Supplemental Methods
Immunohistochemistry
Colon carcinoma and normal colon tissue sections (5 μm) were stained with a
combination of mouse anti–human CD31 (1:100, DAKO) and mouse-anti-human CD34
(1:100, Monosan) or with mouse anti-human HMGB1 (1:50, Sigma) essentially as
described previously (van Beijnum et al., 2006)
Sections of the CAMs were excised, paraformaldehyde-fixed and paraffin
embedded. CAM tumors were excised and stored in
Zn-fixative prior to paraffin
embedding. Tissue sections (5μm) were pretreated with xylene and rehydrated in a series
of ethanol. Endogenous peroxidase activity was blocked using H2O2, and sections were
boiled in citrate buffer (10mM, pH 6.0) for antigen retrieval. CAM sections were incubated
with mouse anti-human HMGB1 (1:50, Sigma) and primary antibodies were detected with
biotinylated rabbit anti-mouse (1:100, DAKO) in combination with streptavidin-HRP
(1:100, DAKO). Antibody binding was detected using DAB. CAM tumors were stained with
mouse anti-human α-smooth muscle actin (1:200, DAKO), in combination with HRPconjugated rabbit anti-mouse Envision staining system (DAKO).
Flow cytometry
Flow cytometry for the detection of HMGB1 in isolated EC was performed by
double labeling of EC in single-cell suspensions form human tumor and normal colon
samples (van Beijnum et al., 2008) using a combination of PE-labeled mouse anti-human
CD31 (1:25, DAKO) and rabbit anti-human HMGB1 (1:50, kind gift of Dr R.G. Roeder,
New York, NY). Cultured EC were trypsinized and stained with mouse anti-human ICAM1
(1:100, Monosan), Cells were fixated with 1% PFA and permeabilized (for HMGB1
detection) with 0.1% Triton X-100 to allow intracellular staining. FITC-labeled secondary
antibodies were used for fluorescent detection. All antibody incubations were performed in
PBS/0.1% BSA. Cells were analyzed on a FACSCalibur (BD Biosciences) and data were
analyzed using CellQuest software (BD Biosciences).
In silico analysis of HMGB1 expression in (colorectal) cancers
The Oncomine database contains a repository of microarray data covering a broad
panel of human cancers (www.oncomine.org). HMGB1 expression values were retrieved
from 24 studies that matched the following criteria: Analysis type, Cancer vs. Normal
Analysis and Multicancer Analysis; Cancer type, Colorectal cancer; Data type, mRNA.
This enabled visualization of HMGB1 mRNA levels in colorectal cancer and normal
tissues.
Using
the
In
silico
Transcriptomics
(IST)
tool,
available
from
www.genesapiens.org, correlations between HMGB1 and VEGFA based on expression
data of >300 gene expression studies concerning cancer were retrieved and plotted.
The Human Protein Atlas (http://www.proteinatlas.org) was mined for examples of
colorectal and ovarian tumors, as well as normal colon and normal ovary with different
intensities of HMGB1 expression. For comparison, sections showing vascular staining for
von Willebrand Factor (vWF) are also shown.
Proliferation assay
Proliferation was measured using the 3H-thymidine incorporation assay. Cells
were plated in gelatin-coated 96-well plates at 5000 (HUVEC) or 15000 (RF24) cells per
well and allowed to adhere for 4 hours. Where applicable, compounds were added and
cells were grown for 3 days. Cultures were pulsed with 0.3µCi of 3H-thymidine for 6 hours.
Subsequently, cells were lysed by freeze-thawing and lysates were transferred to
nitrocellulose filters. Filters were submerged in scintillation fluid and radioactivity was
measured using a liquid scintillation counter (Wallac LSC) (van Beijnum et al., 2006).
Sprouting assay
Bovine capillary endothelial cells (BCE) were seeded on cytodex-3 gelatin-coated
microcarrier beads (500 cells/bead) in suspension. After 24 hours, beads (25/well) were
embedded in collagen gel (2.4 mg/ml) in 96-well flat-bottom plates. BCE were allowed to
sprout into the collagen gel for 24 hrs after which images were taken. Sprouting was
quantified by counting intersections of sprouts with a concentric ring grid overlay on the
image (van Beijnum et al., 2006). Collagen gels and overlay medium were supplemented
with compounds as indicated.
