figure 5. Biological activities of SASPs.

A) Diagrams of direct and indirect epithelial/fibroblast co-culture models, and illustration of quantification methods. Most epithelial cells were transfected with GFP. In both co-culture systems, GFP fluorescent of live cells, or western analysis of GFP protein levels, were used to assess epithelial cell growth. Alternatively, in indirect co-culture, epithelial cell proliferation was quantified by fluorescence of DAPI-stained nuclei , . B-E) SCp2 (gray) and EpH4v (blue) epithelial cells were co-cultured directly with PRE or SEN cells of human (B–C) or mouse (D–E) origin, and monitored for growth. F–I) SCp2 (gray), EpH4v (blue), and MCF10A (green) epithelial cells were co-cultured with CM from PRE or SEN cells of human (F–G) or mouse (H–I) origin, and monitored for growth.

Figure 6. GRO-α/KC is key for the growth promoting effects human and mouse SASPs.

A–B) Epithelial cells were incubated with CM from the indicated fibroblasts and cell number was determined by cell counting, total protein content, or GFP fluorescence as described in the legend to Fig. 5. CM from human (A) or mouse (B) cells was used alone or supplemented with GRO-α (A) or KC (B) recombinant protein or with a blocking antibody. Cell growth was significantly stimulated by recombinant protein and inhibited by blocking antibody (*p<0.02; **p<0.05).

Figure 7. SEN(XRA) but not SEN(OXI) mouse cells promote tumorigenesis in vivo.

A) EpH4-v epithelial cells were injected into the mammary fat pad area of nu/nu mice alone or with mouse breast fibroblasts made senescent in 3% or 20% O₂. Tumor volumes were determined as described in Methods. Number of mice used: PRE, 3% O₂: n = 20; SEN(XRA), 3% O₂: n = 16; SEN (OXI), 20% O₂: n = 6; EpH4-v alone: n = 17. B–C) Comparison of average tumor volumes after injection. B) Significant differences between different tumor populations are graphically represented as -Log10(p) where p = 1. Student t-test value, and significance (p>0.9) is shown by the dashed line (see Fig. S6 for all Student t-test values and tumor population comparisons). C) Average fold-differences 10 d after injection.