Supplemental Materials and Methods
Materials
4-hydroxytamoxifen and BrdU were purchased from Sigma-Aldrich (Sigma-Aldrich, München,
Germany). Erlotinib and gefitinib from LC Laboratories (Woburn, MA, USA) and 10058-F4
was from EMD Biosciences (San Diego, CA, USA).
Murine PDAC cell line
The murine PDAC cell line PPT-6037 was described recently 1. The cell line was cultured in
DMEM medium (Life technologies, Darmstadt, Germany) containing 10% fetal calf serum
(FCS) (Merck Millipore, Biochrom, Berlin, Germany) supplemented with 1% (w/v)
penicillin/streptomycin (Life technologies).
FACS analysis
FACS was performed with a FACSCalibur flow cytometer (BD Biosciences; Heidelberg,
Germany). Minimum 5 × 104 events were counted. Living cells were gated and further
analyzed and illustrated using FlowJo software V10.0.8 (Tree Star). FACS sorting was done
as described 2.
Isolation and propagation of pancreatic ductal epithelial cells (PDECs)
PDECs were isolated and propagated as described
3, 4
. In brief, pancreata from 6 to 8 weeks
old mice with the indicated genotypes were dissected and chopped into small pieces in
Hank´s balanced salt solution ((HBSS), supplemented with 0.9 mg/ml glucose, 1%
penicillin/streptomycin, 2.5 µg/ml fungizone (Sigma-Aldrich) and 47.6 µM CaCl2). Pancreatic
tissue fragments were digested in D-MEM/F12 (Life technologies) containing 1 mg/ml
collagenase type V (Sigma-Aldrich) at 37°C for 20 min with agitation by a magnetic stir bar.
Fragments were digested further for 2 min in trypsin-EDTA and after inactivation of trypsin by
addition of trypsin-inhibitor (Sigma-Aldrich) the pancreatic tissue fragments were washed two
times in HBSS. Isolated PDECs were resuspended in D-MEM/F12-Full medium (D-MEM/F12
supplemented with 5 mg/ml glucose, 1.22 mg/ml Nicotinamide (Sigma-Aldrich), 0.1 mg/ml
soybean trypsin inhibitor (Sigma-Aldrich), 20 ng/ml EGF (BD Biosciences, Heidelberg,
Germany), 0.5% ITS+ Premix (BD Biosciences), 25 µg/ml BPE (BD Biosciences), 5% NU1
Serum IV (BD Biosciences), 1 µM Dexamethasone (Sigma-Aldrich), 5 nM triiodo-L-thyronine
(Sigma-Aldrich), 100 ng/ml cholera toxin (Sigma-Aldrich), 1% penicillin/streptomycin, 2.5
µg/ml fungizone) and plated on rat tail collagen type I (BD Biosciences) coated plastic dishes.
Reaching 70-80% confluency, PDECs were passaged. Only passages 3 to 8 were used for
the experiments. With the exception of PDECs from Pdx1-Flp;FSF-KrasG12D/+;FSF-R26CAGCreERT2
;R26mT/mG;Myclox/lox mice, at least two individual PDEC lines were used for each
experiment.