From freshly excised human colorectal tumor tissue, a blood vessel was prepared
free, cut in 1mm sections and embedded in collagen gel as described above, in the
presence or absence of mouse anti-human HMGB1 antibodies (10 μg/ml), and allowed to
sprout for 5 days. Sprout length was quantified in ImageJ.
Apoptosis assay
HUVEC were plated in gelatin-coated 96-well plates at 5000 cells per well and
allowed to adhere for 4 hours. Where applicable, compounds were added and cells were
grown for 3 days. Cells were harvested by trypsinization and incubated with propidium
iodide (PI) (20 µM) in buffer containing 2.5 µM citric acid, 45 µM Na2HPO4 and 0.1%
Triton-X100, pH7.4 for 20 minutes at 37˚C. Cells were analyzed with a FACSCalibur (BD
Biosciences) in the FL2 channel. Apoptotic cells were defined as those that showed
subG1 DNA staining.
Western blot analysis
Cells were cultured in serum-free medium for 16 hours prior to stimulation with
rHMGB1. Nuclear and cytoplasmic extracts were prepared using NE-PER reagent
(Pierce) according to the manufacturers’ instructions. Protein (25 µg) was loaded on a
standard SDS-PAGE gel and blotted to nitrocellulose. Blots were incubated with antiHMGB1 antibody (1:1000; Sigma) or anti-actin antibody (1:50000; MP Biomedicals)
followed by biotinylated rabbit-anti-mouse antibody (1:1000; DAKO) in combination with
streptavidin-HRP (1:1000; DAKO) and DAB. Blots were scanned and band intensities
were quantified using QuantityOne software (Bio-Rad).
HMGB1 ELISA
RF24 cells were seeded at 15000 cells/well as described above. Cells were grown
for 48 hours, after which cells were treated with rHMGB1 as indicated in serum free
medium for 16 hours. Cells were washed and fresh serum free medium was added.
Media were collected after 8 hours and measured using a commercially available ELISA
kit for the detection of HMGB1 in serum or culture medium (Shino-Test). Wells without
cells were taken along as a control.
NF-B, MMP2 and ITGB1 activity measurement
The proinflammatory adhesion molecules ICAM1, VCAM1 and E-selectin (SELE),
are under control of NF-B. Active p65 NF-B subunit was detected using the EZ-detect
NF-B p65 ELISA assay kit (Pierce), according to the manufacturer’s instructions.
The activity of MMP1 and MMP2 was measured to assess whether the increased
mRNA expression also contributed to more active MMPs that can facilitate matrix
invasion. In fact, HMGB1 itself may mediate MMP activation by promoting plasmin
production on the cell surface through interaction with urokinase-type or tissue-type
plasminogen activator. MMP2 activity was determined with an immunocapture assay
(QuickZyme Biosciences).
EC
were
trypsinized
and
stained
with
an
antibody
against
activated
ITGB1(Chavakis et al., 2007) (1:50, BD Biosciences), and analysed by flow cytometry as
described above.
siRNA transfection
RF24 (1*104) were seeded in 96-well tissue culture plates that were coated with
gelatin and where siRNA (Qiagen; 20 nM) and transfection reagent (HiPerfect; Qiagen,
0.75 µl) were complexed for 20 minutes. Cells were processed for downstream analysis
48-72 hr later. Alternatively, EC were grown for 3 days after transfection with
oligodeoxynucleotides (ODN) (Biognostik; 5µM).
Transfection and cloning of HMGB1 expression constructs
HMGB1 open reading frame was amplified from cDNA (Fw primer: 5’GCGCACTGGGCGACTCTGTG-3’, Rev primer: 5’-ACTGCGCTAGAACCAACTTATTC-3’)
and cloned into pcDNA3.1+-Hygro (pHMGB1) using HindIII and XhoI. RF24 were
transfected using the Amaxa nucleofector system as described (Thijssen et al., 2010).
