Aneuploid senescent cells activate NF‐κB to promote their immune clearance by NK cells
2021; Springer Nature; Volume: 22; Issue: 8 Linguagem: Inglês
10.15252/embr.202052032
ISSN1469-3178
AutoresRuoxi W. Wang, Sonia Viganò, Uri Ben‐David, Angelika Amon, Stefano Santaguida,
Tópico(s)Telomeres, Telomerase, and Senescence
ResumoArticle8 June 2021Open Access Transparent process Aneuploid senescent cells activate NF-κB to promote their immune clearance by NK cells Ruoxi W Wang Ruoxi W Wang Department of Biology, Massachusetts Institute of Technology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Sonia Viganò Sonia Viganò orcid.org/0000-0003-0437-0087 Department of Experimental Oncology at IEO, European Institute of Oncology IRCCS, Milan, Italy Search for more papers by this author Uri Ben-David Uri Ben-David orcid.org/0000-0001-7098-2378 Department of Human Molecular Genetics & Biochemistry, Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Angelika Amon Angelika Amon Department of Biology, Massachusetts Institute of Technology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USADeceased Search for more papers by this author Stefano Santaguida Corresponding Author Stefano Santaguida [email protected] orcid.org/0000-0002-1501-6190 Department of Experimental Oncology at IEO, European Institute of Oncology IRCCS, Milan, Italy Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy Search for more papers by this author Ruoxi W Wang Ruoxi W Wang Department of Biology, Massachusetts Institute of Technology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Sonia Viganò Sonia Viganò orcid.org/0000-0003-0437-0087 Department of Experimental Oncology at IEO, European Institute of Oncology IRCCS, Milan, Italy Search for more papers by this author Uri Ben-David Uri Ben-David orcid.org/0000-0001-7098-2378 Department of Human Molecular Genetics & Biochemistry, Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Angelika Amon Angelika Amon Department of Biology, Massachusetts Institute of Technology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USADeceased Search for more papers by this author Stefano Santaguida Corresponding Author Stefano Santaguida [email protected] orcid.org/0000-0002-1501-6190 Department of Experimental Oncology at IEO, European Institute of Oncology IRCCS, Milan, Italy Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy Search for more papers by this author Author Information Ruoxi W Wang1, Sonia Viganò2, Uri Ben-David3, Angelika Amon1 and Stefano Santaguida *,2,4 1Department of Biology, Massachusetts Institute of Technology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USA 2Department of Experimental Oncology at IEO, European Institute of Oncology IRCCS, Milan, Italy 3Department of Human Molecular Genetics & Biochemistry, Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 4Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy *Corresponding author. Tel: +39 02 9437 5074; E-mail: [email protected] EMBO Reports (2021)22:e52032https://doi.org/10.15252/embr.202052032 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The immune system plays a major role in the protection against cancer. Identifying and characterizing the pathways mediating this immune surveillance are thus critical for understanding how cancer cells are recognized and eliminated. Aneuploidy is a hallmark of cancer, and we previously found that untransformed cells that had undergone senescence due to highly abnormal karyotypes are eliminated by natural killer (NK) cells in vitro. However, the mechanisms underlying this process remained elusive. Here, using an in vitro NK cell killing system, we show that non-cell-autonomous mechanisms in aneuploid cells predominantly mediate their clearance by NK cells. Our data indicate that in untransformed aneuploid cells, NF-κB signaling upregulation is central to elicit this immune response. Inactivating NF-κB abolishes NK cell-mediated clearance of untransformed aneuploid cells. In cancer cell lines, NF-κB upregulation also correlates with the degree of aneuploidy. However, such upregulation in cancer cells is not sufficient to trigger NK cell-mediated clearance, suggesting that additional