media.nature.com · Web viewIn addition, for standard curve calculation five different cell...
Transcript of media.nature.com · Web viewIn addition, for standard curve calculation five different cell...
Canonical WNT signaling in Hodgkin Lymphoma
SupplementarySupplementary Methods
Cell culture
The HL cell lines L428 and KM-H2 were cultured in RPMI1640 medium with 2 mM
glutamine (Lonza, Basel, Switzerland) supplemented with 10 % fetal calf serum
(Biochrom AG, Berlin, Germany), 200 U/ml Penicillin and 200 μg/ml Streptomycin (both
Sigma-Aldrich, St. Louis, USA). Cells were maintained at 37 °C in 5 % CO 2 and seeded
at a density of 5 x 105 cells/ml the day before the experiment.
Stable LEF-1 knockdown KM-H2 cells have been generated by lentiviral transduction of
KM-H2 with the GIPZ plasmid containing either LEFsh or nonsense (ns) sequence (GE
Healthcare Dharmacon, Lafayette, USA). Successfully transfected KM-H2 cells have
been selected using 2 µg/ml puromycin.
Human Umbilical Vein Endothelial Cells (HUVEC) cells were cultured in EBM2 medium
supplemented with EGM-2 MV SingleQuot Kit Supplements & Growth Factors (all
Lonza, Basel, Switzerland). For the experiments HUVEC cells with a confluence of 80-
90% and till passage six have been applied.
All cell lines have been tested regularly for mycoplasma contamination.
HL-cell conditioned medium
For the production of conditioned HL medium L428 or KM-H2 cells have been seeded at
a density of 5 x 105 cells/ml and incubated in RPMI 1640 medium supplemented with 10
% (v/v) FCS, 200 U/ml Penicillin and 200 μg/ml Streptomycin for two days. Cell
supernatant was centrifuged at 300 g for 10 minutes at 4 °C, sterile filtered and stored at
4 °C for a maximum of two weeks.
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VEGF-A ELISA
VEGF-A ELISA (Human VEGF Quantikine ELISA Kit, R&D, Minneapolis, USA) has been
performed with the indicated CMs of siRNA transfected L428 and KM-H2 cells according
to the manufacturer’s instructions or as published before (1).
Inhibitor treatment
For inhibition of the canonical WNT pathway the CKI inhibitor PF670462, the tankyrases
inhibitor XAV939 (both Millipore, Billerica, USA) or DKK1 and DKK2 (both R&D,
Minneapolis, USA) have been added to the cell culture medium at a final concentration
of 50 nM (PF670462) or 5 µM (XAV939) or 100 ng/ml (DKK1, DKK2) in DMSO
(PF670462, XAV939), PBS (DKK2) or PBS with 0.1 % BSA (DKK1) respectively for 24
hours.
Nucleofection
For nucleofection of siRNA the corresponding Cell Line Nucleofector Kit L (for L428) or T
(for KM-H2) (both Lonza, Basel, Switzerland) has been used according to the
manufacturer’s instructions. After the nucleofection, cells were incubated in pre-warmed
RPMI 1640 supplemented with 10 % (v/v) FCS, 200 U/ml Penicillin, 200 μg/ml
Streptomycin, 1 mM Sodium pyruvate and 10 mM Hepes for 48 hours at 37 °C in
5 % CO2. Knock-down quality was verified with qRT-PCR and immunoblotting.
Applied siRNA (2 µg siRNA per two million cells) included LEF-1 (pool of ID108840,
IDs27618, IDs27617, IDs27616) (Life Technologies, Carlsbad, USA), β-catenin (pool of
ID J-003482-09, ID J-003482-12) (GE Healthcare Dharmacon, Lafayette, USA).
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Adhesion assay
Briefly, 96 well plates were coated with 1 mg/ml type-collagen (Trevigen,
Gaithersburg, USA) or seeded with HUVEC cells overnight. Pretreated HL cells were
added to the wells (15.000 cells/well in 320 μl) in quadruplicates and incubated for 1
hour at 37 °C in 5 % CO2. For discrimination between adherent and non-adherent cells
the plate was turned up-side down and incubated for 2 more hours at 37 °C in 5 % CO2.
