Wong C.C.L.,Scripps Research Institute |
Cociorva D.,Scripps Research Institute |
Miller C.A.,Agilent Technologies |
Schmidt A.,University of Basel |
And 3 more authors.
Journal of Proteome Research | Year: 2013
Pyrococcus furiosus (Pfu) is an excellent organism to generate reference samples for proteomics laboratories because of its moderately sized genome and very little sequence duplication within the genome. We demonstrated a stable and consistent method to prepare proteins in bulk that eliminates growth and preparation as a source of uncertainty in the standard. We performed several proteomic studies in different laboratories using each laboratory's specific workflow as well as separate and integrated data analysis. This study demonstrated that a Pfu whole cell lysate provides suitable protein sample complexity to not only validate proteomic methods, work flows, and benchmark new instruments but also to facilitate comparison of experimental data generated over time and across instruments or laboratories. © 2013 American Chemical Society.
Soyka M.B.,University of Zurich |
Treis A.,University of Zurich |
Eiwegger T.,University of Zurich |
Eiwegger T.,Medical University of Vienna |
And 5 more authors.
Allergy: European Journal of Allergy and Clinical Immunology | Year: 2012
Background Activated T lymphocytes and their interaction with resident tissue cells, particularly epithelium, play important roles in inflammatory processes in chronic rhinosinusitis (CRS). IL-32 is a recently described cytokine, which is expressed in a variety of tissue cells and involved in the pathogenesis of several chronic inflammatory diseases. Methods Human sinus epithelial cells were isolated from biopsies and stimulated with different cytokines, which play a role in the pathogenesis of CRS. IL-32 mRNA expression was analyzed using real-time-PCR, IL-32 protein was determined by Western blot and flow cytometry as well as immunofluorescent staining in primary sinus epithelial cells and nasal biopsies from patients with CRS and healthy controls. Results IL-32 mRNA was upregulated by TNF-α and IFN-γ in primary sinus epithelial cells, whereas IL-1 β, IL-4, IL-13, and IL-17 did not influence IL-32 expression. IL-32 mRNA expression was significantly higher in human primary sinonasal epithelial cells (HSECs) cocultured with Th1 cells compared with HSECs cocultured with Th0 or Th2 cells. IL-32 mRNA expression was significantly higher in biopsies from sinus epithelial tissue of CRS patients with nasal polyps compared with healthy subjects (P = 0.01). IL-32 was detected in biopsies from patients with CRS, whereas it was scarcely present in control tissues. Conclusion The induction of IL-32 by TNF-α, IFN-γ and Th1 cells as well as its increased expression in sinus tissues from CRS patients with nasal polyps demonstrated a potential role for IL-32 in the pathogenesis of CRS. © 2012 John Wiley & Sons A/S.
All common chemicals were from Sigma. Pyrrolidinedithiocarbamic acid was from Santa Cruz Biotechnology. Exo-FBS exosome-depleted FBS was purchased from System Biosciences (SBI). PTEN (9188), pAkt(T308) (9275), pAkt(S473) (4060), Pan Akt (4691), and Bim (2933) antibodies were from Cell Signaling. CD9 (ab92726), Rab27a (ab55667), AMPK (ab3759), CCL2 (ab9899), MAP2 (ab11267), and pP70S6K (ab60948) antibodies were from Abcam. Tsg101 (14497-1-AP) and Rab27b (13412-1-AP) antibodies were from Proteintech. CD81 (104901) antibody was from BioLegend. E2F1 (NB600-210) and CCR2 (NBP1-48338) antibodies were from Novus. GFAP (Z0334) antibody was from DAKO. IBA1 antibody was from WAKO. Cre (969050) antibody was from Novagen. NF-κB p65 (SC-109) and CD63 (SC-15363) antibodies were from Santa Cruz. DMA (sc-202459) and CCR2 antagonist (sc-202525) were from Santa Cruz. MK2206 (S1078) was from Selleckchem. PDTC (P8765) was from Sigma-Aldrich. Human breast cancer cell lines (MDA-MB-231, HCC1954, BT474 and MDA-MB-435) and mouse cell lines (B16BL6 mouse melanoma and 4T1 mouse breast cancer) were purchased from ATCC and verified by the MD Anderson Cancer Center (MDACC) Cell Line Characterization Core Facility. All cell lines have been tested for mycoplasma contamination. Primary glia was isolated as described13. In brief, after homogenization of dissected brain from postnatal day (P)0–P2 neonatal mouse pups, all cells were seeded on poly-d-lysine coated flasks. After 7 days, flasks with primary culture were placed on an orbital shaker and shaken at 230 r.p.m. for 3 h. Warm DMEM 10:10:1 (10% of fetal bovine serum, 10% of horse serum, 1% penicillin/streptomycin) was added and flasks were shaken again at 260 r.p.m. overnight. After shaking, fresh trypsin was added into the flask and leftover cells were plated with warm DMEM 5:5:1 (5% of fetal bovine serum, 5% of horse serum, 1% penicillin/streptomycin) to establish primary astrocyte culture. More than 90% of isolated primary glial cells were GFAP+ astrocytes. Primary CAFs were isolated by digesting the mammary tumours from MMTV-neu transgenic mouse. 231-xenograft CAFs were isolated by digesting the mammary tumours from MDA-MB-231 xenograft. For the mixed co-culture experiments, tumour cells were mixed with an equal number of freshly isolated primary glia, CAFs or NIH3T3 fibroblast cells in six-well plate (1:3 ratio). Co-cultures were maintained for 2–5 days before magnetic-bead-based separation. For the trans-well co-culture experiments, tumour cells were seeded in the bottom well and freshly isolated primary glia, CAFs or NIH3T3 cells were seeded on the upper insert (1:3 ratio). Co-cultures were maintained for 2–5 days for the further experiments. Lentiviral-based packaging vectors (Addgene), pLKO.1 PTEN-targeting shRNAs and all siRNAs (Sigma), Human Cytokine Antibody Array 3 (Ray biotech), and lentiviral-based vector pTRIPZ-PTEN and pTRIPZ-CCL2 shRNAs (MDACC shRNA and ORFome Core, from Open Biosystems) were purchased. The human PTEN-targeting shRNA sequences in the lentiviral constructs were: 5′-CCGGAGGCGCTATGTGTATTATTATCTCGAGATAATAATACACATAGCGCCTTTTTT-3′ (targeting coding sequence); 5′-CCGGCCACAAATGAAGGGATATAAACTCGAGTTTATATCCCTTCATTTGTGGTTTTT-3′ (targeting 3′-UTR). The human PTEN-targeting siRNA sequences used were: 5′-GGUGUAAUGAUAUGUGCAU-3′ and 5′-GUUAAAGAAUCAUCUGGAU-3′. The human CCL2-targeting siRNA sequences used were: 5′-CAGCAAGUGUCCCAAAGAA-3′ and 5′-CCGAAGACUUGAACACUCA-3′. The mouse Rab27a-targeting siRNA sequences used were: 5′-CGAUUGAGAUGCUCCUGGA-3′ and 5′-GUCAUUUAGGGAUCCAAGA-3′. Mouse pLKO shRNA (shRab27a: TRCN0000381753; shRab27b: TRCN0000100429) were purchased from Sigma. For lentiviral production, lentiviral expression vector was co-transfected with the third-generation lentivirus packing vectors into 293T cells using Lipo293 DNA in vitro Transfection Reagent (SignaGen). Then, 48–72 h after transfection, cancer cell lines were stably infected with viral particles. Transient transfection with siRNA was performed using pepMute siRNA transfection reagent (SignaGen). For in vivo intracranial virus injection, lentivirus was collected from 15 cm plates 48 h after transfection of packaging vectors. After passing a 0.45 μm filter, all viruses were centrifuged at 25,000 r.p.m (111,000g) for 90 min at 4 °C. Viral pellet was suspended in PBS (~200-fold concentrated). The final virus titre (~1 × 109 UT ml−1) was confirmed by limiting dilution. Cell isolation was performed based on the magnetic bead-based cell sorting protocol according to manufacturer’s recommendation (Miltenyi Biotec Inc.). After preparation of a single-cell suspension, tumour cells (HCC1954 or