mechanisms might be at play during cancer evolution to counteract NF-κB-mediated immunogenicity. SYNOPSIS This study shows that aneuploid cells upregulate NF-κB pathway. This is crucial for promoting an inflammatory state, which in turn leads to NK cell-mediated clearance of aneuploid senescent cells. Natural killer cells engage with and clear aneuploid senescent cells with complex karyotypes. Aneuploidy-induced senescence contributes to cellular cytotoxicity by natural killer cells. NF-κB signaling is central for natural killer cell-mediated clearance of aneuploid senescent cells. Introduction Aneuploidy is defined as a state in which the chromosome number is not a multiple of the haploid complement (Pfau & Amon, 2012). In all organisms analyzed to date, an unbalanced karyotype has detrimental effects (Pfau & Amon, 2012; Santaguida & Amon, 2015). In yeast, aneuploidy leads to proliferative defects and proteotoxic stress (Torres et al, 2010). The impact of aneuploidy on higher eukaryotes is even more severe. Most single autosomal gains and all autosomal losses cause embryonic lethality. Aneuploidies that do survive embryonic development cause significant anatomical and physiological abnormalities (Lindsley et al, 1972; Lorke, 1994; Hassold & Hunt, 2001; Roper & Reeves, 2006). The severe impact of aneuploidy on mammalian physiology is also reflected at the cellular level. Trisomic mouse embryonic fibroblasts (MEFs) and aneuploid human cells proliferate more slowly than their euploid counterparts and experience a variety of cellular stresses (Williams et al, 2008; Stingele et al, 2012; Santaguida et al, 2015; Pfau et al, 2016). Among these, aneuploidy-induced replication stress has been extensively studied. Upon chromosome mis-segregation, cells exhibit slow replication fork progression rate and increased replication fork stalling during the following S phase. Replication stress triggers genomic instability and drives the evolution of highly abnormal karyotypes (Sheltzer et al, 2012; Ohashi et al, 2015; Lamm et al, 2016; Passerini et al, 2016; Santaguida et al, 2017). Although aneuploidy is highly detrimental at both the cellular and organismal levels in untransformed cells, it is a hallmark of cancer, a disease characterized by uncontrolled cell proliferation (Gordon et al, 2012). About 90% of solid tumors and 75% of hematopoietic malignancies are characterized by whole chromosome gains and losses (Weaver & Cleveland, 2006). A high degree of aneuploidy is often associated with poor prognosis, immune evasion, and a reduced response to immunotherapy (Ben-David & Amon, 2020). Given the negative effects of aneuploidy on primary cells, it remains unclear how cells with severe genomic imbalances could gain tumorigenic potential. Furthermore, which aneuploidy-associated molecular features alter immune recognition during tumor evolution remains an active field of research. By inducing high levels of chromosome mis-segregation followed by continuous culturing, we previously generated cells with abnormal complex karyotypes that eventually cease to divide and enter a senescent-like state. We have named such cells arrested with complex karyotypes (ArCK) cells (Santaguida et al, 2017; Wang et al, 2018). Prior work indicated that ArCK cells upregulate gene expression signatures related to an immune response that render them susceptible to elimination by natural killer (NK) cells in vitro (Santaguida et al, 2017). However, the molecular and functional bases for this immune recognition of ArCK cells remained unclear. Several pathways could be involved in this process. Nuclear factor-kappaB (NF-κB) is induced under several stress conditions to elicit a pro-inflammatory response (Hayden & Ghosh, 2012; Liu et al, 2017). In the canonical NF-κB pathway, stress induction causes IκB kinase complex (IKK) to phosphorylate IκB, thereby marking it for proteolytic degradation (Perkins, 2007). As a result of this degradation, RelA-p50 translocates into the nucleus where it activates expression of pro-inflammatory genes. In the non-canonical NF-κB pathway, phosphorylation and cleavage of p100 trigger the nuclear translocation of the RelB-p52 complex to induce