While holding the plate still up-side down the supernatant with the non-adherent cells
was carefully transferred to a second plate. In addition, for standard curve calculation
five different cell concentrations were added to this second plate. To quantify cell
numbers MTT solution to a final concentration of 0,5 mg/ml was added and incubated
with the non-adherent cells, and the cells for the standard curve for 3 hours at 37 °C in 5
% CO2. Optical density was determined at 560 nm using Tecan Infinite F50 Reader
(Tecan Group Ltd., Männedorf, Switzerland). Standard curves were calculated for each
condition and the amount of adherent cells was presented in percentage of the total
number.
Migration, Invasion and Chemotaxis Assays
5x104/50 µl L428 or KM-H2 cells per well in RPMI 1640 medium migrated for 6 hours
towards RPMI 1640 supplemented with 10 % (v/v) FCS and 50 nM CCL19. For analysis
only the migrated cells in the lower chamber were counted. Cell migration towards
RPMI 1640 medium of the control cells was set as one.
For invasion assays the membrane was coated with 1 mg/ml type-1 collagen (Trevigen,
Gaithersburg, USA) and invasion was measured after 16 hours as described above.
For HUVEC invasion assay the Boyden chamber with 8 µm porous membranes coated
with 1 mg/ml type-collagen (Trevigen, Gaithersburg, USA) was used. 1.5x104/50 µl
HUVEC cells per well migrated for 4 hours towards the conditioned media (CM) of cHL
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cell lines as indicated. As positive control EBM2 medium supplemented with EGM-2 MV
SingleQuot Kit Supplements & Growth Factors, and as negative control RPMI medium
(all Lonza, Basel, Switzerland) were used. For analysis, the upper side was cleaned
from resident HUVECs. The specimens were fixed with 4% PFA, stained and
photographed at 100x magnification. In three representative photos per well migrated
cells were counted and the mean calculated.
For monitoring real-time cell movements KM-H2 cells (3 x 106 cells/ml) in 1,5 mg/ml
type-1collagen were added into the µ-Slide chemotaxis3D chamber (Ibidi, Martinsried,
Germany). Chemotaxis against 10 % (v/v) FCS and 50 nM MIP-3β was documented for
6 hours with one photo per 5 minutes by time-lapse microscopy (Olympus IX81 with
Olympus XM-10 camera; Olympus, Shinjuka, Japan). For trajectory analysis of 50 cells
per condition ImageJ 1.45s (National Institutes of Health, Bethesda, USA) and the Ibidi
software “Chemotaxis and Migration Tool” were used. Cell track analysis using shape
fingerprint descriptors is described below.
Cell track analysis using shape fingerprint descriptors – a.k.a. autocorrelation vector analysis
To derive a condensed depiction for the observed track space, we performed an
analysis using 2D shape fingerprints, which were derived from the internal distance
distribution for each track, like it is common for the shape description of chemical
compounds in cheminformatics (2).
Software
We used KNIME version 2.11.3 (3) to organize the analysis of cell tracks and perform
preprocessing, the final analysis steps like deriving the fingerprints, hierarchical
clustering and plotting where done with corresponding R-nodes in KNIME using R
version 3.0.2. (4).
Deriving cell track fingerprints
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To derive the individual track fingerprint we implemented the following steps as an R
function:
1. Generating intra-track distance matrices per track →we transformed the xy-coordinate
collection for each track into an individual distance matrix representation, thus mapping
the occurring internal distances of each track.
2. Estimating the binning parameters → To achieve a comparable scaled binning of the
resulting correlation vectors we scanned the individual track distance matrices for the
global max distance reported, then we used the global maximum of intra-track distances
to set the binning algorithms required max distance to be used for all tracks. The
number of bins was set to n=100; which seems a reasonable choice given that 50 time
steps were recorded per track and the global max distance was ≈ 250 μm, while a
typical eukaryotic cell diameter is ≈ 10 µm.
3. Deriving the shape fingerprints → The use of a binning vector function (using a range
[0; global_max_distance], and #_of_bins = 100), allowed to transform each distance
matrix into a correlation vector representation. For each occurring distance within a track
the corresponding bin position to count up in the n-dimensional correlation vector can be
simply calculated using: bin_position = floor((current_distance –
global_minimal_distance) / (global_max_distance – global_minimal_distance) *
#_of_bins).