BT474) were stained with primary EpCAM-FITC antibody (130-098-113) (50 μl per 107 total cells) and incubated for 30 min in the dark at 4 °C. After washing, the cell pellet was re-suspended and anti-FITC microbeads (50 μl per 107 total cells) were added before loading onto the magnetic column of a MACS separator. The column was washed twice and removed from the separator. The magnetically captured cells were flushed out immediately by firmly applying the plunger. The isolated and labelled cells were analysed on a Gallios flow cytometer (Beckman Coulter). For EpCAM-negative MDA-MB-231 tumour cells, FACS sorting (ARIAII, Becton Dickinson) was used to isolate green fluorescent protein (GFP)+ tumour cells from glia or CAFs. Isolation of primary glia was achieved by homogenization of dissected brain from P0–P2 mouse pups. After 7 days, trypsin was added and cells were collected. After centrifugation and re-suspension of cell pellet to a single-cell suspension, cells were incubated with CD11b+ microbeads (Miltenyl Biotec) (50 μl per 107 total cells) for 30 min at 4 °C. The cells were washed with buffer and CD11b+ cells were isolated by MACS Column. CD11b+ cells were analysed by flow cytometry and immunofluorescence staining. Western blotting was done as previously described. In brief, cells were lysed in lysis buffer (20 mM Tris, pH 7.0, 1% Triton X-100, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA and protease inhibitor cocktail). Proteins were separated by SDS–PAGE and transferred onto a nitrocellulose membrane. After membranes were blocked with 5% milk for 30 min, they were probed with various primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies for 1 h at room temperature, and visualized with enhanced chemiluminescence reagent (Thermo Scientific). In brief, total RNA was isolated using miRNeasy Mini Kit (Qiagen) and then reverse transcribed using reverse transcriptase kits (iScript cDNA synthesis Kit, Bio-rad). SYBR-based qRT–PCR was performed using pre-designed primers (Life Technologies). miRNA assay was conducted using Taqman miRNA assay kit (Life Technologies). For quantification of gene expression, real-time PCR was conducted using Kapa Probe Fast Universal qPCR, and SYBR Fast Universal qPCR Master Mix (Kapa Biosystems) on a StepOnePlus real-time PCR system (Applied Biosystems). The relative expression of mRNAs was quantified by 2−ΔΔCt with logarithm transformation. Primers used in qRT–PCR analyses are: mouse Ccl2: forward, 5′-GTTGGCTCAGCCAGATGCA-3′; reverse: 5′-AGCCTACTCATTGGGATCATCTTG-3′. Mouse Actb: forward: 5′-AGTGTGACGTTGACATCCGT3′; reverse: 5′-TGCTAGGAGCCAGAGCAGTA-3′. Mouse Pten: forward: 5′-AACTTGCAATCCTCAGTTTG-3′; reverse: 5′-CTACTTTGATATCACCACACAC-3′. Mouse Ccr2 primer: Cat: 4351372 ID: Mm04207877_m1 (Life technologies) Synthetic miRNAs were purchased from Sigma and labelled with Cy3 by Silencer siRNA labelling kit (Life Technologies). In brief, miRNAs were incubated with labelling reagent for 1 h at 37 °C in the dark, and then labelled miRNAs were precipitated by ethanol. Labelled miRNAs (100 pmoles) were transfected into astrocytes or CAFs in a 10-cm plate. After 48 h, astrocytes and CAFs containing Cy3-miRNAs were co-cultured with tumour cells (at 5:1 ratio). Genomic DNA was isolated by PreLink genomic DNA mini Kit (Invitrogen), bisulfite conversion was performed by EpiTect Bisulphite Kit and followed by EpiTect methylation-specific PCR (Qiagen). Primers for PTEN CpG island are 5′-TGTAAAACGACGGCCAGTTTGTTATTATTTTTAGGGTTGGGAA-3′ and 5′-CAGGAAACAGCTATGACCCTAAACCTACTTCTCCTCAACAACC-3′. Luciferase reporter assays were done as previously described27. The wild-type PTEN promoter driven pGL3-luciferase reporter was a gift from A. Yung. The pGL3-PTEN reporter and a control Renilla luciferase vector were co-transfected into tumour cells by Lipofectamine 2000 (Life Technologies). After 48 h, tumour cells were co-cultured with astrocytes or CAFs. Another 48 h later, luciferase activities were measured by Dual-Luciferase Report Assay Kit (Promega) on Luminometer 20/20 (Turner Biosystems). The PTEN 3′-UTRs with various miRNA binding-site mutations were generated by standard PCR-mediated mutagenesis method and inserted downstream of luciferase reporter gene in pGL3 vector. The activities of the luciferase reporter with the wild-type and mutated PTEN 3′-UTRs were assayed as described above. Astrocytes or CAFs were cultured for 48–72 h and exosomes were collected from their culture media after sequential ultracentrifugation as described previously. In brief, cells were collected, centrifuged at 300g for 10 min, and the supernatants were collected for centrifugation at 2,000g for 10 min, 10,000g for 30 min. The pellet was washed once with PBS and purified by centrifugation at 100,000g for 70 min. The final pellet containing exosomes was re-suspended in PBS and used for (1) transmission electron microscopy by fixing exosomes with 2% glutaraldehyde in 0.1 M phosophate buffer, pH 7.4; (2) measure of total exosome protein content using BCA Protein Assay normalized by equal number of primary astrocytes and CAF cells; (3) western blotting of exosome marker protein CD63, CD81 and Tsg101; and (4) qRT–PCR by extracting miRNAs with miRNeasy Mini Kit (Qiagen). Fixed samples were placed on 100-mesh carbon-coated, formvar-coated nickel grids treated with poly-l-lysine for about 30 min. After washing the samples on several drops of PBS, samples were incubated on drops of buffered 1% gluteraldehyde for 5 min, and then washed several times on drops of distilled water. Afterwards, samples were negatively stained on drops of millipore-filtered aqueous 4% uranyl acetate for 5 min. Stain was blotted dry from the grids with filter paper and samples were allowed to dry. Samples were then examined in a JEM 1010 transmission electron microscope (JEOL) at an accelerating voltage of 80 Kv. Digital images were obtained using the AMT Imaging System (Advanced Microscopy Techniques Corp.). For exosome detection, 100 μl exosomes isolated from 10-ml conditioned media of astrocytes or CAFs were incubated with 10 μl of aldehyde/sulfate latex beads (4 μm diameter, Life Technologies) for 15 min at 4 °C. After 15 min, PBS was added to make sample volume up to 400 μl, which was incubated overnight at 4 °C under gentle agitation. Exosome-coated beads were washed twice in FACS washing buffer (1% BSA and 0.1% NaN in PBS), and re-suspended in 400 μl FACS washing buffer, stained with 4 μg of phycoerythrin (PE)-conjugated anti-mouse CD63 antibody (BioLegend) or mouse IgG (Santa Cruz Biotechnology) for 3 h at 4 °C under gentle agitation and analysed on a FACS Canto II flow cytometer. Samples were gated on bead singlets based on FCS and SSC characteristics (4 μm diameter). For Annexin V apoptosis assay, after 24 h doxorubicin (2 μM) treatment, the cells were collected, labelled by APC-Annexin V antibody (Biolegend) and analysed on a FACS Canto II flow cytometer. CD11b+ and BV2 cells were stained with CCR2 antibody (Novus) at 4 °C overnight; they were then washed and stained with Alexa Fluor 488 anti-rabbit IgG (Life Technologies) at room temperature for 1 h. The cells were then analysed on a FACS Canto II flow cytometer. All animal experiments and terminal endpoints were carried out in accordance with approved protocols from the Institutional Animal Care and Use Committee of the MDACC. Animal numbers of each group were calculated by power analysis and animals are grouped randomly for each experiment. No blinding of experiment groups was conducted. MFP tumours were established by injection of 5 × 