Mouse lines and orthotopic transplantation
The following mouse strains were described previously: LSL-KrasG12D/+ 5, FSF-KrasG12D 6,
Pdx1-Flp 6, FSF-R26CAG-CreERT2 6, Ai14-R26lox–STOP–lox–tdTom (from here R26Tom) 7, R26YFP 8,
R26mT/mG 9, R26CreERT2
, Cdkn2alox
10
, Hnf1b-CreER
11
, and Myclox
12
13
. The strains were
interbred to obtain mice allowing expression of KrasG12D and deletion of Cdkn2a, Myc, or
expression of reporter genes in PDECs by 4-OHT treatment as described 14-16. Animals were
on a mixed C57Bl/6;129S6/SvEv genetic background. The following primers were used for
genotyping: LSL-KrasG12D 5´-C A C C A G C T T C G G C T T C C T A T T-3´, 5´-A G C T A
A T G G C T C T C A A A G G A A T G T A-3´, 5´-C C A T G G C T T G A G T A A G T C T
G C-3´(300 bp (STOP del), 270 bp (wt), 170 bp (LSL)); R26mT/mG 5´-G T A C T T G G C A T A
T G A T A C A C T T G A T G T A C-3´, 5´-A A A G T C G C T C T G A G T T G T T A T-3´,
5´-G G A G C G G G A G A A A T G G A T A T G-3´ (650 bp (wt), 450 bp (mut)); R26CreERT2
5´-G A A T G T G C C T G G C T A G A G A T C-3´, 5´-G C A G A T T C A T C A T G C G G
A-3´ (330 bp (wt), 190 bp (mut)); Cdkn2alox 5´-C C A A G T G T G C A A A C C C A G G C T
C C-3´, 5´-T T G T T G G C C C A G G A T G C C G A C A T C-3´ (140 bp (wt), 180 bp
(lox)); Myclox 5´-G C C C C T G A A T T G C T A G G A A G A C T G-3’, 5´-C C G A C C G G
G T C C G A G T C C C T A T T-3’ (370 bp (wt), 470 bp (lox)). Genotyping of R26Tom, Hnf1bCreER, FSF-KrasG12D, Pdx1-Flp, FSF-R26CAG-CreERT2 and R26YFP mice was described
6, 8, 17
. To
induce Cre recombinase activation in vivo, Hnf1b-CreER;LSL-KrasG12D/+;R26Tom mice were
injected with Tamoxifen (Sigma, dissolved in Corn Oil) (100 μg/g body weight) once at week
five and once at week six as described
17
. 150.000 PDECs in 50 µl D-MEM/F12 medium
2
were implanted orthotopically into the pancreas of NOD-SCID-Il2r (NSG) mice as
described
14
. For the orthotopic transplantation shown in supplemental figure 2B, PDECs
from LSL-KrasG12D/+;R26YFP mice were isolated and in vitro recombined using lentiviral selfdeactivating Cre-recombinase (Addgene plasmid # 12106) as described previously
3
.
Recombined cells (YFP+) were purified by FACS. 7.5 x 105 YFP+ cells were transplanted
orthotopically into Ncr nude mice (Taconic, Hudson, NY, USA). Pancreata were harvested 21
days after transplantation and processed for histology. All animal studies were conducted in
compliance with European guidelines for the care and use of laboratory animals and were
approved by the Institutional Animal Care and Use Committees (IACUC) of Technische
Universität München, Regierung von Oberbayern, and the University of Pennsylvania.
Cell lysis and western blotting
Whole-cell lysates were prepared in immunoprecipitation buffer (50mM HEPES, 150mM
NaCl, 1 mM EDTA, 0.5% NP-40, 10% glycerol) supplemented with protease and
phosphatase inhibitors (Protease inhibitor cocktail cOmplete EDTA free, Roche Diagnostics,
Mannheim, Germany and Phosphatase-Inhibitor-Mix I, Serva, Heidelberg, Germany).
