Chromosomal instability drives metastasis through a cytosolic DNA response methods
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CIN is a driver of metastasis
We injected MDA-MB-231 cells into the left cardiac ventricles of athymic mice to enable systemic dissemination while tracking metastatic colonization using a bioluminescence reporter. Altering chromosome missegregation rates had a dramatic effect on colonization, whereby mice harboring CIN-high cells rapidly succumbed to metastatic disease with a median survival of 70 days. Conversely, mice injected with CIN-low cells exhibited lower metastatic burden and a median survival of 207 days. Many metastases from CIN-low cells waxed-and-waned and, at times, spontaneously resolved, whereas metastases from CIN-high cells involved multiple organs and rapidly progressed leading to death. Similar results were obtained after injection of lung adenocarcinoma H2030 cells ( and ). Overexpression of the spindle assembly checkpoint protein, Mad2 in MCAK-expressing cells partially rescued chromosome mis...
CIN generates cytosolic DNA
To determine whether missegregated chromosomes provide a source of cytosolic DNA, we employed an inducible Y chromosome-specific missegregation system established in chromosomally stable DLD-1 colorectal cancer cells. Whole-chromosome FISH probes targeting the Y chromosome or an independent autosome (chr15) revealed selective incorporation of the Y chromosome into micronuclei two days following chromosome missegregation induced by doxycycline and auxin (Dox/IAA) treatment. Strikingly, Y chromosome-specific fragments were found dispersed within the cytosol 2-3 days after Dox/IAA addition, whereas the control autosome remained confined to the nucleus ( ), demonstrating that cytosolic DNA is generated from chromosomes undergoing high rates of missegregation
Single-cell karyotyping
Cultures were treated with colcemid at a final concentration of 0.1µg ml -1. Following 45 min incubation at 37°C, the cultures were trypsinized, resuspended in pre-warmed 0.075M KCl, incubated for an additional 10 minutes at 37°C and fixed in methanol-acetic acid (3:1). The fixed cell suspension was then dropped onto slides, stained in 0.08µg/ml DAPI in 2xSSC for 5 minutes and mounted in antifade solution (Vectashield, Vector Labs). Metaphase spreads were captured using the Nikon Eclipse E800 epifluorescence microscope equipped with GenASI Cytogenetic suite (Applied Spectral Imaging, Carlsbad). For each sample a minimum of 20 inverted DAPI-stained metaphases were fully karyotyped and analyzed according to the International System of Human Cytogenetic Nomenclature (ISCN) 2013.
RhoA and Rac1 pull-down assays
The activity of RhoA and Rac1 was determined according to the bead-based pull-down assay kits (Cytoskeleton Inc., RhoA: BK036S, Rac1: BK035S). Cells were lysed on the tissue culture dish and rapidly snap frozen until further processing. cGAMP was added for 18 hours prior to lysis. In addition to His-tagged RhoA and Rac1, the positive and negative controls were total cell lysates supplanted with non-hydrolysable GTP or GDP, respectively.
Cell culture
Cell lines were purchased from the American Type Culture Collection (ATCC). Tumor (MDA-MB-231, 4T1, HEK293, and H2030), cells were cultured in DMEM or RPMI (4T1) supplemented with 10% FBS and 2mM of L-Glutamine in the presence of penicillin (50 Uml -1 ) and streptomycin (50 µgml -1 ). All cells tested negative for mycoplasma. Cell confluence was measured using IncuCyte live-cell analysis system (Essen Bioscience).
Immunofluorescence microscopy
Cell fixation and antibody staining were performed as previously described. Briefly, cells were fixed with ice-cold (-30 C) methanol for 15 minutes - when staining for centromeres, centrosomes, cGAS, Vimentin, β-actin, IRF3, or a-tubulin - or 4% paraformaldehyde - when staining for RelB, p65, STING, ssDNA, dsDNA, CoxIV, or β-catenin. Subsequently, cells were permeabilized using 1% triton for 4 minutes. See for antibody information. For selective plasma membrane permeabilization used for cytosolic dsDNA and ssDNA staining, cells were treated with 0.02% saponin for 5 minutes after fixation. For single-stranded (Thermo Fisher FEREN0321) and double stranded (Life Technologies - EN0771)-specific nuclease treatment, cells were also permeabilized with 0.02% saponin for 2 minutes and treated with either nucleases for 10 minutes before fixation using 4%...