106 tumour cells in 100 μl of PBS:Matrigel mixture (1:1 ratio) orthotopically into the MFP of 8-week-old Swiss nude mice as done previously28. Brain metastasis tumours were established by ICA injection of tumour cells (250,000 cells in 0.1 ml HBSS for MDA-MB-231, HCC1954, MDA-MB-435, 4T1 and B16BL6, and 500,000 cells in 0.1 ml HBSS for BT474.m1 into the right common carotid artery as done previously29). Mice (6–8 weeks) were randomly grouped into designated groups. Female mice are used for breast cancer experiments, both female and male are used for melanoma experiments. Since the brain metastasis model does not result in visible tumour burdens in living animal, the endpoints of in vivo metastasis experiments are based on the presence of clinical signs of brain metastasis, including but not limited to, primary central nervous system disturbances, weight loss, and behavioural abnormalities. Animals are culled after showing the above signs or 1–2 weeks after surgery based on specific experimental designs. Brain metastasis lesions are enumerated as experimental readout. Brain metastases were counted as micromets and macromets. The definition of micromets and macromets are based on a comprehensive mouse and human comparison study previously published30. In brief, ten haematoxylin and eosin (H&E)-stained serial sagittal sections (300 μm per section) through the left hemisphere of the brain were analysed for the presence of metastatic lesions. We counted micrometastases (that is, those ≤ 50 μm in diameter) to a maximum of 300 micrometastases per section, and every large metastasis (that is, those > 50 μm in diameter) in each section. Brain-seeking cells from overt metastases and whole brains were dissected and disaggregated in DMEM/F-12 medium using Tenbroeck homogenizer briefly. Dissociated cell mixtures were plated on tissue culture dish. Two weeks later, tumours cells recovered from brain tissue were collected and expanded as brain-seeking sublines (Br.1). For the astrocyte miR-19 knockout mouse model, Mirc1tm1.1Tyj/J mice (Jax lab) (6–8 weeks) were intracranially injected with Ad5-GFAP-Cre virus (Iowa University, Gene Transfer Vector Core) 2 μl (MOI ~108 U μl−1) per point, total four points at the right hemisphere (n = 9). Control group (n = 7) was injected with the same dose Ad5-RSV-βGLuc (Ad-βGLuc) at the right hemisphere. All intracranial injections were performed by an implantable guide-screw system. One week after virus injection, mice were intracarotidly injected with 2 × 105 B16BL6 tumour cells. After two weeks, whole brains were dissected and fixed in 4% formaldehyde, and embedded in paraffin. Tumour formation, histological phenotypes of H&E-stained sections, and IHC staining were evaluated. Only parenchymal lesions, which are in close proximity of adenovirus injection, were included in our evaluation. Tumour size was calculated as (longest diameter) × (shortest diameter)2/2. For the intracranial tumour model, Mirc1tm1.1Tyj/J mice (Jax lab) (6–8 weeks) were intracranially injected as described above. Seven mice were used in the experiment. One week later, these mice were intracranially injected with 2.5 × 105 B16BL6 tumour cells at both sides where adenoviruses were injected. After another week, whole brains were dissected and fixed in 4% formaldehyde, and embedded in paraffin. Tumour formation and phenotype were analysed as above. For the Rab27a/b knockdown mouse model, seven C57BL6 mice (Jax lab) (6–8 weeks) were intracranially injected with concentrated lentivirus containing shRab27a and shRab27b (ratio 1:2) 2 μl per point, total three points at the right hemisphere; concentrated control lentivirus containing pLKO.1 scramble were injected at the left hemisphere. All intracranial injections were performed by an implantable guide-screw system. One week later, mice were intracranially injected with 5 × 104 B16BL6 tumour cells at both sides where they had been infected. After one week, whole brains were dissected and fixed in 4% formaldehyde, and embedded in paraffin. Tumour formation, histological phenotypes of H&E-stained sections, IHC staining were evaluated. When performing metastases size quantification, only parenchymal lesions that were in close proximity to the adenovirus injection sites were included in the analyses. Tumour size was calculated as (longest diameter) × (shortest diameter)2/2. For exosome rescue experiments, eight C57BL6 mice (Jax lab) (6–8 weeks) were intracranially injected with concentrated lentivirus containing shRab27a and shRab27b (ratio 1:2) 2 μl per point, total 3 points at both hemispheres. One week later, these mice were intracranially injected with 5 × 104 B16BL6 tumour cells with 10 μg exosome isolated from astrocyte media at the right sides where they had been injected with lentivirus; 5 × 104 B16BL6 tumour cells with vehicle were injected at the left sides where lentivirus had been injected. After another week, whole brains were dissected and fixed in 4% formaldehyde, and embedded in paraffin. Tumour formation and phenotype were analysed as above. For in vivo extravasation assay, equal numbers of cells labelled with GFP-control shRNA and RFP-PTEN shRNA (Open Biosystems) were mixed and ICA injected. After cardiac perfusion, brains were collected and sectioned through coronal plan on a vibrotome (Leica) into 50-μm slices. Fluorescent cells were then counted. For inducible PTEN expression in vivo, mice were given doxycycline (10 μg kg−1) every other day. To quantify brain metastasis incidence and tumour size, brains were excised for imaging and histological examination at the end of experiments. Ten serial sagittal sections every 300 μm throughout the brain were analysed by at least two pathologists who were blinded to animal groups in all above analyses. Reverse-phase protein array of PTEN-overexpressing cells was performed in the MDACC Functional Proteomics core facility. In brief, cellular proteins were denatured by 1% SDS, serial diluted and spotted on nitrocellulose-coated slides. Each slide was probed with a validated primary antibody plus a biotin-conjugated secondary antibody. The signal obtained was amplified using a Dako Cytomation-catalysed system and visualized by DAB colorimetric reaction. The slides were analysed using customized Microvigene software (VigeneTech Inc.). Each dilution curve was fitted with a logistic model (‘Super curve fitting’ developed at the MDACC) and normalized by median polish. Differential intensity of normalized log values of each antibody between RFP (control) and PTEN-overexpressed cells were compared in GenePattern (http://genepattern.broadinstitute.org). Antibodies with differential expression (P < 0.2) were selected for clustering and heat-map analysis. The data clustering was performed using GenePattern. Two studies in separate cohorts were conducted. The first one was a retrospective evaluation of PTEN in two cohorts. (1) Archived formalin-fixed and paraffin-embedded brain metastasis specimens (n = 131) from patients with a history of breast cancer who presented with metastasis to the brain parenchyma and had surgery at the MDACC (Supplementary Information). Tissues were collected under a protocol (LAB 02-486) approved by the Institutional Review Board (IRB) at the MDACC. (2) Archived unpaired primary breast cancer formalin-fixed and paraffin-embedded specimens (n = 139) collected under an IRB protocol (LAB 02-312) at the MDACC (Supplementary information). Formal consent was obtained from all patients. The second study was a retrospective evaluation of PTEN, CCL2 and IBA1 in