Chromosomal instability drives metastasis through a cytosolic DNA response methods
Aim. Evidence-backed execution summary for Chromosomal instability drives metastasis through a cytosolic DNA response methods from Chromosomal instability drives metastasis through a cytosolic DNA response.
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human
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Metastasis from cytosolic DNA response
reagent used in the protocol.
- Use
- Cytosolic DNA, however, can activate the noncanonical NF-κB pathway in a STING-dependent and TBK1-independent manner. We found evidence for noncanonical NF-κB activation in CIN-high cells as revealed by lower levels of the precursor protein, p100, a trend toward higher ratios of p52 and phosphorylated p10...
Single-cell karyotyping
reagent used in the protocol.
- Use
- 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 suspensio...
RhoA and Rac1 pull-down assays
reagent used in the protocol.
- Use
- 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...
Cell culture
reagent used in the protocol.
- Use
- 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 test...
Immunofluorescence microscopy
reagent used in the protocol.
- Use
- 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...
Immunoblotting
reagent used in the protocol.
- Use
- Cells were pelleted and lysed using RIPA buffer. Protein concentration was determined using BCA protein assay and 20-30µg of total protein were loaded in each lane. Proteins were separated by gradient SDS-PAGE and transferred to PVDF or nitrocellulose membranes. Full blots are shown in. See for antibody...
Y chromosome missegregation and fluorescent in situ hybridization (FISH)
reagent used in the protocol.
- Use
- 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...
Cell migration in microfluidic devices
reagent used in the protocol.
- Use
- Microfluidic migration devices with precisely defined constrictions were prepared as described previously,. Devices were coated with 50 µg/mL of type-I rat tail collagen (BD Biosciences) in 0.02N acetic acid overnight at 4°C. Approximately 80,000 cells were seeded (in DMEM supplemented with 10% FBS and 1...
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 succumb...
- Use
- 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 succumb...
CIN enriches for mesenchymal traits
Bulk RNA sequencing (RNA-seq) of CIN-low and CIN-high MDA-MB-231 cells revealed 1,584 differentially expressed genes. Principle component analysis (PCA) and unsupervised clustering accurately separated samples according to their CIN status. Metastasis-related and epithelial-to-mesenchymal (EMT) gene sets were relati...
- Use
- Bulk RNA sequencing (RNA-seq) of CIN-low and CIN-high MDA-MB-231 cells revealed 1,584 differentially expressed genes. Principle component analysis (PCA) and unsupervised clustering accurately separated samples according to their CIN status. Metastasis-related and epithelial-to-mesenchymal (EMT) gene sets were relati...
CIN enriches for mesenchymal traits
We then performed single-cell RNA sequencing (scRNA-seq) on two CIN-low (Kif2b and MCAK) and one CIN-high (dnMCAK) MDA-MB-231 cell lines comprising a total of 6,821 cells. Clustering of single cells using EMT genes successfully classified most cells based on their CIN-status and revealed a fraction of cells (primari...
- Use
- We then performed single-cell RNA sequencing (scRNA-seq) on two CIN-low (Kif2b and MCAK) and one CIN-high (dnMCAK) MDA-MB-231 cell lines comprising a total of 6,821 cells. Clustering of single cells using EMT genes successfully classified most cells based on their CIN-status and revealed a fraction of cells (primari...
CIN generates cytosolic DNA
Instead, CIN-high cells and those derived from metastases exhibited a higher preponderance of micronuclei compared with CIN-low or primary tumor-derived cells, respectively (, ). To examine if rupture-prone micronuclei correlated with increased cytosolic DNA, we stained cells using two different anti-dsDNA antibodi...
- Use
- Instead, CIN-high cells and those derived from metastases exhibited a higher preponderance of micronuclei compared with CIN-low or primary tumor-derived cells, respectively (, ). To examine if rupture-prone micronuclei correlated with increased cytosolic DNA, we stained cells using two different anti-dsDNA antibodi...
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) reveale...
- Use
- 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) reveale...
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 suspensio...
- Use
- 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 suspensio...
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 test...
- Use
- 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 test...
Immunoblotting
Cells were pelleted and lysed using RIPA buffer. Protein concentration was determined using BCA protein assay and 20-30µg of total protein were loaded in each lane. Proteins were separated by gradient SDS-PAGE and transferred to PVDF or nitrocellulose membranes. Full blots are shown in. See for antibody...