4. Resulting correlation vector table containing fingerprints →having derived the shape
fingerprint vector for each track we used the vector representations for our similarity
calculations; e.g., hierarchical clustering, heat map representations
Similarity analysis using cell track fingerprints
We used the fingerprints to explore the track space. The analysis can be divided into the
following sequential steps:
1. Hierarchical clustering of the tracks → We used the fingerprints to derive dendrogram
representations of the track space using Manhattan distance for all distance calculations
and complete linkage method for the tree construction.
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2. Identifying the main clusters → we used the cut-tree function to derive the main
clusters in track space.
3. Identifying cluster representatives → for each cluster we derived the representative
track, by identifying the track fingerprint with the smallest cumulative distance to the
other cluster members.
4. Mapping tracks from different experiments to the clusters → after successful
inspection of the cluster representatives to represent the required distinction level, we
mapped the tracks respective experiment type to the dendrogram leafs. Extracting the
cluster group members we thus could derive the count of the tracks per experiment per
cluster. Finally scaling the count per experiment resulted in the relative distribution of
track types per experiment.
Scratch Assay
HUVECs were grown to 80% density and scratched using a pipette tip. Cells were
washed twice with PBS and incubated with pGIPZ-KM-H2 conditioned medium in equal
parts with EBM2 medium supplemented with EGM-2 MV SingleQuot kit, supplements
and growth factors (all Lonza, Basel, Switzerland). Photos of the scratch were taken for
each well and cells incubated for 16 hours at 37 °C in 5% CO2. HUVECs of the positive
controls (complete EBM2 medium supplemented with EGM-2 MV SingleQuot kit,
supplements and growth factors) closed the scratch completely. Photos were taken for
each well at the previously marked positions. For each well the relation between the
width of the scratch after 0 hours and 16 hours was measured and calculated using
ImageJ software 1.45s (National Institutes of Health, Bethesda, USA).
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Sprouting Assay
Sprouting was studied with endothelial spheroids. For spheroid generation, HUVECs
were mixed with 1.2% methyl cellulose in EC medium and incubated in 96 well plates for
24 hours Spheroids formed, which were harvested and seeded with Matrigel (BD
Bioscience, Franklin Lakes, USA) mixed with one part matrigel and two parts KM-H2
conditioned medium in 96-well plates for 48 hours. At least 12 spheroids were used per
condition. For positive controls spheroids were seeded with EBM2 medium
supplemented with EGM-2 MV SingleQuot Kit supplements and growth factors (Lonza,
Basel, Switzerland). Photos of each spheroid were taken after 48 hours and sprouting
frequency was calculated as relation between sprouted spheroids and totally seeded
spheroids per condition. From each spheroid sprout numbers and total sprout length
were calculated with ImageJ software 1.45s (National Institutes of Health, Bethesda,
USA).
Tube Formation Assay
HUVECs were seeded as single cell suspension in Matrigel (BD Bioscience) mixed with
two parts of the particular cHL conditioned medium and one part EC medium in 96-well
plates. Four wells per condition were used. After 24 hours, tubes formed throughout the
well. Three photos per well were taken and total tube lengths was measured using
ImageJ software 1.45s (National Institutes of Health, Bethesda, USA).
Western Blot
Cells were lysed in NP-40 lysis buffer (50mM tris (pH 7.4), 150mM NaCl, 1mM EDTA,
0.5% NP-40) and 20 µg of total protein was used for SDS page. SDS page was used for
size-fractionation of the proteins, which were transferred to Immobilon PVDF
membranes (Millipore, Billerica, USA). Blots were air-dried, rehydrated and incubated in 7
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blocking buffer (5 % BSA or milk powder, 0.1 % Tween in tris-buffered saline) at room
temperature for one hour and afterwards incubated with primary antibody in blocking
buffer overnight at 4 °C. After washing, blots were incubated with the respective
peroxidase-coupled secondary antibody for 1h at room temperature. Enhanced
chemiluminescence detection was used to visualize the immune reaction (Image Reader
LAS-4000mini, Fujifilm). For re-probing with other antibodies blots were incubated in
Reblot Mild Plus Buffer (Millipore, Billerica, USA) at room temperature for 15 minutes.