the matched primary breast tumours and brain metastatic samples from 35 patients, of which there are 12 HER2-positive, 14 triple-negative and nine oestrogen-receptor-positive tumours according to clinical diagnostic criteria (Supplementary Information). Formalin-fixed, paraffin-embedded primary breast and metastatic brain tumour samples were obtained from the Pathology Department, University of Queensland Centre for Clinical Research. Tissues were collected with approval by human research ethics committees at the Royal Brisbane and Women’s Hospital (2005/022) and the University of Queensland (2005000785). For tissue microarray construction, tumour-rich regions (guided by histological review) from each case were sampled using 1-mm cores. All the archival paraffin-embedded tumour samples were coded with no patient identifiers. Standard IHC staining was performed as described previously28. In brief, after de-paraffinization and rehydration, 4 μm sections were subjected to heat-induced epitope retrieval (0.01 M citrate for PTEN). Slides were then incubated with various primary antibodies at 4 °C overnight, after blocking with 1% goat serum. Slides underwent colour development with DAB and haematoxylin counterstaining. Ten visual fields from different areas of each tumour were evaluated by two pathologists independently (blinded to experiment groups). Positive IBA1 and Ki-67 staining in mouse tumours were calculated as the percentage of positive cells per field (%) and normalized by the total cancer cell number in each field. TUNEL staining was counted as the average number of positive cells per field (10 random fields). We excluded necrotic areas in the tumours from evaluation. Immunofluorescence was performed following the standard protocol recommended by Cell Signaling. In brief, after washing with PBS twice, cells were fixed with 4% formaldehyde. Samples were blocked with 5% normal goat serum in PBS for 1 h before incubation with a primary antibody cocktail overnight at 4 °C, washed, then incubated with secondary antibodies before examination using confocal microscope. Pathologists were blinded to the group allocation during the experiment and when assessing the outcome. Publicly available GEO data sets GSE14020, GSE19184, GSE2603, GSE2034 and GSE12276 were used for bioinformatics analysis. The top 2 × 104 verified probes were subjected to analysis. Differentially expressed genes between metastases from brain and other sites (primary or other metastatic organ sites) were analysed by SAM analysis in R statistical software. The 54 commonly downregulated genes in brain metastases from GSE14020 and GSE19184 were depicted as a heat-map by Java Treeview. For staining of patient samples, we calculated the correlation by Fisher’s exact test. For survival analysis of GSE2603, the patient samples were mathematically separated into PTEN-low and -normal groups based on K-means (K = 2). Kaplan–Meier survival curves were generated by survival package in R. Multiple group IHC scores were compared by Chi-square test and Mantelhaen test in R. All quantitative experiments have been repeated using at least three independent biological repeats and are presented as mean ± s.e.m. or mean ± s.d.. Quantitative data were analysed either by one-way analysis of variance (ANOVA) (multiple groups) or t-test (two groups). P < 0.05 (two-sided) was considered statistically significant.
No statistical methods were used to determine sample size. Foxp3CNS3-fl-gfp mice were generated using ES cell line CY2.4 (C56BL/6) as previously described11. Cd4Cre, LckCre, UbcCre-ERT2 and Rosa26-stop-YFP (R26Y) mice were obtained from the Jackson Laboratories. DO11.10 TCRβ transgenic and Aire-knockout mice were provided by P. Marrack, and D. Mathis and C. Benoist, respectively. Heterozygous females carrying Foxp3ΔCNS3-gfp and Foxp3gfp were crossed with B6 males to generate hemizygous Foxp3ΔCNS3-gfp and wild-type Foxp3gfp littermates. Foxp3DTR, Foxp3-null, Rag1−/−, CD45.1+ Foxp3gfp and Tcrb−/− Tcrd−/− mice were maintained in our animal facility. To study the genetic interactions between CNS3 and Aire, heterozygous females of Foxp3ΔCNS3-gfp/gfp were first crossed with AireKO/WT, and F harbouring AireKO/WT and Foxp3ΔCNS3-gfp or Foxp3gfp were then intercrossed to generate AireKO/KO or AireKO/WT mice carrying Foxp3ΔCNS3-gfp or Foxp3gfp. To examine TCR diversity with restricted repertoire, Foxp3ΔCNS3-gfp/gfp heterozygous females were crossed to the DO11.10 TCRβ transgenic and Tcra−/+ males. F males of Foxp3ΔCNS3-gfp or Foxp3gfp mice carrying the DO11.10 TCRβ transgene and Tcra−/+ were used for T-cell isolation and TCR sequencing. To induce deletion of CNS3 in vivo, tamoxifen solution (40 mg ml−1 in olive oil) was administered by gavage to UbcCre-ERT2 Foxp3CNS3-fl-gfp R26Y mice more than 3 days before lymphocyte isolation. All mice were maintained in the MSKCC animal facility under SPF conditions, and the experiments were approved by the Institutional Review Board (IACUC 08-10-023). The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. Statistical tests were performed with Prism (GraphPad), Excel (Microsoft) or R statistical environment. Box-and-whisker plots show minimum, maximum, first and third quartiles and median. For in vitro T cell differentiation, naive CD4+ T cells (GFP−CD25−CD44loCD62Lhi) or mature CD4+CD8− SP (TCRβhiGFP−CD25−CD62LhiCD69lo) T cells were sorted from Foxp3gfp, Foxp3ΔCNS3-gfp or Foxp3CNS3-fl-gfp mice after the enrichment of CD4+ T cells or depletion of CD8+ T cells using Dynabeads FlowComp Mouse CD4 or CD8 kits, respectively (Life Technologies), and then cultured with lethally irradiated (20 Gy) antigen-presenting cells (splenocytes depleted of T cells with Dynabeads FlowComp Mouse CD90.2 kit, Life Technologies) or on plates pre-coated with CD3 and CD28 antibodies in RPMI1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 2 × 10−5 M 2-mercaptoethanol, 100 U ml−1 penicillin, 100 mg ml−1 streptomycin, 500 U ml−1 IL-2 and 1 ng ml−1 TGFβ. Sodium butyrate (water solution) or iBET solution in dimethylsulfoxide (a gift from R. Prinjha) was added to the culture to block histone deacetylase or bromodomain-containing proteins, respectively. T cells were sorted on the basis of Foxp3gfp reporter expression. Assessment of the stability of Foxp3 expression in vitro was performed as previously described31. Briefly, T cells were activated in culture in the presence of CD3 and CD28 antibody-coated beads (Life Technologies) with the following recombinant pro-inflammatory cytokines: IL-2 (250 U ml−1), IL-4 (20 ng ml−1), IL-6 (10 ng ml−1), IFNγ (100 ng ml−1) and IL-12 (20 ng ml−1). To assess T -cell suppressor capacity in vivo we conducted adoptive T-cell transfers into T-cell-deficient recipients as previously described31. Briefly, ~2.5 × 106–3.0 × 106 Foxp3−CD4+ and/or CD8+ T cells isolated from Foxp3-null or Foxp3ΔCNS3-gfp AireKO/KO mice were transferred to congenic and gender-matched Tcrb−/− Tcrd−/− recipients alone or at a 10:1 ratio with sorted T cells from Foxp3gfp or Foxp3ΔCNS3-gfp littermates. Similar numbers of effector T cells and T cells were used for in vivo evaluation of T suppressor function after acute ablation of CNS3. Recipient mice were monitored for body weight change regularly and lymphocytes were analysed by flow cytometry at least 4 weeks after the transfer. Around 8–10 weeks after bone marrow reconstitution of CD45.1+ Foxp3gfp and CD45.2+ Foxp3ΔCNS3-gfp in Tcrb−/− Tcrd−/− recipients, CD45.1+ and CD45.2+ T cells (CD4+GFP+) were sorted, mixed at a 1:1 ratio and co-transferred into