Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Immunohistochemistry For antibody details see Supplementary Table 1. Immunohistochemical staining for CD31 (ab28364, Abcam, Cambridge, United Kingdom) was performed on 4 μm thick paraffin sections of formalin-fixed liver. The sections were dewaxed, rehydrated, and antigen was retrieved using citrate buffer (pH 6.0). Avidin/biotin blocking (00-4303, Invitrogen, Carlsbad, USA) was followed by 30-minute incubation in 4% milk to prevent nonspecific binding. CD31 antibody diluted 1:50 in Dako Antibody Diluent (S3022, Dako, Glostrup, Denmark) was incubated overnight at 4°C. Next day, sections were incubated with the relevant biotinylated secondary antibody (ab97067, Abcam) followed by a 30 minute incubation with ABC reagent (PK-6100, Vector Laboratories, Burlingame, USA) at room temperature. For visualization of the antigen, the sections were immersed in diaminobenzidine (K4368, Dako) and counterstained with haematoxylin (41-5136-00, MEDITE, Burgdorf, Germany). The Benchmark XT instrument (Ventana, Tucson, USA) has been used to perform the Factor VIII immunohistochemistry using the Optiview protocol according to the manufacturer’s instruction. Prediluted primary antibody against Factor VIII (760-2642, Ventana) incubation has been carried out for 12 minutes, followed by secondary antibody DAB-conjugated exposure for 3 minutes. Vascular Casting Vascular casts of the mouse liver were prepared similarly to the previously described method (21). In brief, the vasculature of a mouse liver harbouring an AS was perfused via the portal vein with a freshly prepared solution of PU4ii polymer (vasQtec, Zurich, Switzerland). One hour after perfusion, the liver was transferred to 7.5% potassium hydroxide for dissolution of tissue, which was accomplished after a period of 2 to 3 weeks. After washing, the casts were dehydrated in ethanol and dried in a vacuum desiccator. The sample was mounted on an aluminium stab and then sputtered with gold to a coating thickness of 10 nm. The resulting cast was examined in a Philips XL-30 SFEG scanning electron microscope. Transmission Electron Microscopy Small tissue fragments (as well as isolated cells) were fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h at 4°C. Cells were washed with the same buffer and post-fixed with 1% OsO4 in 0.1 M sodium cacodylate buffer for 1 h. Samples were dehydrated in a graded ethanol series and embedded in Epon 812. Ultrathin sections were mounted on copper grids, air-dried, and stained for 10 minute with 4.7% uranyl acetate and for 2 minute with lead citrate. The sections were examined and photographed using a JEOL JEM 1011 electron microscope (Jeol, Tokyo, Japan). Isolation and generation of AS cell lines Hepatic endothelial cells were isolated from Notch1 KO mice harbouring a visible AS as previously described with modifications. (17) Briefly, livers were perfusiondigested with collagenase (Liberase TM Research Grade, Roche, Basel, Switzerland) followed by separation of hepatocytes and non-parenchymal cells by low-speed centrifugation. Non-parenchymal cells were applied on a two-step Percoll gradient resulting in three fractions, which were all preserved for further processing. After selective adherence, freshly isolated endothelial cells were plated on fibronectin-coated dishes and cultured in endothelial cell medium (ECM, ScienCell, Carlsbad, CA). Fibroblast fractions present in some of the isolated cells were removed by differential trypsinization and repeated subculturing of cells onto plastic dishes. Longterm culturing of the primary cells selected malignant AS cells based on their atypical growth capacity and eventually lead to a uniform cell population. For controls, liver sinusoidal endothelial cells (LSEC) were also isolated from mice 12 weeks after Notch 1 deletion and from wildtype mice, as described above. But after the two-step Percoll gradient centrifugation only the intermediate fraction was used for further processing. Freshly isolated LSEC were allowed to attach to fibronectincoated dishes for 40 minutes and were directly used for further processing. Scanning electron microscopy (SEM) AS cells were fixed in 2.5% glutaraldehyde for one hour, dehydrated, critical-point dried, coated with a 10 nm thick gold layer, and examined at 5kV with a Nova NanoSEM 230 scanning electron microscope (FEI, Hillsboro, USA). Immunocytofluorescence Cells were grown in 24-well plates, fixed with 4% Formaldehyde, permeabililzed