For Wnt5a immunoblotting lysates have not been boiled for SDS page but transferred
semi-dry onto a nitrocellulose membrane (Life Technologies, Carlsbad, USA).
Micro-CT scans of CAM tumors
CT scans of the PTA stained and agarose gel embedded samples have been performed
with the Phoenix Nanotom CT (GE Measurement & Control, USA) . The system was
operated with the following parameters: tube voltage 100 kVp, tube current 135 µA, 360°
rotation with 1800 projection images, exposure time per projection 250ms, detector
binning 2x2 resulting in a pixel size of ~ 5 x 5 x 5 μm3. In order to increase the signal-to-
noise ratio every projection data has been acquired six times and averaged.
Vascularization Score of CAM tumors
Vascularization index is based on three criteria: appearance of hemorrhages, intensity
of hemorrhages and relative area covered by hemorrhages. For each criterion, zero to
three points were given, leading to a maximum of nine points per tumor. For the
evaluation process light microscopic pictures of each tumor were blinded and then
evaluated by a group member, who was not involved in the experiments.
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Microarray data analyses
Analysis of available microarray mRNA expression data was conducted using the
Oncomine web platform (Life Technologies) (5). The Steidl Lymphoma, Brune
Lymphoma and the Basso Lymphoma datasets were used (6–8). In the Steidl study 130
HL samples were analyzed using Human Genome U133 Plus 2.0 Array (19 574 genes,
54 675 reporters). To determine the cut-off for high and low VEGFA gene expression
levels the software Cut-off Finder was used (9). In the Brune study 42 malignant
lymphoma samples and 25 normal B-cell samples of various types were analyzed using
as well the Human Genome U133 Plus 2.0 Array. In the Basso study 336 B-cell samples
(normal, tumor, cell line, and cell line perturbation samples) were analyzed using the
Affymetrix U95Av2 Array (8,603 genes, 12,651 reporters). Data were analyzed using
GraphPad Prism 6.04 (GraphPad Software Inc., La Jolla, USA).
Preparing FFPE tissue blocks
Hodgkin and other lymphoma cases from all age groups that were routinely diagnosed
between 2001 and 2014 were identified within the files of the Lymph Node Registry Kiel.
Lymphoma biopsy specimens were routinely fixed in 10% buffered formalin, embedded
in paraffin (FFPE), cut into 3-5 µm sections and mounted on coated glass slides for
conventional immunohistochemistry.
Preparing Tissue Micro Arrays
Cases with sufficient FFPE tissue were arranged in a tissue microarray (TMA) with
duplicate cores 0.6 mm in diameter for each lymphoma. TMA and whole tissue sections
were subsequently subjected to immunohistochemistry.
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Immunohistochemistry on FFPE sections
The 3-5 µm paraffin TMA and whole tissue sections were placed onto coated glass
slides and dried over night at 37°C. Deparaffinization steps were followed by hydration.
The conventional immunohistochemical staining for LEF-1 was carried out according to
standard procedures including endogen peroxidase blocking (10 min in methanol/H2O2)
and antigen retrieval (3 min at 100°C in 10 mM citrate buffer, pH 6.0) prior to incubation
with the rabbit monoclonal anti-LEF-1 antibody (#2230S, Cell Signalling, Frankfurt,
Germany) at a dilution of 1:50 for 1 h. The detection was performed using the
ZytoChemPlus (HRP) Polymer Bulk Kit (Zytomed Systems GmbH, Berlin, Germany).
The counterstaining was performed with Haemalaun after Meyer (1:4) (Merck,
Darmstadt, Germany) for 3-5 min and rinsing in water for 10 min. After dehydration the
section was covered with the mounting medium Pertex (Histolab Products AB;
Göteborg, Sweden).
The double-staining for LEF-1 and CD3 was performed according to the standard
procedure described above for LEF-1. The mouse monoclonal anti-CD3 antibody
(#NCL-L-CD3-565, NovoCastra, XXX) was applied at a dilution of 1:100 for 1 h together
with the anti-LEF-1 antibody further following the described protocol.
Scoring system of immunohistochemical stainings
Whole tissue slides and Tissue Microarrays of lymphoma samples were scored
according to their percentage content of LEF-1 positive tumor cells from 0 to 4 (0=0%;
1=1-25%; 2=26-50%; 3=51-75%; 4=76-100%).