Tcrb−/− Tcrd−/− male mice with tenfold naive CD4+ T cells (CD25−CD44loCD62Lhi) isolated from wild-type CD45.2+ B6 males. To block TCR stimulation by pMHC-II complexes, 0.5 mg of I-Ab-specific monoclonal antibody Y3P (IgG2a) or control IgG2a (Bio X Cell) was injected intravenously every other day before and after T-cell transfer32, 33. The lymphocyte subsets were analysed by flow cytometry 9 days later. Tissue lymphocytes were prepared as previously described31. The following fluorophore-conjugated antibodies were used for cell-surface staining: CD4 (RM4-5, eBioscience), CD8 (5H10, Life Technologies), CD25 (PC61.5, eBioscience), CD3e (145-2C11, eBioscience), CD44 (IM7, eBioscience), CD62L (MEL-14, eBioscience), CTLA4 (UC10-4B9, eBioscience), TCRβ (BioLegend), CD45.1 (A20, eBioscience) and CD45.2 (104, eBioscience). Antibodies used for intracellular staining were: Foxp3 (FJK-16 s, eBioscience), Ki-67 (B56, BD Biosciences), IL-17 (eBio17B7, eBioscience), IFNγ (XMG1.2, eBioscience) and IL-2 (JES6-5H4, eBioscience). To stain endogenous Nur77, cells were incubated with rabbit-anti-Nur77 antibody (Cell Signaling) after fixation and permeabilization with a Foxp3/transcription-factor-staining buffer set (eBioscience), followed by phycoerythrin-conjugated donkey anti-rabbit antibody (eBioscience). For the flow cytometric analysis of cytokine production, lymphocytes were first stimulated in vitro with 10 mg ml−1 of CD3 antibody in the presence of monensin (BD Biosciences) at 37 °C for 5 h, then stained with antibodies against indicated cell-surface markers followed by staining of cytokines with an intracellular staining kit (BD Biosciences). All flow cytometric analyses were performed using live-cell gate defined as negative by staining with the LIVE/DEAD Fixable Dead Cell Stain Kit (Life Technologies). Flow cytometric analysis was performed with FlowJo (Treestar). The Cre coding region was subcloned into MigR1-IRES-Thy1.1 vector (A. Levine, unpublished data) to generate MigR1-Cre-IRES-Thy1.1. Retroviral packaging with regular Phoenix-ECO cells and transduction of T cells were performed following standard protocols5. Analysis of autoantibody reactivity against a panel of 95 autoantigens was conducted using the autoantigen microarrays developed by University of Texas Southwestern Medical Center34. Briefly, serum samples pretreated with DNase-I and diluted at 1:50 were incubated with the auto-antigen arrays. After a second incubation with Cy3-conjugated anti-mouse IgG, the arrays were scanned with a Genepix 4200A scanner (Molecular Device). The fluorescent signals for individual autoantigens were extracted from the resulting images with Genepix Pro 6.0 (Molecular Devices), followed sequentially by subtraction of local background, average of duplicates, normalization with total IgG, and subtraction of a negative PBS control. Cell isolation and RNA extraction. Lymphocytes were collected from the peripheral lymphoid organs or thymi of 6–8-week-old male Foxp3gfp or Foxp3ΔCNS3-gfpTcra−/+ littermates bearing the DO11.10 TCRβ transgene, and were enriched for CD4+ T cells (Dynabeads FlowComp Mouse CD4 kit, Life Technologies) or depleted of CD8+ T cells (Dynabeads FlowComp Mouse CD8 kit, Life Technologies), respectively, and T cells (CD4+GFP+), mature Foxp3−CD4 SP thymocytes (CD4+CD8−GFP−CD25−CD62LhiCD69lo), peripheral naive (CD4+GFP−CD25−CD44loCD62Lhi) and effector (CD4+GFP−CD44hiCD62Llo) CD4+ T cells were isolated using a FACSAria II sorter (BD) gated on TCR-Vβ8hi. Extraction of total RNA from TRIzol-preserved cell lysates was performed according to the manufacturer’s instructions (Life Technologies). mRNA was purified from total RNA with Dynabeads mRNA DIRECT Kit (Life Technologies) and used for reverse transcription. cDNA synthesis. To maximize the priming efficiency of reverse transcription, a mixture of oligo(dT) and eight DNA oligonucleotides corresponding to the mouse TCRα constant region was used. The oligonucleotides used in this study were synthesized by Integrated DNA Technologies, Inc. To label the 5' end of TCRα mRNA, a DNA–RNA hybrid oligonucleotide with 12 random nucleotides serving as barcodes to tag individual mRNA molecules was synthesized as previously reported35. Hybrid oligonucleotide: AAGCAGTGGTATCAACGCAGAGUNNNNUNNNNUNNNNUCTTrGrGrGrGrG (r, ribonucleotide). cDNA was synthesized in SMARTScribe reverse-transcription buffer (Clontech) with 1.0 μM each of reverse transcription oligonucleotide, 0.5 mM of each dNTP, 5.0 mM of dithiothreitol (DTT), 2.0 U μl−1 recombinant RNase inhibitor (Takara), 1 μM hybrid oligonucleotide, 1 M betaine (Affymetrix), 6 mM MgCl and 5 U μl−1 SMARTScribe reverse transcriptase by incubating at 42 °C for 90 min, followed by 10 cycles of incubation at 50 °C for 2 min, 42 °C for 2 min, and then one step of incubation at 70 °C for 15 min. After removal of hybrid oligonucleotide with Uracil-DNA Glycosylase (New England BioLabs), cDNA was purified with Agencourt AMPure XP beads (Beckman Coulter) according the manufacturer’s manual. Sequencing library preparation. Purified cDNA was used as templates for a four-step PCR amplification, in which sequencing adaptors and sample indices were introduced. The first PCR reaction was performed with purified cDNA, 0.2 μM universal primer (5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3', Clontech), 0.2 μM TRAC reverse primer 8 (5'-TTTTGTCAGTGATGAACGTT-3'), 0.2 mM each dNTP, 1.5 mM MgCl and 0.02 U μl−1 KOD Hot Start DNA Polymerase (EMD Millipore). PCR parameters were as follows: initial denature at 95 °C for 2 min; 10 cycles of 95 °C for 20 s, 70 °C for 10 s with an increment of −1 °C per cycle, and 70 °C for 30 s; 15 cycles of 95 °C for 20 s, 60 °C for 10 s and 70 °C for 30 s; and final cycle at 70 °C for 3.5 min. Amplified DNA was purified with Agencourt AMPure XP magnetic beads for the subsequent reaction. The second PCR reaction used the same reactants except that the reverse primer was replaced by a nested primer (5'-CAATTGCACCCTTACCACGACAGTCTGGTACACAGCAGGTTCTGGGTTCTGGA-3'). Cycling parameters were: 95 °C for 2 min; 6 cycles of 95 °C for 20 s, 60 °C 10 s and 70 °C 30 s; and a final cycle at 70 °C for 3.5 min. DNA from individual samples was extracted with Agencourt AMPure XP magnetic beads and used for the third round of amplification with 5RACE TCR forward primer (5'-AATGATACGGCGACCACCGAGATCTACACCTAATACGACTCACTATAGGGC-3') and indexed reverse primer (5'-CAAGCAGAAGACGGCATACGAGATXXXXXXAGTCAGTCAGCCCAATTGCACCCTTACCACGA-3', XXXXXX for 6-nucleotide barcode). The cycling parameters were: 95 °C for 2 min; 6 cycles at 95 °C for 20 s, 55 °C for 10 s and 70 °C for 30 s; and a final cycle at 70 °C for 3.5 min. The PCR products were purified with Agencourt AMPure XP magnetic beads and used for the fourth PCR amplification with primers P1 (5'-AATGATACGGCGACCACCGAG-3') and P2 (5'-CAAGCAGAAGACGGCATACGA-3'), and the following cycling parameters: 95 °C for 2 min; 5 cycles at 95 °C for 20 s, 57 °C for 10 s and 70 °C for 30 s; and a final cycle at 70 °C for 3.5 min. The final PCR products were separated by agarose gel electrophoresis and a single band around 600 base-pairs was cut and extracted with Gel Extraction and PCR Clean-Up kits (Takara). High-throughput sequencing. Samples were quantified with Kapa Library Quantification kits (Kapa Biosystems) and sequenced on a MiSeq sequencer (Illumina) using 200 cycles of read 1, 6 cycles of index read and 200 cycles of read 2 with the following customized primers: read 1: 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3'; index read: 5'-TCGTGGTAAGGGTGCAATTGGGCTGACTGACT-3'; read 