a pro-inflammatory response (Perkins, 2007). Recent studies further suggest that cytosolic nucleic acids lead to cGAS/STING activation in senescent cells, which induces an interferon response via JAK-STAT signaling pathway (Glück et al, 2017). Here, we investigate which innate immune pathways contribute to NK cell-mediated elimination of aneuploid cells and show that the NF-κB pathway elicits pro-inflammatory signals in ArCK cells. Inactivating both canonical and non-canonical NF-κB pathways in cells with an unbalanced karyotype prevents NK cell-mediated killing in vitro. Furthermore, we find that the NF-κB signature is upregulated in cancer cell lines possessing a higher degree of aneuploidy. However, this activation no longer enhanced NK cell-mediated killing of cancer cells, raising the possibility that aneuploidy-induced immunogenicity might be present only at the early stage of tumorigenesis and aneuploid cancer cells evolve mechanisms to evade immune clearance. Results An assay to assess elimination of ArCK cells by natural killer (NK) cells in vitro To address the molecular basis for immune recognition of ArCK cells, we established a co-culture system to monitor the interactions between NK cells and ArCK cells. In this setup, we utilized human, untransformed RPE1-hTERT cells in which chromosome segregation errors were forced by inhibiting the function of the spindle assembly checkpoint (SAC; Santaguida et al, 2010). To generate ArCK cells, we synchronized RPE1-hTERT cells at the G1/S boundary and released them into the cell cycle in the presence of the SAC kinase Mps1 inhibitor reversine (Fig 1A). We removed the drug once the cells had undergone one round of aberrant mitosis due to SAC inhibition. 72 h after inducing chromosome mis-segregation, we exposed cells to the spindle poison nocodazole for 12 h, which allowed us to remove dividing cells by mitotic shake-off (Santaguida et al, 2017; Wang et al, 2018). We repeated the mitotic shake-off 4 more times to ensure the removal of all cycling cells. Cells that remained on the tissue culture plate by the end of this procedure were highly enriched for the ArCK population (Fig 1A; Wang et al, 2018). Importantly, such cell cycle arrest was not due to the prolonged nocodazole treatment since euploid control cells were completely removed after two consecutive rounds of shake-offs (Santaguida et al, 2017). We co-cultured ArCK cells with an immortalized NK cell line activated by constitutive IL2 expression (NK92-MI; Tam et al, 1999) and monitored their interactions by live cell imaging (Fig 1B). Figure 1. ArCK cells are recognized by natural killer (NK) cells in vitro Schematic representation for the generation of ArCK cells. Time 0 is defined by the estimated onset of Mps1 inhibitor-induced chromosome mis-segregation. Representative images of euploid control or ArCK cells interacting with NK cells. The NK cell-mediated killing was measured at a 2.5:1 (NK cells: target cells) ratio and was recorded by live cell imaging for 36 h at a 30-min interval. TO-PRO-3 (1 μM) was added to the medium at the same time of NK cell addition to measure cell membrane integrity. Phase contrast (top) and TO-PRO-3 signal (bottom) from the same field were presented. Arrowheads indicate ArCK cell death. All images were acquired at the same exposure time and light intensity. Scale bar 20 μM. Measurement of NK cell-mediated killing of ArCK and euploid control cells (Ctrl) at a 2.5:1 (NK cells: target cells) ratio. 50 randomly chosen target cells were followed for 36 h by live cell imaging per condition per replicate. The cumulative cell death was calculated. n = 3 biological replicates; mean ± SEM. The statistical significance was determined using nonparametric Kolmogorov–Smirnov test (KS test) as described in the method section; P < 0.0001. Measurement of euploid control (Ctrl) and ArCK cell proliferation without NK cells. Live cell imaging of target cells without NK cells was performed using the same condition as described in the method section. For each condition, 50 cells were randomly chosen at the beginning of the movie as the initial population (indicated by the dashline, ninitial = 50). The