Lysates were normalized for protein, heated at 95oC for 5 min in protein loading buffer (45.6
mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 0.01% bromophenol
blue) and loaded onto 10% SDS-polyacrylamide gels. Proteins were transferred to
Immobilon-FL (Merck-Millipore) as described
15
. To detect phosphorylated MYC, cells were
directly boiled in protein loading buffer. Membranes were blocked in PBS supplemented with
5% skim milk and 0.1% Tween, and incubated at room temperature with the following
antibodies: pan-Ras (clone 10, #05-516, Millipore), pan-ERK (#4696, Cell Signaling
Technology, Danvers, MA, USA), phospho-(Thr202/Tyr204)-ERK (#4370, Cell Signaling
Technology), phospho-(Tyr1068)-EGFR (#2234, Cell Signaling Technology), pan-EGFR
((1005):sc-03, Santa Cruz Biotechnology, Dallas, Tx, USA), cyclin D1 ((HD11):sc-246, Santa
Cruz Biotechnology), cyclin A ((H-432):sc-751, Santa Cruz Biotechnology), phospho(S807/S811)-Rb (#8516, Cell Signaling Technology), phospho-(T308)-AKT (#9275, Cell
Signaling Technology), phospho-(S473)-AKT (#9271, Cell Signaling Technology), pan-AKT
3
((C67E7), #4691, Cell Signaling Technology), phospho-(S9)-GSK3β (#9323, Cell Signaling
Technology), phospho-(Tyr705)-Stat3 ((D3A7), #9145, Cell Signaling Technology), pan-Stat3
((C-20):sc-482, Santa Cruz Biotechnology), phospho-(T58/S62)-MYC (#9401, Cell Signaling
Technology), and pan-MYC (#9402, Cell Signaling Technology). Anti-β-actin (Sigma-Aldrich)
or anti-tubulin (Sigma-Aldrich) were used as loading controls. DyLightTM 680 or 800
conjugated secondary antibodies (Cell Signaling Technology) were detected by the Odyssey
Infrared Imaging System (Licor, Bad Homburg, Germany). At least two biological replicates
were analyzed and one representative western blot is shown.
Raf-Ras pull down assay
PDEC samples were lysed in MLB-buffer (Millipore, Schwalbach, Germany) supplemented
with 10% glycerol (Sigma-Aldrich), phosphatase-inhibitor (Serva) and protease-inhibitor
(Roche). 0.5 mg protein was incubated with Raf-RBD Protein GST beads (Cytoskeleton;
Denver, CO, USA) at 4°C for 1 hour with rotation. Beads were pelleted, washed twice with
MLB-buffer, resuspended in 2x protein loading buffer and boiled at 95oC for 5 min. Pulldowns were loaded onto a 12% SDS-gel and transfered to a Immobilon-FL membrane
(Merck-Millipore). Activated Ras was detected with a pan-Ras antibody (clone 10, #05-516,
Merck-Millipore).
Histochemistry, Immunohistochemistry and Immunofluorescence
For histopathological analysis, murine specimens were fixed in 4% formaldehyde (Carl Roth,
Karlsruhe, Germany), embedded in paraffin and sectioned (3 μm or 6 μm thick). Tumors
were stained with haematoxylin and eosin as described
15, 18
. For BrdU and K19
immunodetection, formalin-fixed paraffin-embedded tissue sections were dewaxed and
placed in a microwave (10 min., 600 watt) to recover antigens before incubation with the
primary antibody (1:300, anti-K19, (TROMAIII), Developmental Studies Hybridoma Bank,
Iowa City, IA, USA); 1:250, anti-BrdU (MCA2060), AbD Serotec, Düsseldorf, Germany).
Antibodies conjugated to biotin (Vector Laboratories, Peterborough, UK) were used as
secondary
antibodies.
Peroxidase
conjugated
4
streptavidin
was
used
with
3,3’-
diaminobenzidine tetrahydrochloride (Vectastain® elite ABC Kit and DAB Peroxidase
Substrat Kit, Vector Laboratories) as chromogen for detection. Sections were counterstained
with haematoxylin. Immunofluorescence was performed as described previously
19
. In brief,
paraffin sections were stained using chicken anti-GFP (1:250, ab13970, Abcam, Cambridge,
MA, USA) and rat anti-K19 (1:100, TROMAIII). Nuclear counterstain was achieved using
DAPI (4',6-Diamidino-2-Phenylindole, Dilactate, Thermo Fisher Scientific, Waltham, MA,
USA). High-resolution images were captured and analyzed using AxioVision 4.3 software
(Carl Zeiss, Jena, Germany).