Cells were processed for analysis three days after transfection.
Supplemental Table 1: qPCR primer sequences
Gene
Forward
Reverse
ACTB
CATTCCAAATATGAGATGCATT
CCTGTGTGGACTTGGGAGAG
ANG1
AGCTACCACCAACAACAGTG
GCAAAGATTGACAAGGTTGTGG
ANG2
TGCCACGGTGAATAATTCAG
TTCTTCTTTAGCAACAGTGGG
ANG3
AACAGCGCGCTCGAGAAG
GCTTCGCCTTCTTGCTGA
B2M
TCCATCCGACATTGAAGTTG
CGGCAGGCATACTCATCTT
bFGF
AGGAGTGTGTGCTAACCGTTAC
ACTCATCCGTAACACATTTAGAA
EGFR
TGCCCATGAGAAATTTACAGG
ATGTTGCTGAGAAAGTCACTGC
FGFR1
ATCACGGCTCTCCTCCAGTG
AAGACCAGTCTGTCCCGAGG
FGFR3
ATGATCATGCGGGAGTGCTG
AGTACTGCTCGAAAGGCGCC
HMGB1
TTCATTTCTCTTTCATAACGGG
TCTAAGAAGTGCTCAGAGAGGTG
ICAM1
GGCCGGCCAGCTTATACAC
TAGACACTTGAGCTCGGGCA
ITGA2
GGAACGGGACTTTCGCAT
GGTACTTCGGCTTTCTCATCA
ITGB1
GAAGGGTTGCCCTCCAGA
GCTTGAGCTTCTCTGCTGTT
MMP1
CCAAATGGGCTTGAAGCT
GTAGCACATTCTGTCCCTAA
MMP2
AGATGCCTGGAATGCCAT
GGTTCTCCAGCTTCAGGTAAT
MMP9
TACTGTGCCTTTGAGTCCG
TTGTCGGCGATAAGGAAG
NRP1
CCCGAGAGAGCCACTCATG
GTCATCACATTCATCCACCAA
NRP2
CCGGTCGTTTGGGCTGGA
AACTGCAACTTTGATTTTCCG
PDGFRA
TTCATTGAAATCAAACCCACC
TTTCCTGAATCTTTTCCACATC
PDGFRB
AGAGCTGCCCATGAACGAG
CGAATCTCAAACTTCTCTTCCC
PlGF
ACGTCTGAGAAGATGCCGGTC
ACCACTTCCACCTCTGACGA
PPIA
CTCGAATAAGTTTGACTTGTGTTT CTAGGCATGGGAGGGAACA
RAGE
AACTCTAGCCCTGGCCCTGG
TTCTGGGGCCTTCCTCTCCT
SELE
CCCGAAGGGTTTGGTGAG
CAAATCCCTCCTCACAGCTG
Tie1
CCCCGCTGGTCTCGTTCTC
CACAATGGTCGACCAGTCC
Tie2
TTGAAGTGGAGAGAAGGTCTG
GTTGACTCTAGCTCGGACCAC
TLR2
ATTCCCCAGCGCTTCTGCA
AATCCTTCCCGCTGAGCCTC
TLR4
TTGAAAGTGTGTGTGTCCGC
CAAGCATAAGGGATAAGGGG
VCAM1
TCAGATTGGAGACTCAGTCATGT
ACTCCTCACCTTCCCGCTC
VEGFA
AAGGAGGAGGGCAGAATCAT
CAGAAGGAGAGCAGAAGTCC
VEGFR1
CCAGCAGCGAAAGCTTTGCG
CTCCTTGTAGAAACCGTCAG
VEGFR2
ATGACATTTTGATCATGGAGC
CCCAGATGCCGTGCATGAG
Supplemental Table 2: Species-specific qPCR primer sequences
Gene
Forward
Reverse
ACTB
CATTCCAAATATGAGATGCATT
CCTGTGTGGACTTGGGAGAG
B2M
TGTAGACGGCTTCGCTGC
AGGAGTGTGTGCTAACCGTTAC
bFGF
AGGAGTGTGTGCTAACCGTTAC
ACTCATCCGTAACACATTTAGAA
CD31
ACTCCAGTGGCATGAAAACT
ACAGAGCAGAGAAAAGTGGTC
EGFR
AGAGCTGCCAATGAAACG-G
AAGTACTGTGAGAGGCTTCCT
HMGB1
AAGAATATTCTTCCGTAACTGG
TTCTCAACTCTCCAAGACTGG
PDGFRA
GTTCATTCATCTAGAGCCTCAAT
TTTCCTGGACTCTGTTTGAACT
PDGFRB
CATGGCGGTGAACAGCAA
TCTGTGTGGGCTCCAGGCT
PPIA
AAGGAGGGGATGAACGTG
AGCTGCCCGCAGTTGGA
VCAM