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Chick Chorio-Allantoic Membrane (CAM) Assay
Chicken eggs (Valo BioMedia GmbH, Osterholz-Scharmbeck, Germany) have been
bred and regularly turned for 4 days at 37.8 °C and 80 % air humidity. For the in ovo
assay, an approximately 0.75 cm² window has been sawed into the egg shell at the
fourth day of chicken embryo development. For the ex ovo assay, eggs have been
cracked into small bowls and incubated in cell culture flasks for the following days. At
day eleven of chicken embryo development 2x106 L428 cells or 3x106 KM-H2 cells each
embedded in 20 µl Matrigel (BD Bioscience, Franklin Lakes, USA) were inoculated onto
the CAM. After 4 days of incubation on the CAM tumors have been cut out, evaluated,
photographed (Motic SMZ-161, Motic Asia, Hong Kong, China; camera: Moticam 3.0)
and fixed either in Bouin’s solution or 4 % paraformaldehyde solution with a subsequent
ascending series of saccharose. For histological analysis tumors were embedded in
paraffin or Tissue Tek (Poysciences Inc., Warrington, USA) and cut into 3 µm or 11 µm
thick sections. Tumor areas have been measured using the light microspcopy photos
and ImageJ software 1.45s (National Institutes of Health, Bethesda, USA). CAM tumors
smaller than 0.1 cm2 were excluded from the evaluation because those “tumors” were
considered as matrigel rest and not as grown tumors.
Quantification of intracellular metabolites using NMR spectroscopy
Sample preparation: Acetonitrile extraction (10) was employed to quench cell
metabolism and to quantitatively extract low molecular weight compounds from HL cell
lines. Acetonitrile fraction was subjected to overnight lyophilisation. Dried extracts were
resuspended in 550 µl of D2O (Sigma-Aldrich) containing 0.005% sodium 3-
(trimethylsilyl)propionate-2,2,3,3-d4 (TSP) (Sigma-Aldrich) used as both chemical shift
reference and internal standard for metabolite quantification.
NMR spectroscopy: Information on concentration of metabolites in individual samples
was derived from volumes of corresponding signals in 1D 1H NMR spectrum.
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Assignment of signals in NMR spectra of individual samples to particular metabolite was
achieved via comparison of a sample spectrum with spectra of pure metabolites (Sigma-
Aldrich). The 1D 1H spectra were measured at 700 MHz using a Bruker Avance III NMR
spectrometer equipped with a triple resonance room temperature probe using the
WATERGATE pulse sequence (11), including 1-ms rectangular selective pulses to
suppress the residual signal of water. All spectra were acquired at 20ºC and processed
using TopSpin 3.2 (Bruker, USA). To make comparison of metabolite concentration
profiles among various samples possible, the signals intensities in individual samples
were normalized to total protein concentration.
Flow cytometry
Cells were stained with saturating amounts of antibody for 20 min at 4°C in PBS
supplemented with 1% FCS and washed two times with PBS. Samples were collected
on a FACSCantoII flow cytometer (BD Bioscience, Franklin Lakes, USA) and data
analyzed using the FlowJo software (FlowJo LLC, Ashland, USA). Cells were gated
based upon their forward and side scatter parameters for size, shape and granularity.
FSC-A/FSC-H plots were used to gate on single cells. Mean fluorescence intensity ratios
(MFIRs) were calculated by dividing the mean fluorescence intensity for CCR7 by the
mean fluorescence intensity of the isotype control.
Statistical Analyses
Results are shown as mean or as mean ± standard deviation (mean ± SD) of the
indicated number of samples. The statistical significance of the values was determined
using the Student's t-test. If applicable group results were compared using the ANOVA-
method (One-way or Two-way Analysis of Variance) with a subsequent Bonferroni’s
post-hoc test to correct for multiple comparisons as indicated. Normal distribution and
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homogeneity of variance has been tested using the Kolmogorov–Smirnov Test and the
F-test. For nonparametric group results Kruskal-Wallis test with Dunn’s post-hoc test has
been performed. Significance levels are indicated as *=p<0.05, **=p<0.01, ***=p<0.001.
All statistical analyses and plots were done with GraphPad Prism 6.04 (GraphPad
Software Inc., La Jolla, USA).