with 0.5% Saponin and 0.5% Triton 100 (for vWF), blocked with 3% bovine serum albumin (BSA), and incubated with antibodies to CD31 (1:100; ab24590, Abcam) and vWF (1:200; A 0082, Dako) overnight at 4°C. The binding of primary antibodies was detected using FITC donkey anti-mouse or Cy3 donkey anti-rabbit (1:100; 715-095150; 711-165-152, Jackson ImmunoResearch, West Grove, USA). Mounting and counterstaining was performed using Roti®-Mount FluorCare DAPI (HP20.1, Roth, Karlsruhe, Germany). Vascular Tube Formation Assay Tube formation assay was performed in 4-well chamber slides. Each well was coated with 200 µl of Matrigel (BD Biosciences) and allowed to solidify in the incubator at 37°C for 30 minute. AS cells were trypsinized and resuspended in FBS-free ECM. In a total volume of 1 ml, 50.000 cells per well were placed onto the matrix. Cells were treated with various concentrations of sorafenib (1, 3, and 5 µM) or temsirolimus (1, 10, and 100 ng/ml) resulting in a total dimethyl sulfoxide (DMSO) concentration of 0.1%. Controls were treated with 0.1% DMSO. Pictures of the entire well were taken after 46 h. Total vascular tube length per well was analyzed digitally using the software Cell^P (Olympus, Münster, Germany). Uptake of acetylated Low-Density Lipoprotein, Dil-AcLDL The cells were split in uncoated 24-well plates at a density of 50% confluence. On the next day, cells were washed and incubated with 10 μg/mL Dil-labeled actelyated LDL (L3484, Invitrogen) in complete medium for 4h at 37°C. After the incubation period, cells were washed with PBS, fixed with 4% formaldehyde, and mounted with Roti®-Mount FluorCare DAPI (HP20.1, Roth). Uptake was examined by fluorescence microscopy. RNA Extraction, Microarray analysis Affymetrix Mouse Gene ST 1.0 was used for microarray analysis of AS cells. Total RNA was isolated from AS cells and LSEC by Trizol (Invitrogen) and then by use of Zymo-Spin IIC columns (Zymo Research, Irvine, USA) according to the manufacturer’s instructions. The extracted RNA was used for microarray analysis and qRT-PCR analysis. RNA was reverse-transcribed, biotin labelled (Ambion), purified, and fragmented. After the 3’ end TdT labeling (Affymetrix), the samples were hybridized overnight to the Affymetrix Mouse Gene ST 1.0 Array (Affymetrix). The arrays were scanned on the Affymetrix GeneChip Scanner 3000 7G. All original array data are deposited at the National Center for Biotechnology Information Gene Expression Omnibus database. Microarray analysis was performed with Bioconductor packages of R statistical environment (22). Microarray data were preprocessed using standard robust multiarray averaging. Probe sets with very low expression intensities (<80 in the highest-expressing sample), as well as the control probe sets were excluded from the subsequent analyses. Differential gene expression was assessed using the limma package (23) with fold-change cut-off value of 2 and a false discovery rate cut-off value of 0.05. Gene Set Enrichment Analysis was performed with Java gene set enrichment analysis software version 2.07 (Broad Institute, Cambridge, MA), using the signal-to-noise ratio as a ranking metric. On the gene set enrichment analysis plot (Figure 4C), the x-axis represents a list of all genes on the array rank-ordered according to their decreasing correlation with AS phenotype (red, genes overexpressed in AS; blue, genes overexpressed in LSEC). Black ticks along the xaxis show positions of genes that are part of the tested gene set. The y-axis unit is the enrichment score defined as a running-sum statistic calculated walking down the ranked gene list. The running-sum increases when a gene in the ordered list is present in the gene set in question and decreases when it is absent. The increment of the enrichment score depends on the value of the ranking metric. Real-time quantitative reverse transcription-PCR (RT-PCR). RNA was processed into complementary DNA using moloney murine leukemia virus reverse transcriptase (Promega, Madison, USA). Quantitative RT-PCR was performed with SYBR green PCR Master Mix (Applied Biosystems, Carlsbad, California) using the 7500 Real Time PCR System (Applied Biosystems). All reactions were run in duplicate. Exon-spanning primers were designed and are listed in Supplemetary Table 2. To quantify the results, the difference between the Ct values (ΔCt) of the gene of interest and of the Ribosomal protein L19 (RPL19), which served as endogenous control, was