Y chromosome missegregation and fluorescent in situ hybridization (FISH)
Flp-In T-REx DLD-1 cells were engineered to express the TIR1 auxin-dependent plant E3 ligase, an auxin-inducible degron (AID)-tagged CENP-A modified at the endogenous allele (CENP-A AID/- ), and a doxycycline-inducible CENP-A C-H3 rescue gene integrated into the Flp-In locus as previously described. 4.0 × 10 4 cells were seeded into 4-well chamber slides and treated with doxycycline (DOX, Sigma) and indole-3-acetic acid (IAA, Sigma) for up to 3 days to induce Y chromosome missegregation and micronuclei. Slides were washed in PBS, fixed in 3:1 methanol:acetic acid for 15 minutes at room temperature, and dehydrated with 80% ethanol. Chromosome paint FISH probes targeting chromosome Y and 15 (MetaSystems) were mixed at equal ratios, applied to cells, sealed with a coverslip, and co-denatured at 75°C for 2 minutes followed by overnight hybridization at 37°C in...
Knockdown and overexpression constructs
Luciferase expression was achieved using pLVX plasmid (expressing tdTomato) and cells stably expressing luciferase were selected using hygromycin and sorted for tdTomato expression. Kinesin-13 expression was achieved using plasmid (pEGFP) transfection or lentiviral (pLenti-GIII-CMV-GFP-2A-Puro) expression where cells were selected using G418 (0.5mgml -1 ) or puromycin (5µgml -1 ), respectively. Dnase2 overexpression was achieved using a pLenti-GIII-CMV-RFP-2A-Puro plasmid with puromycin used for selection. Plasmids containing kinesin-13 or Lamin B2 (pQCXIB-mCherry-lmnb2) constructs were kindly offered by the Compton and Hetzer Laboratories, respectively. Blasticidin was used to select for lmnb2 expressing cells at 10µgml -1. All other plasmids were purchased from Applied Biological Materials Inc. ( www.abmgood.com ). Stable knockdown of STING, NFKB2, RelB,...
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Evidence quotes (8)
We injected MDA-MB-231 cells into the left cardiac ventricles of athymic mice to enable systemic dissemination while tracking metastatic colonization using a bioluminescence reporter. Altering chromosome missegregation rates had a dramatic effect on colonization, whereby mice harboring CIN-high cells rapidly succumbed to metastatic disease with a median survival of 70 days. Conversely, mice injected with CIN-low cells exhibited lower metastatic burden and a median survival of 207 days. Many metastases from CIN-low cells waxed-and-waned and, at times, spontaneously resolved, whereas metastases from CIN-high cells involved multiple organs and rapidly progressed leading to death. Similar results were obtained after injection of lung adenocarcinoma H2030 cells ( and ). Overexpression of the spindle assembly checkpoint protein, Mad2 in MCAK-expressing cells partially rescued chromosome missegregation, and correspondingly augmented metastasis ( and ).
To determine whether missegregated chromosomes provide a source of cytosolic DNA, we employed an inducible Y chromosome-specific missegregation system established in chromosomally stable DLD-1 colorectal cancer cells. Whole-chromosome FISH probes targeting the Y chromosome or an independent autosome (chr15) revealed selective incorporation of the Y chromosome into micronuclei two days following chromosome missegregation induced by doxycycline and auxin (Dox/IAA) treatment. Strikingly, Y chromosome-specific fragments were found dispersed within the cytosol 2-3 days after Dox/IAA addition, whereas the control autosome remained confined to the nucleus ( ), demonstrating that cytosolic DNA is generated from chromosomes undergoing high rates of missegregation
Cultures were treated with colcemid at a final concentration of 0.1µg ml -1. Following 45 min incubation at 37°C, the cultures were trypsinized, resuspended in pre-warmed 0.075M KCl, incubated for an additional 10 minutes at 37°C and fixed in methanol-acetic acid (3:1). The fixed cell suspension was then dropped onto slides, stained in 0.08µg/ml DAPI in 2xSSC for 5 minutes and mounted in antifade solution (Vectashield, Vector Labs). Metaphase spreads were captured using the Nikon Eclipse E800 epifluorescence microscope equipped with GenASI Cytogenetic suite (Applied Spectral Imaging, Carlsbad). For each sample a minimum of 20 inverted DAPI-stained metaphases were fully karyotyped and analyzed according to the International System of Human Cytogenetic Nomenclature (ISCN) 2013.