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Knowledge Organization. GfKL 2007. Springer International Publishing; 2007.
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6. Steidl C, Lee T, Shah SP, Farinha P, Han G, Nayar T, et al. Tumor-Associated
Makrophages and Survival in Classic Hodgkin’s Lymphoma. N Engl J Med.
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8. Brune V, Tiacci E, Pfeil I, Döring C, Eckerle S, van Noesel CJM, et al. Origin and
pathogenesis of nodular lymphocyte-predominant Hodgkin lymphoma as revealed
by global gene expression analysis. J Exp Med. 2008;205:2251–68.
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9. Budczies J, Klauschen F, Sinn B V., Gyorffy B, Schmitt WD, Darb-Esfahani S, et
al. Cutoff Finder: A Comprehensive and Straightforward Web Application Enabling
Rapid Biomarker Cutoff Optimization. PLoS One. 2012;7:1–7.
10. Dietmair S, Timmins NE, Gray PP, Nielsen LK, Krömer JO. Towards quantitative
metabolomics of mammalian cells: development of a metabolite extraction
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Supplementary Figures
Suppl Fig S1
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Suppl Fig S9
Case Processing Summarya,b,c
Cases
Valid Missing Total
N Percent N Percent N Percent
40 88,9 5 11,1 45 100,0
a. Protein factor = 1
b. Squared Euclidean Distance used
c. Average Linkage (Between Groups)
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Supplementary Figure Legends
Supplementary Figure S1: Inhibition of canonical WNT signaling inhibits CCL19 guided chemotaxis of L1236 cells.
(A) Migration of L1236 cells for 6 hours after 24 h pre-treatment with 100ng/ml DKK1,
DKK2 or DKK1 plus DKK2 was performed towards CCL19 in Boyden chamber assays.
(mean ± SD, n=3; Kruskal-Wallis test,). (***P < 0.001)
(B) Migration of L1236 cells for 6 hours after 24 h pre-treatment with 5 µM XAV939 or
0.05 µM PF670462 was performed towards CCL19 in Boyden chamber assays. (mean
± SD, n=3; 1-way ANOVA and Bonferroni’s post-hoc test). (***P < 0.001)
Supplementary Figure S2: Inhibition of canonical WNT signaling does not affect cHL cell viability.
(A) Relative cell numbers of L428 and KM-H2 after 24 hours treatment with DKK1 and
DKK2. (mean ± SD, n=3, 2-way ANOVA with Bonferroni’s post-hoc test).
(B) Relative cell numbers of L428 and KM-H2 after 24 hours treatment PF670462.
(mean ± SD, n=3, 2-way ANOVA with Bonferroni’s post-hoc test)
(C) Relative cell numbers of L428 and KM-H2 after 24 hours treatment XAV939. Of note,
no significant difference in cell viability has been caused by inhibitor treatments. (mean ±
SD, n=3, 2-way ANOVA with Bonferroni’s post-hoc test)
Supplementary Figure S3: CCR7 cell surface expression is not affected by inhibitor treatment.
(A) Gating scheme for CCR7 cell surface expression by flow cytometry. Representative
pictures of CCR7 expression on DMSO treated L428 cells.
(B) No differences in CCR7 cell surface expression between DMSO or inhibitor treated
L428 cells (mean ± SD, n=3). 25
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(C) No differences in CCR7 cell surface expression between DMSO or inhibitor treated
KMH2 cells (mean ± SD, n=3).
Supplementary Figure S4: KM-H2 cells show constitutive active canonical WNT signaling. Western Blot of cytosolic and nuclear fractions of KM-H2 cells stimulated for
24 hour or 48 hours with conditioned medium (CM). Note the increased nuclear β-
catenin fraction after CM stimulation. As controls, nuclear HDAC and cytosolic HSP90
was used. Relative β-catenin intensities compared to the respective cytosolic and
nuclear controls are shown beneath.
Supplementary Figure S5: Stable LEF-1 knockdown reduces cHL migration.
(A) Migration of L428 cells after stable LEF-1 knockdown is significantly reduced. (mean
± SD, n=3, unpaired, 2-tailed t-test).
(B) Migration of KM-H2 cells after stable LEF-1 knockdown is significantly reduced.