TGATGTGGTCACAACCCTTAA
TCCAAATTTGTTAGTGAATGTGC
VEGFA
GACCTGTAAATGTTCCTGCAA
AGAAATCAGGCTCCAGAAACA
VEGFR1
TCGACACTATCTTCACAGCGG
GCTTCTGCAGTTTGGGCT
VEGFR2
TCACGCCTTACAGACACCCT
AGGGAGATGTTACGGAGAATG
ACTB
TTCCTATGTGGGCGACGAG
TCCTCGGGAGCCACACG
B2M
TCCATCCGACATTGAAGTTG
ACACGGCAGGCATACTCAT
bFGF
CGTAAGTGCAAACCGCTTT
CGTAAGTGCAAACCGCTTT
CD31
TTCCCACGCCAAAATGTTA
CACAGCACATTGCAGCACA
EGFR
TGCCCATGAGAAATTTACAGG
ATGTTGCTGAGAAAGTCACTGC
HMGB1
ACATCCAAAATCTTGATCAGTTA
AGGACAGACTTTCAAAATGTTT
PDGFRA
TTCATTGAAATCAAACCCACC
TTTCCTGAATCTTTTCCACATC
PDGFRB
AGAGCTGCCCATGAACGAG
CGAATCTCAAACTTCTCTTCCC
PPIA
AGCATGTGGTGTTTGGCAAA
TCGAGTTGTCCACAGTCAGC
VCAM
TAACGGGGAGCTACAGCC
CAGCCTGGTTAATTCCTTCAC
VEGFA
CCATCGACAGAACAGTCCTT
CGAATCCAATTCCAAGAGGG
VEGFR1
CAATGCCATACTGACAGGAA
CAGAGCTTCCTGAATTAAACTT
VEGFR2
ACAGCCTCTGCCAATCCATG
AAGGATGCATTCTTAAGCTCC
Chicken
Human
Supplemental Figure legends
Supplementary Figure 1: HMGB1 expression in human tumors
Proteinatlas.org was mined for examples of human colorectal tumors and normal colon
with different levels of overall HMGB1 expression. Endothelial expression of HMGB1 is
highlighted in the boxed sections using arrows. For comparison, endothelial von
Willebrand factor (vWF) staining is also shown. Endothelial expression of HMGB1 is also
evident in human ovarian cancer and normal ovary tissue samples.
Supplementary Figure 2: Expression of and reactivity to HMGB1 in different
endothelial cells
(a) RF24 and HUVEC were treated with bFGF (20 ng/ml) or rHMGB1 ( 50 an 500 ng/ml)
derived from different sources (see materials and methods), and was assessed using a
wound healing assay. (b) qPCR was used to determine expression of HMGB1 in primary
(HUVEC) and immortalized (RF24) endothelial cells. In addition, HMGB1 expression
levels were determined in A2780 human ovarian carcinoma and LS174T human
colorectal carcinoma cell lines.
Supplementary Figure 3: HMGB1 expression correlates with VEGFA expression in
clinical samples
The In silico Transcriptomics (IST) data repository was queried for expression of HMGB1
in gastrointestinal tumor types. Correlation of HMGB1 with VEGFA was significant and
positive for CRC and pancreas tumors (left), as well as in renal cancer (right).