- Use
- Cells were pelleted and lysed using RIPA buffer. Protein concentration was determined using BCA protein assay and 20-30µg of total protein were loaded in each lane. Proteins were separated by gradient SDS-PAGE and transferred to PVDF or nitrocellulose membranes. Full blots are shown in. See for antibody...
RNA sequencing and analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Bulk RNA was extracted from cells using the QIAShredder (Qiagen - 79654) and the RNA extraction kit (Qiagen - 74106) and sequenced using HiSeq2500 or HiSeq4000 (Illumina Inc.). The quality of the raw FASTQ files were checked with FastQC ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). For...
Code availability
Software used for acquisition, scoring, statistics, or reporting.
- Use
- All custom code, statistical analysis, and visualizations were performed in Python or R, and used Nextflow to manage some of the computational pipelines. Code for the RNA sequencing analysis is available online at: https://github.com/murphycj/manuscripts/tree/master/BakhoumEtAl2017. The live-cell tracking MATLAB 2...
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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,...
Measurement outputs
What raw and processed outputs should exist?
All available breast adenocarcinoma cases in the Mitelman database were analyzed. Primary literature was reviewed to determine the source of the sample (primary tumor or metasta...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Next, karyotype analysis of primary breast tumors and metastases archived in the Mitelman Database of chromosomal translocations revealed a predilection for near-diploid (2n) ka...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Finally, histologic analysis of primary tumors from patients with locally advanced head and neck squamous cell carcinoma revealed a significant association between anaphase chro...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
We then assessed chromosome missegregation in the injected cells as well as cells derived from primary tumors and metastatic colonies ( ). We performed this analysis using MDA-M...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
Finally, histologic analysis of primary tumors from patients with locally advanced head and neck squamous cell carcinoma revealed a significant association between anaphase chromosome missegregation and the incidence of lymph node metastasis (, ).
from paperScoring or quantification
Quantify the primary readouts for this experiment: All available breast adenocarcinoma cases in the Mitelman database were analyzed. Primary literature was reviewed to determine the source of the sample (primary tumor or metasta...; Next, karyotype analysis of primary breast tumors and metastases archived in the Mitelman Database of chromosomal translocations revealed a predilection for near-diploid (2n) ka...; Finally, histologic analysis of primary tumors from patients with locally advanced head and neck squamous cell carcinoma revealed a significant association between anaphase chro...; We then assessed chromosome missegregation in the injected cells as well as cells derived from primary tumors and metastatic colonies ( ). We performed this analysis using MDA-M....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Finally, histologic analysis of primary tumors from patients with locally advanced head and neck squamous cell carcinoma revealed a significant association between anaphase chro...; Interestingly, there was no evidence for robust activation of downstream canonical NF-κB or type I interferon signaling in CIN-high cells as evidenced by the lack of a sign...; Bulk RNA was extracted from cells using the QIAShredder (Qiagen - 79654) and the RNA extraction kit (Qiagen - 74106) and sequenced using HiSeq2500 or HiSeq4000 (Illu...; All custom code, statistical analysis, and visualizations were performed in Python or R, and used Nextflow to manage some of the computational pipelines. Code for the RNA seque...
from paperReporting output
Report representative outputs alongside summary comparisons for All available breast adenocarcinoma cases in the Mitelman database were analyzed. Primary literature was reviewed to determine the source of the sample (primary tumor or metasta..., Next, karyotype analysis of primary breast tumors and metastases archived in the Mitelman Database of chromosomal translocations revealed a predilection for near-diploid (2n) ka..., Finally, histologic analysis of primary tumors from patients with locally advanced head and neck squamous cell carcinoma revealed a significant association between anaphase chro..., We then assessed chromosome missegregation in the injected cells as well as cells derived from primary tumors and metastatic colonies ( ). We performed this analysis using MDA-M....
inferred from protocolStructured statistical methods
Finally, histologic analysis of primary tumors from patients with locally advanced head and neck squamous cell carcinoma revealed a significant association between anaphase chro...; Interestingly, there was no evidence for robust activation of downstream canonical NF-κB or type I interferon signaling in CIN-high cells as evidenced by the lack of a sign...; Bulk RNA was extracted from cells using the QIAShredder (Qiagen - 79654) and the RNA extraction kit (Qiagen - 74106) and sequenced using HiSeq2500 or HiSeq4000 (Illu...; All custom code, statistical analysis, and visualizations were performed in Python or R, and used Nextflow to manage some of the computational pipelines. Code for the RNA seque...
source structuredSource and audit
What supports the facts on this page?