2: 5'-AGTCAGTCAGCCCAATTGCACCCTTACCACGA-3'. Data analysis. Barcoded sequencing data were analysed with MIGEC software22. Briefly, unique molecular identifier sequences were extracted from raw sequencing data (read 1) with MIGEC/Checkout routine. Reads (≥5) bearing the same unique molecular identifier were grouped and assembled to generate consensus sequences with MIGEC/Assemble. Variable (V) and joining (J) segment mapping, CDR3 extraction, and error correction were performed with MIGEC/CdrBlast as previously described22, which eliminates PCR and sequencing errors, as well as normalizes the output data as cDNA counts that represent the TCR clonotypes in a population36. Comparison of TCRα repertoires between CNS3-deficient and -sufficient mice at protein level was evaluated using VDJtools post-analysis framework (https://github.com/mikessh/vdjtools)23. Pearson correlation of clonotype frequencies for the shared TCR clones was used for the generation of the dendrogram. Clonal diversities of TCRα repertoires were evaluated using inverse Simpson index computed separately for individual samples after downsampling the repertoires to the size of the smallest sample from the same organ. Similar downsampling strategy, not weighted by clonotype frequencies, was used to compute the average size of added nucleotides in CDR3. A mathematical model24 was used to assess the strength of CDR3 amino acid interactions with pMHC complexes. Numbers of strongly interacting amino acid residues (LFIMVWCY) were calculated for the V-segment part of TCRα CDR3 and V–J segment junction. Those numbers were then weighted by the corresponding clonotype frequencies and the resulting sums were used for the comparisons between samples. Mature Foxp3−CD4+CD8− SP (TCRβ+GFP−CD62LhiCD69lo) thymocytes, Foxp3+ CD4 SP thymocytes (thymic T cells), peripheral resting (CD44loCD62Lhi) and activated (CD44hiCD62Llo) T cells were FACS-sorted from ~6–8-week-old male Foxp3gfp and Foxp3ΔCNS3-gfp littermates. RNA was extracted and cDNA libraries were generated after SMART amplification (Clontech). Libraries were sequenced using a HiSeq 2000 platform (Illumina) according to a standard paired-end protocol. Reads were first processed with Trimmomatic37 to remove TruSeq adaptor sequences and bases with quality scores below 20, and reads with less than 30 remaining bases were discarded. Trimmed reads were then aligned to mm10 mouse genome with the STAR spliced-read aligner38. For each gene from the RefSeq annotations, the number of uniquely mapped reads overlapping with the exons was counted with HTSeq (http://www-huber.embl.de/users/anders/HTSeq/). Genes with fewer than 50 read counts were considered as not expressed and filtered out. Principal component analysis (PCA) was performed (n = 11,962) for clustering gene expression. Differential gene expression was estimated using DESeq package39. To determine activation-related transcriptional signatures in T cells, the differences between read counts of peripheral activated versus resting T cells from wild-type Foxp3gfp mice were evaluated by fold-change and Benjamini–Hochberg corrected P values (false discovery rate < 0.001) (Supplementary Data 1 and 2). For gene expression comparisons, previously published transcriptional signatures of TCR-dependent genes in T cells were used7. The distribution of gene expression changes is shown for transcriptional signature genes and the rest of all expressed genes. One-tailed Kolmogorov–Smirnov test is used to determine the significance between the distributions of signature genes and the rest of expressed genes. We cross-linked 1 × 106 cells with 1% formaldehyde for 5 min at room temperature. Cross-linked cells were lysed and nuclei were resuspended in 250 μl nuclear lysis buffer containing 1% SDS. Chromatin input samples were prepared by sonication of cross-linked nuclear lysates. For histone ChIPs, nuclear lysates were subjected to micrococcal nuclease (MNase) digestion before sonication. Nuclei were resuspended in 100 μl MNase (New England Biolabs) at 12,000 U ml−1 for 1 min at 37 °C. The reaction was stopped by addition of 10 μl of 0.5 M EDTA. Chromatin input samples were incubated overnight at 4 °C with antibodies against H3K4me1 (Abcam), H3K4me3 (Millipore) or H3K27ac (Abcam), and precipitated for 90 min at 4 °C using protein A Dynabeads (Life Technologies). After thorough washing, bead-bound chromatin was subjected to proteinase K digestion and decrosslinking overnight at 65 °C. DNA fragments were isolated using a Qiagen PCR purification kit. Relative abundance of precipitated DNA fragments was analysed by qPCR using Power SYBR Green PCR Master Mix (Applied Biosystems). The following primers were used for qPCR: Gm5069: forward: 5'-TAAGCAATTGGTGGTGCAGGATGC-3', reverse: 5'-AAAGGGTCATCATCTCCGTCCGTT-3'; Hspa2: forward: 5'-TCGTGGAGAGTTGTGAGAAGCGA-3', reverse: 5'-AACGTTAGGACGAAAGCGTCAGGA-3'; Hsp90ab: forward: 5'-TTACCTTGACGGGAAAGCCG AGTA-3', reverse: 5'-TTCGGGAGCTCTCTTGAGTCACC-3'; Rpl30: forward: 5'-TCGGCTTCACTCACCGTCTTCTTT-3', reverse: 5'-TG TCCTCTGTGTATGCTAGGTTGG-3'; Foxp3 promoter: forward: 5'-TAATGTGGCAGTTTCCCACAAGCC-3', reverse: 5'-AATACCTC TCTGCCACTT TCGCCA-3'; CNS1: forward: 5'-AGACTGTCTGGAACAACCTAGCCT-3', reverse: 5'-TGGAGGTACAGAGAGGTTAAGAGCCT-3'; CNS2: forward: 5'-ATCTGGCCAAGTTCAGGTTGTGAC-3', reverse: 5'-GGGCGTTCCTGTTTGACTGTTTCT-3'; CNS3: forward: 5'-TCTCCAGGCTTCAGAGATTCAAGG-3', reverse: 5'-ACAGTGGGATGAGGATACATGGCT-3'. Relative enrichment was calculated by normalizing to background binding to the control region (Gm5069). Quantification of serum Ig isotypes was performed by ELISA as previously described40. Tissue sections from gender matched Rag1−/− mice were used to detect mouse autoantibodies. Briefly, organs from the Rag1−/− mice were dissected, fixed with neutral buffered formalin, embedded with paraffin and sectioned. After deparaffinization with EZPrep buffer (Ventana Medical Systems) and antigen retrieval with cell conditioning solution (Ventana Medical Systems) the sections were blocked for 30 min with Background Buster solution (Innovex), followed by avidin/biotin blocking for 8 min, mouse serum (1:50 dilution) incubation for 5 h and biotinylated horse anti-mouse IgG (Vector Labs) incubation for 1 h. The detection was performed with streptavidin–horseradish peroxidase (Ventana Medical Systems) followed by incubation with Tyramide Alexa Fluor 488 (Invitrogen). The slides were then counterstained with DAPI (Sigma Aldrich) for 10 min, mounted, scanned with a Mirax scanner and visualized with Pannoramic Viewer (3DHISTECH). Scanned images were scored and representative snapshots were processed with Photoshop (Adobe) to switch the green and red channels for presentation purpose. Mixed bone marrow chimaeras were generated as previously described31. Briefly, recipient mice were irradiated (9.5 Gy) 24 h before intravenous injection of 10 × 106 bone marrow cells from CD45.1+ Foxp3gfp and CD45.2+ Foxp3ΔCNS3-gfp mixed at a 1:1 ratio. After bone marrow transfer, the recipient mice were administrated with 2 mg ml−1 neomycin in drinking water for 3 weeks and analysed 8–10 weeks later. Tissue samples were fixed in 10% neutral buffered formalin and processed for haematoxylin and eosin staining. Stained slides were scored for tissue inflammation as previously described41. Experimental autoimmune encephalomyelitis was induced by immunization with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55, GenScript) in complete Freund’s adjuvant (CFA, Sigma) and mice were monitored for disease as previously described42.