cumulative cell number was recorded. Dot plot of individual data points and mean was presented; n = 2 biological replicates. NK cell-mediated cytotoxicity across various NK cell-to-target cell ratios. Either euploid control (Ctrl) or ArCK cells were co-cultured with NK cells at the indicated NK cell: target cell ratios. The cumulative killing of target cells was measured. n = 3 biological replicates; mean ± SEM. P < 0.0001 for all four NK cell: target cell ratios, KS test. Download figure Download PowerPoint To quantify the degree of NK cell killing of target cells, we first defined a killing event as a target cell that was (i) engaged by one or multiple NK cells, and (ii) lifted from the tissue culture plate (Fig 1B). We chose these criteria because they coincided with target cell membrane permeabilization as judged by the ability of the nucleic acid dye TO-PRO3 to enter a cell (Figs 1B and EV1A). We tracked individual target cells and recorded the time when each of them was killed during the 36-h live cell imaging. If a target cell divided during the time course, we followed only one of the resulting two cells for the remainder of the assay. We then calculated the cumulative cell death for each condition and generated killing curves at hourly resolution. We found that at a ratio of 2.5 NK cells to 1 target cell, ArCK cells were consistently killed twice as effectively as euploid control cells, during a 36-h co-culture experiment (Fig 1C). Click here to expand this figure. Figure EV1. Characterization of the NK cell killing assay Side-by-side comparison analyzing NK cell-mediated killing on euploid control or ArCK cells by phase contrast image (Phase) or TO-PRO3 signal. Cells were cultured as described in Fig 1A. Statistical analyses were performed as in Fig 1C. Individual points and mean were presented; n = 2 biological replicates. Ctrl-Phase vs. Ctrl-TOPRO3, P = 0.89, n.s.; ArCK-Phase vs. ArCK-TOPRO3, P = 0.89, n.s.; KS test. Measurement of NK cell-mediated killing of ArCK cells in two consecutive 18-h time lapse experiments. After the first 18 h of the analysis, the cell suspension was collected and co-cultured with a second set of target cells. NK cell-mediated killing was measured in the first (black and dark red curves) and the second (gray and light red curves) 18-h time lapse and plotted on the same graph. The killing assay was performed at a NK cell-to-target cell ratio of 2.5:1 (left panel) and 5:1 (right panel); n = 2 biological replicates. NK:Target = 2.5:1, Ctrl-1st movie vs. Ctrl-2nd movie, P = 1.00, n.s.; ArCK-1st movie vs. ArCK-2nd movie, P = 0.21, n.s.; NK:Target = 5:1, Ctrl-1st movie vs. Ctrl-2nd movie, P = 0.28, n.s.; ArCK-1st movie vs. ArCK-2nd movie, P = 0.97, n.s.; KS test. Cell proliferation measurements in the absence of NK cells. RPE1-hTERT (passage 4), human normal neonatal or adult human dermal fibroblasts (NHDF-Neo, passage 5 or NHDF-Ad, passage 5), and human embryonic lung fibroblast (IMR90, passage 3) were plated side by side in NK cell medium, and cell proliferation rate was recorded using live cell imaging as described in Fig 1D. The dashed line indicates the starting cell number (ninitial = 50). Dot plot of individual data points and mean is shown; n = 2 biological replicates. NK cell-mediated cytotoxicity across different cell types. The killing of RPE1-hTERT, human normal neonatal or adult human dermal fibroblasts (NHDF-Neo or NHDF-Ad), and human embryonic lung fibroblast (IMR90) were measured as described in Fig 1 using a NK cell-to-target cell ratio of 2.5–1; n = 2 biological replicates. Individual data and mean are shown. Human normal adult dermal fibroblasts (NHDF-Ad) were treated with either DMSO or the Mps1 inhibitor reversine (500 nM) for 24 h. Drugs were washed out, and NK cell-mediated killing was compared between DMSO-treated (NHDF-Ad Ctrl) and Mps1 inhibitor-treated (NHDF-Ad Mps1 inhibitor) cells as described in Fig 1C; n = 2 biological replicates. NHDF-Ad Ctrl vs. NHDF-Ad Mps1 inhibitor, P = 0.001; KS test. Download figure Download PowerPoint Arrested with complex karyotypes cells hardly divided during the 36-hour time lapse employed in our NK cell killing assay whereas euploid control