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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"@type": "HowTo",
"name": "Chromosomal instability drives metastasis through a cytosolic DNA response methods",
"description": "Evidence-backed execution summary for Chromosomal instability drives metastasis through a cytosolic DNA response methods from Chromosomal instability drives metastasis through a cytosolic DNA response.",
"totalTime": "PT39060M",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "CIN is a driver of metastasis",
"text": "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..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "CIN generates cytosolic DNA",
"text": "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"
},
{
"@type": "HowToStep",
"position": 3,
"name": "Single-cell karyotyping",
"text": "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."
},
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"@type": "HowToStep",
"position": 4,
"name": "RhoA and Rac1 pull-down assays",
"text": "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."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Cell culture",
"text": "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)."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Immunofluorescence microscopy",
"text": "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%..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Y chromosome missegregation and fluorescent in situ hybridization (FISH)",
"text": "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..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Knockdown and overexpression constructs",
"text": "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,..."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "CIN is a driver of metastasis"
},
{
"@type": "HowToTool",
"name": "CIN enriches for mesenchymal traits"
},
{
"@type": "HowToTool",
"name": "CIN enriches for mesenchymal traits"
},
{
"@type": "HowToTool",
"name": "CIN generates cytosolic DNA"
},
{
"@type": "HowToTool",
"name": "CIN generates cytosolic DNA"
},
{
"@type": "HowToTool",
"name": "Single-cell karyotyping"
},
{
"@type": "HowToTool",
"name": "Cell culture"
},
{
"@type": "HowToTool",
"name": "Immunoblotting"
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],
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{
"@type": "HowToSupply",
"name": "Metastasis from cytosolic DNA response"
},
{
"@type": "HowToSupply",
"name": "Single-cell karyotyping"
},
{
"@type": "HowToSupply",
"name": "RhoA and Rac1 pull-down assays"
},
{
"@type": "HowToSupply",
"name": "Cell culture"
},
{
"@type": "HowToSupply",
"name": "Immunofluorescence microscopy"
},
{
"@type": "HowToSupply",
"name": "Immunoblotting"
},
{
"@type": "HowToSupply",
"name": "Y chromosome missegregation and fluorescent in situ hybridization (FISH)"
},
{
"@type": "HowToSupply",
"name": "Cell migration in microfluidic devices"
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"headline": "Chromosomal instability drives metastasis through a cytosolic DNA response",
"datePublished": "2018",
"author": [
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"@type": "Person",
"name": "Samuel F. Bakhoum"
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{
"@type": "Person",
"name": "Bryan Ngo"
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"@type": "Person",
"name": "Ashley M. Laughney"
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{
"@type": "Person",
"name": "Julie-Ann Cavallo"
},
{
"@type": "Person",
"name": "Charles J. Murphy"
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{
"@type": "Person",
"name": "Peter Ly"
},
{
"@type": "Person",
"name": "Pragya Shah"
},
{
"@type": "Person",
"name": "Roshan K Sriram"
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{
"@type": "Person",
"name": "Thomas B. K. Watkins"
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{
"@type": "Person",
"name": "Neil K. Taunk"
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"@type": "Person",
"name": "Mercedes Duran"
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"name": "Chantal Pauli"
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"@type": "Person",
"name": "Kalyani Chadalavada"
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"@type": "Person",
"name": "Vinagolu K. Rajasekhar"
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"@type": "Person",
"name": "Giulio Genovese"
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{
"@type": "Person",
"name": "Subramanian Venkatesan"
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"@type": "Person",
"name": "Nicolai J. Birkbak"
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"@type": "Person",
"name": "Nicholas McGranahan"
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{
"@type": "Person",
"name": "Mark Lundquist"
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{
"@type": "Person",
"name": "Quincey LaPlant"
},
{
"@type": "Person",
"name": "John H. Healey"
},
{
"@type": "Person",
"name": "Olivier Elemento"
},
{
"@type": "Person",
"name": "Christine H Chung"
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{
"@type": "Person",
"name": "Nancy Y. Lee"
},
{
"@type": "Person",
"name": "Marcin Imielenski"
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"@type": "Person",
"name": "Gouri Nanjangud"
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"@type": "Person",
"name": "Dana Pe'er"
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