(mean ± SD, n=3, unpaired, 2-tailed t-test with Welch correction).
(C) Relative cell numbers of L428 after LEF-1 and β-catenin knockdown. Of note, no
significant difference in cell viability has been caused by the knockdowns. (mean ± SD,
n=3, 1-way ANOVA with Bonferroni’s post-hoc test)
(D) Relative cell numbers of L428 and KM-H2 after stable LEF-1 knockdown. Of note, no
significant difference in cell viability has been caused by the knockdowns. (mean ± SD,
n=3, 2-way ANOVA with Bonferroni’s post-hoc test)
Supplementary Figure S6: CCR7 cell surface expression is not affected by LEF-1 or β-catenin knockdown.
(A) Gating scheme for CCR7 cell surface expression by flow cytometry. Representative
pictures of CCR7 expression on L428 cells scr si control.
(B) No differences in CCR7 cell surface expression between L428 scr si control and
LEF-1 or β-catenin knockdown cells (mean ± SD, n=3). 26
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(C) No differences in CCR7 cell surface expression between KMH2 scr si control and
LEF-1 or β-catenin knockdown cells (mean ± SD, n=3).
Supplementary Figure S7: Cell tracks of KM-H2 LEF-1si and β-catenin si cluster separately from scr control cells. Heat map displaying the results of the hierarchical
clustering of movement patterns of scr si, LEF-1si and β-catenin si KM-H2 cells based
on shape fingerprints according to the form and length of each track. The x-axis of the
heat map corresponds to the internal distances within single tracks, with short distances
mapped to the left and longer distances to the right. The color of the heat map tiles
represents the number of counts for specific distances per track ('square root
transformed' to enhance contrast). Experimental group membership is encoded by
colors on the left side of the heat map with black (scr si), green (LEF-1si) and orange
(β-catenin).
Supplementary Figure S8: LEF-1 and β-catenin modify cHL adhesion and the influence of cHL cells on endothelial cell viability and migration.
(A) Migration of HUVEC cells towards CM of L428, ns control and LEF-1 sh cells. As
positive control HUVEC migration towards EBM2 medium supplemented with EGM-2
MV SingleQuot Kit Supplements & Growth Factors and as negative control RPMI
medium have been used. (mean ± SD, n=3, Kruskal-Wallis test with Bonferroni’s post-
hoc test).
(B-C) Scratch assay of HUVEC cells towards CM of L428, ns control and LEF-1 sh cells.
As positive control HUVEC migration towards EBM2 medium supplemented with EGM-2
MV SingleQuot Kit Supplements & Growth Factors and as negative control RPMI
medium have been used. Note the decrease in HUVEC migration towards CM of L428
after LEF-1 knockdown (KD). (mean ± SD, n=3; Kruskal-Wallis test with Bonferroni’s
post-hoc test).
(D) Adhesion of scr si control, LEF-1si and β-catenin si L428 cells on either HUVEC
(white) or collagen I (grey). Note the decrease of adhesion in LEF-1si and β-catenin si
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cells compared to scr si cells. (mean ± SD, n=4, 2-way ANOVA and Bonferroni’s post-
hoc test).
(E) MTT test of HUVEC cells treated CM of ns control and LEF-1 KD KM-H2 cells (mean
± SD, n=4, Kruskal-Wallis test with Bonferroni’s post-hoc test).
(F) MTT test of HUVEC cells treated CM of ns control and LEF-1 KD L428 cells (mean ±
SD, n=4, 1-way ANOVA and Bonferroni’s post-hoc test). Note decrease of HUVEC
viability in LEF-1 knockdown CM. (*P < 0.05, **P < 0.01 and ***P < 0.001)
Supplementary Figure S9: NMR analyses reveal no significant differences in metabolites after LEF-1 KD.
Metabolomics NMR analysis data from three independent experimental sets
(KM-H2/L428; parental, LEF1sh, ns ctr) were normalized by total protein amount in each
sample and their metabolic profile (based on 25 metabolites) was analyzed using
Cluster analysis (Euclidean distance; Ward´s method; link distance scaled as
percentage of maximal link distance). The LEF-1 knockdown does not cause any
obvious change in the metabolism of Hodgkin lymphoma cell lines KM-H2 and L428.
Burkitt lymphoma line BL41 was used as an external control.
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