calculated. mRNA expression levels of the transcripts were calculated relative to the housekeeping gene with the formula 2- ΔCt. Time-lapse microscopy AS cells were pre-treated for 5 hours with 3 μM sorafenib (no treatment for controls) before being subjected to the tube formation assay. For time-lapse imaging, AS cells were seeded on matrigel-coated glass bottom dishes and grown in FBS-free ECM in the presence or absence of sorafenib (3 μM). Controls were exposed to DMSO as vehicle control. Cells were allowed to attach for 30 minutes, followed by a washing step to remove non-adherent cells. Imaging started around 45 minutes after plating the cells. During time-lapse imaging, cells were kept in a plexiglass chamber at 37°C and 5% CO2. Time-lapse live cell images were captured every 7 minutes over 20 hours with a 20x objective on an inverted microscope (Nikon Eclipse Ti) equipped with a charge-coupled device camera (ORCA-R2, Hamamatsu Photonics) and NIS Elements software (Nikon). Filopodia quantification The average number of filopodia per cell was determined on still images at various time points (0, 2, 4, 6, 8, h) obtained from time-lapse series. Within each picture frame, 6 – 9 cells were assessed at the different time points and the average number of filopodia per cell was calculated. In each condition (untreated versus sorafenibtreated) a total of 7 time-lapse series (captured from the same plate) was analysed. Paired student’s t-test was used to assess statistical significance of differences between time points in the same condition. Cell Proliferation Assay AS cell proliferation with/without sorafenib was measured using the xCELLigence System RTCA DP analyzer (ACEA Biosciences, San Diego, CA). AS cells (5.000 cells/well) were seeded in septuplicate in the E-Plate 16 (ACEA Biosciences) and cell proliferation was monitored in real time using the xCELLigence analyzer, which was placed in a humidified incubator at 37°C and 5% CO2. Cells were grown in complete ECM and 24 hours after cell seeding sorafenib (final concentration of 1, 3, 5, 10, and 20 μM) or solvent control (DMSO; 0.1% v/v) was added. Cell growth was assayed for 72 hours. Data analysis and data normalization to the time before drug addition was carried out using the RTCA Software 1.2.1.1002. Cell index values from 2 replicate experiments were normalized to DMSO, which was as set as 100%. Apoptosis To study the effect of sorafenib on cell apoptosis, the activity of caspase 3 was assessed by immunohistochemistry using an anti-activated caspase 3 antibody (AF835, R&D Systems, Minneapolis, USA). In brief, AS cells were treated with sorafenib at the indicated doses for 24 hours. Cells were trypsinized, attached onto glass slides by cytospin centrifugation, and allowed to air dry. Cytospins were fixed in periodate-lysine-paraformaldehyde, treated for antigen retrieval with citrate buffer (pH 6) for 5 minute at 80°C, and then immunostained with a primary anti-Caspase 3 antibody using standard avidin biotin complex method. Eventually, the slides were counterstained with hemalaun. Immunoblotting Cells were exposed to 3, 5, and 10 μM sorafenib or 0.1% DMSO (controls) in complete medium for 8 hours. Cells were serum-starved overnight in the presence of the drug/vehicle and then ERK activation was triggered by incubation with PMA (100 ng/mL) for 15 minutes after a total incubation time of 24 h. Afterwards, cells were collected and lysed in a buffer containing 100 mM NaCl, 50mM Tris (pH 7.5), 1mM EDTA, 0.1% Tx-100, 10mM NaF, 1mM phenylmethylsulfonyl fluoride, 1mM vanadate, 1x protease inhibitor cocktail tablets (Roche Diagnostics GmbH, Mannheim, Germany), and 1% (v/v) phosphatase inhibitor cocktail (Sigma). The lysates were kept on ice at 4°C for 30 minutes and centrifuged at 15,000 rpm for 15 minutes at 4°C. Protein concentrations were measured using the Bio-Rad protein assay dye reagent (Bio-Rad Laboratories AG, Cressier, Switzerland) with bovine serum albumin as the standard. Proteins (20 μg per lane) were separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to a nitrocellulose membrane (Whatman, Dassel, Germany). Membranes were probed with specific antibodies against phospho-ERK1/2 (Thr202/Tyr204) (4370, Cell Signaling Technology, Danvers, USA) and panERK (E17120, BD Biosciences, San Jose, USA). Bands were visualized by incubating infrared fluorescent secondary antibodies (LI-COR Biosciences, Lincoln, USA) and analyzed by Odyssey Infrared Imaging System from LI-COR.