cells continued to divide (Fig 1D). It was thus possible that the difference in NK cell-mediated cytotoxicity toward euploid and ArCK cells was affected by the fact that NK cells became limiting when co-cultured with euploid cells but not aneuploid cells. To test this possibility, we analyzed the effect of changing the NK cell-to-target cell ratio. We found that even at high NK cell-to-target cell ratio (5:1 and 10:1), ArCK cells were still more effectively killed than euploid controls (Fig 1E). We conclude that NK cells were not limited in our assay. We further note that when a cell divided during observation, we followed only one of the two cells after cell division, which corrected for the bias in target cell number. To address the possibility that NK cells became exhausted during the course of the co-culture experiment, we divided the 36-h assay into two time courses, where the same population of NK cells was consecutively co-cultured with target cells for 18 h each. NK cells were equally effective in killing the target cells (Fig EV1B) in this experimental setup, indicating that NK cell exhaustion did not occur within the time course of the analysis. We propose that the eventual plateauing of the killing curve as the assay proceeds is likely due to NK cells taking longer to find their targets. We next set out to test why euploid control cells are readily killed by NK cells in our in vitro assay. One possible explanation was that RPE1-hTERT cells express human telomerase reverse transcriptase (hTERT) and harbor a KRAS mutation (Nicolantonio et al, 2008), which could generate oncogenic transformation-associated NK cell stimulatory signals (Chiossone et al, 2018; Shimasaki et al, 2020). To test this possibility, we assessed NK cell-mediated cytotoxicity across three different types of early passage euploid primary fibroblasts derived from normal donors (human embryonic lung fibroblast, IMR90, and normal neonatal or adult human dermal fibroblasts, NHDF-Neo or NHDF-Ad). The analysis of these primary cells revealed large variations in both cell proliferation and NK cell-mediated killing (Fig EV1C and D). Adult human dermal fibroblasts were not readily eliminated by NK cells, whereas both neonatal human dermal fibroblasts and IMR90 cells were highly immunogenic. Thus, it appears that NK cell-mediated killing differs significantly between primary cultured cells. Importantly, we also observed a consistent twofold increase in killing on the Mps1 inhibitor reversine-induced aneuploid NHDF-Ad cells compared with their euploid controls (Fig EV1E), indicating that NK cell-mediated immune clearance of aneuploid cells is not a cell type-specific phenotype. We conclude that in the assay we developed here, highly aneuploid RPE1-hTERT cells are more effectively recognized and eliminated by NK92-MI cells in vitro than their euploid counterparts. Since we had developed robust protocols to generate the aneuploid cell population using RPE1-hTERT cells (Santaguida et al, 2017; Wang et al, 2018), we decided to focus on this cell line to investigate the effects of karyotype alterations on NK cell-mediated immune clearance. Prolonged cell cycle arrest associated with features of senescence elicits NK cell-mediated cytotoxicity Arrested with complex karyotypes cells are largely arrested in G1 and exhibit features of senescence (Santaguida et al, 2017; Wang et al, 2018). Permanent cell cycle arrest has been shown to elicit an immune response (Gorgoulis et al, 2019). To determine whether G1 arrest per se is sufficient to cause immune recognition, we assessed NK cell-mediated cytotoxicity toward G1-arrested cells induced by three different methods. We treated RPE1-hTERT cells for 7 days with (i) the topoisomerase II inhibitor, doxorubicin, to induce high levels of DNA damage (Pommier et al, 2010); (ii) the cyclin-dependent kinases CDK4/6 inhibitor, palbociclib; or (iii) the imidazoline analog, nutlin3, to disrupt the interaction between p53 and its ubiquitin ligase Mdm2, thereby stabilizing p53. All three conditions have been shown to cause features associated with cellular senescence (Sliwinska et al, 2009; Oliveira & Bernards, 2018; Wiley et al, 