Download Immunohistochemistry For antibody details see Supplementary

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.