The activity of RhoA and Rac1 was determined according to the bead-based pull-down assay kits (Cytoskeleton Inc., RhoA: BK036S, Rac1: BK035S). Cells were lysed on the tissue culture dish and rapidly snap frozen until further processing. cGAMP was added for 18 hours prior to lysis. In addition to His-tagged RhoA and Rac1, the positive and negative controls were total cell lysates supplanted with non-hydrolysable GTP or GDP, respectively.
Cell lines were purchased from the American Type Culture Collection (ATCC). Tumor (MDA-MB-231, 4T1, HEK293, and H2030), cells were cultured in DMEM or RPMI (4T1) supplemented with 10% FBS and 2mM of L-Glutamine in the presence of penicillin (50 Uml -1 ) and streptomycin (50 µgml -1 ). All cells tested negative for mycoplasma. Cell confluence was measured using IncuCyte live-cell analysis system (Essen Bioscience).
Cell fixation and antibody staining were performed as previously described. Briefly, cells were fixed with ice-cold (-30 C) methanol for 15 minutes - when staining for centromeres, centrosomes, cGAS, Vimentin, β-actin, IRF3, or a-tubulin - or 4% paraformaldehyde - when staining for RelB, p65, STING, ssDNA, dsDNA, CoxIV, or β-catenin. Subsequently, cells were permeabilized using 1% triton for 4 minutes. See for antibody information. For selective plasma membrane permeabilization used for cytosolic dsDNA and ssDNA staining, cells were treated with 0.02% saponin for 5 minutes after fixation. For single-stranded (Thermo Fisher FEREN0321) and double stranded (Life Technologies - EN0771)-specific nuclease treatment, cells were also permeabilized with 0.02% saponin for 2 minutes and treated with either nucleases for 10 minutes before fixation using 4% paraformaldehyde. TBS-BSA was used as a blocking agent during antibody staining. DAPI was added together with secondary antibodies. Cells were mounted with Prolong Diamond Antifade Mountant (Life Technologies - P36961 ). cGAMP (Invivogen - tlrl-nacga23) was transfected into cells at a c...
Flp-In T-REx DLD-1 cells were engineered to express the TIR1 auxin-dependent plant E3 ligase, an auxin-inducible degron (AID)-tagged CENP-A modified at the endogenous allele (CENP-A AID/- ), and a doxycycline-inducible CENP-A C-H3 rescue gene integrated into the Flp-In locus as previously described. 4.0 × 10 4 cells were seeded into 4-well chamber slides and treated with doxycycline (DOX, Sigma) and indole-3-acetic acid (IAA, Sigma) for up to 3 days to induce Y chromosome missegregation and micronuclei. Slides were washed in PBS, fixed in 3:1 methanol:acetic acid for 15 minutes at room temperature, and dehydrated with 80% ethanol. Chromosome paint FISH probes targeting chromosome Y and 15 (MetaSystems) were mixed at equal ratios, applied to cells, sealed with a coverslip, and co-denatured at 75°C for 2 minutes followed by overnight hybridization at 37°C in a humidified chamber. Slides were washed in 0.4x saline-sodium citrate (SSC) buffer for 2 minutes at 72°C, followed by a 30 second wash in 2x SCC, 0.05% Tween-20 buffer at room temperature. Cells were counterstained with DAPI and captured on a DeltaVision Elite (GE Healthcare) microscope...
Luciferase expression was achieved using pLVX plasmid (expressing tdTomato) and cells stably expressing luciferase were selected using hygromycin and sorted for tdTomato expression. Kinesin-13 expression was achieved using plasmid (pEGFP) transfection or lentiviral (pLenti-GIII-CMV-GFP-2A-Puro) expression where cells were selected using G418 (0.5mgml -1 ) or puromycin (5µgml -1 ), respectively. Dnase2 overexpression was achieved using a pLenti-GIII-CMV-RFP-2A-Puro plasmid with puromycin used for selection. Plasmids containing kinesin-13 or Lamin B2 (pQCXIB-mCherry-lmnb2) constructs were kindly offered by the Compton and Hetzer Laboratories, respectively. Blasticidin was used to select for lmnb2 expressing cells at 10µgml -1. All other plasmids were purchased from Applied Biological Materials Inc. ( www.abmgood.com ). Stable knockdown of STING, NFKB2, RelB, and cGAS were achieved using shRNAs in pRRL (SGEP or SGEN) plasmids and were obtained from the MSKCC RNA Interference Core. 2-4 distinct shRNA hairpins were screened per target. Targeted shRNA sequences are listed in. To visualize primary nuclear rupture, cells were stably modified with retr...
Machine-readable layer
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