2018). DNA content analysis by flow cytometry and EdU incorporation showed that after 7 days, all 3 treatments caused the cells to arrest in G1 (Fig 2A–C). With the exception of nutlin3 treatment, these G1 arrests were irreversible: Most cells did not resume proliferation following drug washout as judged by cell proliferation assays (Fig 2D). Co-culturing these G1-arrested cells with NK cells revealed that irrespective of the means by which the arrest was induced, NK cells exhibited a twofold increase in killing on these G1-arrested cells compared with the untreated proliferating control cells (Fig 2E). Figure 2. Prolonged cell cycle arrest associated with features of senescence elicits NK cell-mediated cytotoxicity DNA content analysis of various G1 arrests. RPE1-hTERT cells were treated for 7 days with doxorubicin (Doxo; 100 ng/ml), palbociclib (Palbo; 5 μM), or nutlin3 (Nutlin; 10 μM). Total number of cells analyzed is indicated by n in each condition. Results were comparable between 2 biological replicates. Schematics of EdU incorporation assay. Drugs were applied to RPE1-hTERT cells 12 h after initial cell plating. 6 days later (144 h), cells were switched to drug medium containing 5-ethynyl-2'-deoxyuridine (EdU; 10 μM) for 24 h before fixation and analysis. The percentage of EdU-positive cells after doxorubicin (Doxo), palbociclib (Palbo), or nutlin3 (Nutlin) treatment. EdU incorporation was performed as described in (B). At least 100 cells were analyzed per condition per replicate. Individual data points and mean are shown; n = 2 biological replicates. Cell proliferation (in the absence of NK cells) after 7 days of doxorubicin (Doxo), palbociclib (Palbo), or nutlin3 (Nutlin) treatment. The drugs were washed out after 7 days, and the cells were re-plated for live cell imaging. Cell proliferation was measured as described in Fig 1D. The dashed line indicates the starting cell number (ninitial = 50). Dot plot of individual data points and mean are shown; n = 2 biological replicates. NK cell-mediated killing for doxorubicin (Doxo), palbociclib (Palbo), and nutlin3 (Nutlin) treated samples (NK cell: target cell = 2.5:1). n = 3 biological replicates; mean ± SEM. Ctrl vs. Doxo, P < 0.0001; Ctrl vs. Palbo, P < 0.0001; Ctrl vs. Nutlin, P < 0.0001; KS test. The percentage of EdU-positive cells after 7 days of torin1 treatment was determined as described in (B) and (C). Individual data points and mean are shown; n = 2 biological replicates. NK cell-mediated cytotoxicity toward torin1-treated cells. Torin1-treated (Torin) cells were generated as described in (F), and the NK cell killing assay was performed as described in Fig 1. n = 3 biological replicates; mean ± SEM. Ctrl vs. Torin, P = 0.79, not significant (n.s.); KS test. Download figure Download PowerPoint Inactivation of the TORC1 pathway also causes cell cycle arrest (Sousa-Victor et al, 2015), but cells enter a quiescent state instead of senescence (Sousa-Victor et al, 2015; Kucheryavenko et al, 2019). RPE1-hTERT cells were mostly arrested in cell cycle upon treatment with 1 μM of the mTOR kinase inhibitor torin1 after 7 days (Fig 2F). Yet NK cell recognition and killing were not enhanced in cells treated with torin1 (Fig 2G). We conclude that G1 arrest in target cells contributes to NK cell engagement, but only when accompanied by features of senescence. Mechanisms triggering senescence contribute to NK cell recognition in ArCK cells The observation that senescence triggered by multiple mechanisms led to NK cell recognition begged the question of what features in aneuploid cells elicit NK cell-mediated clearance. To address this, we compared a collection of cellular markers contributing to senescence in ArCK cells to those of cells treated with doxorubicin, palbociclib, or nutlin3 for 7 days. First, we assessed DNA damage levels across all conditions by measuring nuclear γ-H2AX foci (Figs 3A and EV2A). DNA damage can increase the expression of NK cell-activating receptor (NKG2D) ligands such as MICA and ULBP2, thereby triggering NK cell-mediated clearance (Raulet & Guerra, 2009). In untreated proliferating control cells, more than 80% of the cells harbored fewer than 10 