Wild-type C57BL/6 and FVB/n mice, and transgenic mice with ACTB–tdTomato–eGFP (stock no. 007676), Fsp1–Cre (stock no. 012641), MMTV–PyMT (stock no. 002374), and MMTV–Neu (stock no. 002376) were obtained from The Jackson Laboratory. The vimentin–CreER mouse was a kind gift from the laboratory of R. F. Schwabe at Columbia University. CB-17 SCID mice were obtained from Charles River Laboratories. All mouse strains obtained were bred in the animal facility at Weill Cornell Medical College. All animal work was conducted in accordance with a protocol approved by the Institutional Animal Care and Use Committee at Weill Cornell Medical College. The ACTB–tdTomato–EGFP and Fsp1–Cre mice were bred together to obtain double transgenic mice and then bred with MMTV–PyMT or MMTV–Neu mice to obtain the tri-PyMT and tri-Neu triple-transgenic mice, respectively. Double transgenic male mice carrying ACTB–tdTomato–eGFP and MMTV–PyMT were crossed with the vimentin–CreER mice to obtain the tri-PyMT/Vim triple-transgenic mice. Genotyping for each transgenic line was performed following the standardized protocols as described in the website of The Jackson Laboratory. Genotyping for vimentin–CreER was done using forward primer 5′-CCCCTTCCTCACTTCTTTCC and reverse primer 5′-ATGTTTAGCTGGCCCAAATG. To induce vimentin–CreER activity in the tri-PyMT/Vim mice, Tamoxifen (Sigma-Aldrich, 2 mg per mouse, dissolved in corn oil) was administered through intraperitoneal injections, three times per week starting when the primary tumours appear (at 8 weeks of age) and continuing for 6 weeks until metastasis developed in the lung. The primary tumour of the tri-PyMT mouse (12-week-old female) was surgically removed under sterile conditions. Tumour tissue was sliced into ~1 mm3 blocks and implanted into the fat pad (no. 4 on the right side) of CB-17 SCID mice. The secondary tumour was used to establish the tri-PyMT cell line, eliminating the contamination of fluorescent positive stromal cells in the tumour tissue from tri-PyMT transgenic mice. Tumour tissue was minced and digested with an enzyme cocktail (Collagenase A, elastase, and DNase I, Roche Applied Science) in HBSS buffer at 37 °C for 30 min. The cell suspension was strained through a 40-μm cell strainer (BD Biosciences). Cells were washed with PBS three times and uploaded in the Aria III cell sorter (BD Biosciences). The sorted RFP+ cells were cultured in DMEM supplemented with 10% fetal bovine serum. The PyMT oncogene expression in the established cell line was confirmed by RT–PCR (Extended Data Fig. 4c). The tumorigenic ability of these cells was confirmed throughout the study. To determine EMT induction by TGF-β, cells were cultured for one week in DMEM with 2% FBS and 2 ng ml−1 TGF-β1 (R&D Systems). The GFP+ cell ratio was quantified by flow cytometry. To generate the miR-200 overexpressing cell line, a pLenti 4.1 Ex miR-200b-200a-429 construct20, was obtained from Addgene. To eliminate the contamination of fluorescent marker expression in targeted cells, the GFP gene in this construct was removed by BstBI/XbaI digestion followed by blunted self-ligation. Lentivirus was packaged by co-transfection of the pLenti-miR-200 construct and packaging plasmids into HEK293T cells. tri-PyMT cells (passage 2) were infected with the lentivirus. Infected cells (tri-PyMT miR-200) were selected by culturing with puromycin (2 μg ml−1) for 14 days. A control tri-PyMT cell line was generated by infecting cells with lentivirus carrying the puromycin resistance gene, following the same procedure in parallel. To establish an orthotopic breast tumour model, we first purified RFP+ cells from passages 10–15 of tri-PyMT cell culture by FACS. The purified RFP+ tri-PyMT cells (1 × 106 cells with purity >99%, Extended Data Fig. 5a) were injected into the mammary fat pad of 8-week-old female CB-17 SCID mice. The growth of the primary tumour was monitored by external calliper measurement once a week. In approximately 4 weeks, the primary tumour was surgically removed and the incision was closed with wound clips. The tumour size did not exceed 5% of total body weight as permitted in the IACUC protocol. Animals were euthanized 4 weeks after primary tumour removal to analyse the development of pulmonary metastasis. For animals subjected to chemotherapy, Cyclophosphamide (CTX, Sigma-Aldrich, 100 mg kg−1) was administered once per week, for 2 weeks prior and 2 weeks after surgery. The harvested primary tumours and PBS-perfused lungs bearing metastases were fixed in 4% paraformaldehyde overnight, followed by 30% sucrose for 2 days, and then embedded in Tissue-tek O.C.T. embedding compound (Electron Microscopy Sciences). Serial sections (10 μm, at least 10 sections) were prepared for histological analysis by haematoxylin and eosin staining, and immunofluorescent staining following standardized protocols. Primary antibodies used in this study include CD45 (30-F11, BioLegend), E-cadherin (DECMA-1, BioLegend), vimentin (sc-7557, Santa Cruz), PyMT (ab15085, Abcam), Neu (sc-284, Santa Cruz), Ki67 (ab15580, Abcam), and active caspase-3 (C92-605, BD Pharmingen). Primary antibodies were directly conjugated to Alexa Fluor 647 using an antibody labelling kit (Invitrogen) performed as per manufacturer’s instructions and purified over BioSpin P30 columns (Bio-Rad). GFP+ and RFP+ cells were detected by inherent fluorescence. Fluorescent images were obtained using a computerized Zeiss fluorescent microscope (Axiovert 200M), fitted with an apotome and an HRM camera. Images were analysed using Axiovision 4.6 software (Carl Zeiss). For the metastatic lungs and primary tumours, cell suspensions were prepared by digesting tissues with an enzyme cocktail (collagenase A, elastase, and DNase I, Roche Applied Science) in HBSS buffer at 37 °C for 30 min. For cultured cells, cells were collected through trypsinization. A single-cell suspension was prepared by filtering through a 30-μm cell strainer (BD Biosciences). Then cells were stained following a standard immunostaining protocol. In brief, cells