Quantitative reverse-transcriptase PCR
Total RNA was isolated from PDECs using the RNeasy kit (Qiagen). Quantitative mRNA
analysis by qPCR was performed as previously described (StepOnePlus, PE Applied
Biosystems) 15. Primer sequences (forward / reverse): cypA 5´-A T G G T C A A C C C C A C
C G T G T-3´ / 5´-T T C T G C T G T C T T T G G A A C T T T G T C-3´; beta-actin 5´-G T C
G A G T C G C G T C C A C C-3´ / 5´-G T C A T C C A T G G C G A A C T G G T-3´; areg
5´-T G G C A G T G A A C T C T C C A C A G-3´ / 5´-T G G G C T T A A T C A C C T G T T
C A-3´; ereg 5´-C A C T C C G C A A G C T G C A C C G A-3´ / 5´-T G C A C T T G A G C C
A C A C G G G G-3´; p16INK4a 5´-C C C A A C G C C C C G A A C T-3´ / 5´-G T G A A C G T
T G C C C A T C A T C A-3´; Odc1 5´-A C A T C C A A A G G C A A A G T T G G-3´ / 5´-A G
C C T G C T G G T T T T G A G T G T-3´; Skp2 5´-G G C A A A G G G A G T G A C A A A
G A-3´ / 5´-C A G C T C A T C T G G A A G G G A G T-3´; E2F1 5´-G C A A C T G C T T T
C G G A G G A C T-3´ / 5´-A T C A C T A T G A C C A T C T G T T C T G C-3´; Ncl 5´ -T G
C T C C A G A G T C G T C G G T A-3´ / 5´-G G T T T T G C C A G C C T T T G C G A -3´;
Hspe1 5´ -T C T C C C A G A A T A T G G A G G C A C-3´ / 5´-A T C A G T G G A A T G G
C A G C T T C A-3´; eIF4e 5'- T C T A A G A T G G C G A C T G T G G A -3’ / 5’- C C C A C
C T G T T C T G T A G A G G G - 3’. Results represent the mean and standard error of the
mean (S.E.M) of at least 3 biological replicates performed as technical triplicate unless
otherwise indicated.
Microarray analysis and gene set enrichment analysis (GSEA)
5
Gene expression profiling was performed as recently described
15
. In brief, PDECs were
treated for 3 days with 200nM 4-OHT or were treated with an equivalent volume of ethanol
as a vehicle-treated control. Afterwards, total RNA was prepared and labeled sense strand
DNA was produced and hybridized onto the GeneChip® Mouse Gene 1.0 ST arrays
(Affymetrix, Santa Clara, CA, USA) according to Affymetrix standard protocols. For
expression profiles of PDEC cells treated with the EGFR inhibitors, cells were incubated for 6
days with 500 nM 4-OH-tamoxifen or were treated with an equivalent volume of ethanol as a
vehicle-treated control. For the last 24 hours of the experiment erlotinib and gefitinib were
added or an equivalent volume of DMSO as a vehicle-treated control. mRNA expression was
analyzed with Illumina mousewg-6_v2 Gene expression BeadChip arrays (Illumina, San
Diego, CA, USA). Microarrays can be accessed via EMBL-EBI ArrayExpress (Accession
number: E-MTAB-2592). To investigate gene expression changes induced by KrasG12D in the
pancreas in vivo, published expression profiles from duct and duct-like cells of control (n=3)
and Pdx1-Cre;LSL-KrasG12D/+ (n=6) mice, aged 10 to 20 weeks, were used
19
. For gene set
enrichment analysis, the GSEA software (www.broadinstitute.org) was used and microarray
data were analyzed with the gene set matrix composed files c2.all.v4.0.symbols.gmt, and
c3.tft.v4.0.symbols.gmt
20
. Significant enriched sets were identified using a FDR (false
discovery rate) q-value < 0.05 and a nominal p-value < 0.05.
Statistical methods
Unless otherwise indicated, all data were determined from at least three independent
experiments and presented as mean and standard error of the mean (S.E.M). A two-sided
Student`s t-test or one-way ANOVA was used to investigate statistical significance. p-values
were corrected for multiple testing according to Bonferroni and calculated with GraphPad
Prism5. * in the figures denotes a p-value of at least < 0.05.
6
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9