γ-H2AX foci per nucleus. As expected, doxorubicin caused significantly higher levels of DNA damage in the euploid cells, such that approximately 90% of the cells displayed more than 20 foci and ˜50% of this population had 50 foci or more (Fig 3A, panel 2). In contrast, the DNA damage levels in palbociclib- and nutlin3-treated cells were comparable to untreated control cells (Fig 3A, panels 3 and 4). About one third of the ArCK cells harbored more than 10 foci (Fig 3A, panel 5), likely caused by replication stress and/or endogenous reactive oxygen species (ROS) associated with aneuploidy (Li et al, 2010; Passerini et al, 2016; Santaguida et al, 2017). Figure 3. Mechanisms triggering senescence contribute to NK cell recognition in ArCK cells γ-H2AX foci were analyzed in ArCK- and G1-arrested cells (generated as described in Fig 2). At least 50 cells were analyzed per condition per replicate. n = 2 biological replicates; individual values and mean are shown. The distribution of the foci number in each treated condition was compared to that of control using Kolmogorov–Smirnov test. Ctrl vs. Doxo, P < 0.0001; Ctrl vs. Palbo, P = 0.07, n.s.; Ctrl vs. Nutlin, P = 0.11, n.s.; Ctrl vs. ArCK, P = 0.0007. p53 and p21 levels were determined by Western blot analysis. Vinculin was used as loading control. Results were comparable between 2 biological replicates. The degree of senescence was measured by senescence-associated β-galactosidase (β-gal) activity. The graph shows the percentage of β-gal-positive cells. At least 100 cells were analyzed per condition per replicate. n = 3 biological replicates; mean ± SEM. Ctrl vs. Doxo, ***P = 0.0006; Ctrl vs. Palbo, *P = 0.025; Ctrl vs. Nutlin, *P = 0.016; Ctrl vs. Torin, P = 0.806, n.s.; Ctrl vs. ArCK, ***P = 0.0001; unpaired t-test. Secreted cytokine and interferon levels were determined in cell supernatants. Media were collected after 36 h of incubation with cells grown as described in Figs 1 and 2. Cytokine and interferon levels were shown as fold change normalized to euploid control cells. Individual values and mean are shown; n = 2 biological replicates. NK cell medium was incubated with either euploid control or ArCK cells for 12 h. At the time of NK cell addition, media were switched between ArCK and euploid control cells (Ctrl). NK cell killing was measured as described in Fig 1C. For reference, NK cell killing of ArCK and euploid control cells (Ctrl) without medium switch were performed side by side and plotted on the graph. Black, euploid control cells without medium switch; red, ArCK cells without medium switch; blue, euploid control cells in ArCK cell condition medium; green, ArCK cells in euploid control cell condition medium. n = 3 biological replicates; mean ± SEM. Ctrl vs. ArCK, P < 0.0001; Ctrl vs. Ctrl in ArCK med, P < 0.0001; Ctrl vs. ArCK in Ctrl med, P < 0.0001; ArCK vs. ArCK in Ctrl med, P = 0.0002; KS test. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Characterization of aneuploid- and G1-arrested cells Representative images of γ-H2AX staining in the indicated samples. γ-H2AX is in red and DNA in blue. Scale bar 10 μm. Representative image of senescence-associated β-galactosidase staining in the indicated samples. Scale bar 100 μm. Analysis of all cytokines secreted by the indicated cells. Cytokine levels are shown as fold change in euploid control cells; n = 2 biological replicates. Individual values and mean are plotted. Gene set enrichment analysis (GSEA) for doxorubicin-treated (Doxo), palbociclib-treated (Palbo), nutlin3-treated (Nutlin), torin1-treated (Torin) and ArCK cells relative to euploid proliferating control cells. Only the top 10 ranked hallmarks are presented in Doxo, Palbo, and ArCK conditions. The normalized enrichment score (NES) is plotted. The numbers on the NES score bar indicate the corresponding p-values for each hallmark (FDR q value ≤ 0.05). Download figure Download PowerPoint Induction of the DNA damage response genes p53 and p21 agreed with the presence of γ-H2AX foci with the obvious exception of nutlin3-treated cells (as nutlin3 inhibits Mdm2 to stabilize p53 but does not cause endogeno
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