were pre-blocked with 2% FBS plus Fc block (CD16/CD32, 1:30, BD Biosciences) and then incubated with the primary antibody against E-cadherin (DECMA-1, BioLegend). SYTOX Blue (Invitrogen) was added to the staining tube in the last 5 min to facilitate the elimination of dead cells. GFP+ and RFP+ cells were detected by their intrinsic signals. The stained samples were analysed using the LSRII flow cytometer coupled with FACS Diva software (BD Biosciences). Flow cytometry analysis was performed using a variety of controls including isotype antibodies, unstained and single-colour stained samples for determining appropriate gates, voltages and compensations required in multivariate flow cytometry. For sorting live cells back for further culturing or injection into animals, we used the Aria II cell sorter coupled with FACS Diva software (BD Biosciences). The preparation of cells for sorting was performed under sterile conditions. The purity of subpopulations after sorting was confirmed by analysing post-sort samples in the sorter again. Total RNA was extracted by using the RNeasy Kit (Qiagen), and miRNA via the mirVana miRNA isolation kit (Life Technologies), and converted to cDNA using qScript cDNA SuperMix (Quanta Biosciences) and RT–PCR. qPCR was performed with the appropriate primers (sequences shown in the table) and iQTM SYBR Green master mix (Bio-Rad). PCR protocol: initial denaturing at 95 °C for 3 min, 40 cycles of 95 °C for 20 s, 60 °C for 30 s, and 72 °C for 30 s, followed by final extension at 72 °C for 5 min and melt curve analysis was applied on a Bio-Rad CFX96 Real Time System (Bio-Rad) coupled with Bio-Rad-CFX Manager software. Primers used are as follows: GAPDH, forward, 5′-GGTCCTCAGTGTAGCCCAAG-3′; reverse 5′-AATGTGTCCGTCGTGGATCT-3′; Cdh1 (E-cadherin), forward, 5′-ACACCGATGGTGAGGGTACACAGG-3′; reverse, 5′-GCCGCCACACACAGCATAGTCTC-3′; Ocln, forward, 5′-TGCTAAGGCAGTTTTGGCTAAGTCT-3′, reverse, 5′-AAAAACAGTGGTGGGGAACGTG-3′; Vim, forward, 5′-TGACCTCTCTGAGGCTGCCAACC-3′; reverse, 5′-TTCCATCTCACGCATCTGGCGCTC-3′; Cdh2 (N-cadherin), forward, 5′-AAAGAGCGCCAAGCCAAGCAGC-3′; reverse, 5′-TGCGGATCGGACTGGGTACTGTG-3′; FSP-1, forward, 5′-CCTGTCCTGCATTGCCATGAT-3′, reverse, 5′-CCCACTGGCAAACTACACCC-3′; Snai1, forward, 5′-ACTGGTGAGAAGCCATTCTCCT-3′; reverse, 5′-CTGGCACTGGTATCTCTTCACA-3′; Snai2, forward, 5′-TTGCAGACAGATCAAACCTGAG-3′; reverse, 5′-TGTTTATGCAGAAGCGACATTC-3′; Twist1, forward, 5′-AGCTACGCCTTCTCCGTCTG-3′; reverse, 5′-CTCCTTCTCTGGAAACAATGACA-3′; Zeb-1, forward, 5′-GATTCCCCAAGTGGCATATACA-3′; reverse, 5′-TGGAGACTCCTTCTGAGCTAGTG-3′; Zeb-2, forward, 5′-TGGATCAGATGAGCTTCCTACC-3′; reverse, 5′-AGCAAGTCTCCCTGAAATCCTT-3′; PyMT, forward, 5′-ACTGCTACTGCACCCAGACA-3′; reverse, 5′-CTGGAAGCCGGTTCCTCCTA-3′; GFP, forward, 5′-CCACATGAAGCAGCACGACT-3′; reverse, 5′-GGGTCTTGTAGTTGCCGTCG-3′; RFP, forward, 5′-AGCGCGTGATGAACTTCGAG-3′; reverse, 5′-CCGCGCATCTTCACCTTGTA-3′. Total RNA was extracted from sorted RFP+ and GFP+ tri-PyMT cells with the RNeasy Kit (Qiagen). RNA-seq libraries was constructed and sequenced following standard protocols (Illumina). Single-end RNA-seq reads were mapped to UCSC mouse genome (GRCm38/mm10) using Tophat2. FPKM values for each gene were estimated by Cufflinks and statistical analysis was done using Cuffdiff2. Heat maps for differentially expressed genes with adjusted P values <0.05 were drawn using gplots R package. Cells were homogenized in 1× RIPA lysis buffer (Millipore) with protease inhibitors (Roche Applied Science). Samples were boiled in 1× Laemmli buffer and 10% β-mercaptoethanol, and loaded onto 12% gradient Tris-glycine gels (Bio-Rad). Western blotting was performed using antibodies specific for E-cadherin (clone DECMA-1), vimentin (clone RV202, BD Pharmingen), and β-actin (clone AC-15, Sigma-Aldrich). To determine apoptosis of RFP+ and GFP+ cells, tri-PyMT cells (Passage 10) were seeded on adherent six-well plates (1 × 106 cells), and treated with 4-hydroperoxy cyclophosphamide (Santa Cruz) for 48 h. After treatment, cells were trypsinized and stained with APC-conjugated Annexin V (BD Biosciences) and SYTOX Blue (Invitrogen) for apoptotic-cell labelling. The stained cells were analysed in the LSRII flow cytometer to quantify the percentage of apoptotic, dead, and live RFP+ and GFP+ cells by FACS Diva software. To determine the viability of tri-PyMT control and miR-200-expressing cells treated with CTX, cells were plated in 96-well adherent black-walled plates (1 × 104 cells), and treated with 4-hydroperoxy cyclophosphamide for 48 h. After treatment, cell viability was measured with the CellTiter-Glo Luminescent Cell Viability Assay (Promega). 1 × 105 tri-PyMT cells were seeded in a six-well plate. Real-time images of cells (including phase, GFP and RFP channels) were taken under a computerized Zeiss microscope (Axiovert observation) every 10 min for 10 h. Movement of individual cells (>10 RFP+ and >10 GFP+ cells in each field, >2 fields were analysed) were tracked with ImageJ software, and the distance that was travelled during that time was measured as indicated. RFP+ and GFP+ tri-PyMT cells (1 × 106 cells each) were freshly sorted from culture by FACS and then homogenized in cold ALDH Assay buffer provided in the ALDH Activity Colorimetric Assay Kit (Biovision Inc.) Following the protocol, ALDH substrate and acetaldehyde were added. ALDH activities in samples were measured by OD at 450 nm in kinetic mode (every 3 min for 60 min). To determine the sample size of animal experiments, we used power analysis assuming . Therefore, all animal experiments were conducted with ≥5 mice per group to ensure adequate power between groups by two-sample t-test comparison. Animals were randomized within each experimental group. No blinding was applied in performing experiments. Results are expressed as mean ± s.e.m. Data distribution in groups and significance between different treatment groups was analysed by using the Mann–Whitney U-test in GraphPad Prism software. P values <0.05 were considered significant. Error bars depict s.e.m., except where indicated otherwise.