Supplementary Figure 4: rHMGB1 activates primary endothelial cells
HUVEC were treated for 3 days with bFGF (20 ng/ml) or rHMGB1 (500 ng/ml; Sigma). (a)
The amount of active MMP2 was measured in HUVEC using ELISA in the conditioned
medium of the cells (left). The amount of activated form of ITGB1 was determined by flow
cytometry, and normalized for total ITGB1 expression (right). (b) HUVEC were treated as
described above, and expression of genes was profiled by qPCR. (c) HUVEC were
treated as described above and NF-B subunit p65 activity was determined by target
sequence binding ELISA. (d) NF-B target genes ICAM, VCAM and E-selectin (SELE)
were determined by qPCR. (e) Relative cytoplasmic HMGB1 content in HUVEC treated
with bFGF or rHMGB1 was determined as described. *P<0.05; **P<0.01 Mann-Whitney
U-test.
Supplementary Figure 5: rHMGB1 stimulates angiogenesis in ovo
CAMs were treated with bFGF (20 ng/ml) or rHMGB1 (500 ng/ml; Sigma) or control saline
solution from EDD10-14. CAMs were treated daily and 100 μl solution was applied
topically. CAMs were photographed after injection of a suspension of vegetable oil with
zincoxide powder to provide contrast. An overlay grid was applied on the photos (5x
magnification) and intersections of vessels with the grid were counted as a measure of
vessel density. *P<0.05; **P<0.01 Mann-Whitney U-test.
Supplementary Figure 6: HMGB1 receptor expression in endothelial cells
(a, b) RAGE and TLR4 expression were profiled by qPCR in HUVEC and RF24 (a) and in
endothelial cells isolated from colon tumors (TEC) and normal colon (NEC) (b).
Supplementary Figure 7: Overexpression of HMGB1 stimulates EC migration
(a) Immortalized HUVEC (EVLC2) were transfected with pcDNA3-HMGB1 (pHMGB1) and
expression levels were measured. (b) No effect of increased HMGB1 expression was
seen on proliferation of EC (middle), though migration was markedly induced (right).
Supplementary Figure 8: HMGB1 antibodies inhibit angiogenesis in vitro and in
vivo
(a, b) HUVEC were treated for 3 days with the indicated concentration of anti-HMGB1 Ab.
Proliferation was determined by 3H-labeled thymidine incorporation (a), apoptotic cells
were detected by PI DNA staining and flow cytometry (b). (c) Color representation of
Figure 5e. Fragments of tumor blood vessel were embedded in collagen gel and cells
were allowed to sprout for 5 days in the presence or absence of anti-HMGB1 antibodies.
(d) Cross sections of paraffin embedded CAMs were stained with anti-HMGB1 antibodies
to determine reactivity with chick HMGB1. Clear staining of vascular structures is seen.
Scale bar = 100 μm. (e) CAMs were treated with anti-HMGB1 antibodies for 4 days
(EDD8-12), after which images were taken after injection of contrast as described in
Figure S5, and vessel density adjacent to the tumor was quantified using the overlay grid
as described. HET-CAM software was used to determine capillary branching. Clear
avascular areas are seen after anti-HMGB1 treatment. Scale bar = 500 μm. (f) LS147T
cell spheroids were grown in non-adhesive culture plates and placed on top of the chick
chorioallantoic membrane (CAM). qPCR was used to determine expression of HMGB1 in
the endothelium of control CAMs and LS174T containing CAMs (left panel). CAMs were
treated with anti-HMGB1 antibodies or control antibodies applied topically for 4
consecutive days. CAMs were photographed and analyzed as described. Scale bar = 1
mm.
Supplementary Figure 9: Species-specific primer validation
(a) Alignment of human (hs) and chicken (gg) HMGB1 sequences. Asterisks indicate
identical nucleotides. In bold are the primer sequences used for qPCR. (b, c) qPCR was
performed with both primer pairs on both human cDNA and chicken cDNA. No
amplification was observed when chicken primers were used in combination with human
cDNA and vice versa, whereas specific products were formed with species-matching
primers. (e) Primer sensitivity is comparable for both species HMGB1 primer sets.
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