VEGFA amplification predicts sorafenib sensitivity in HCC

730 | CANCER DISCOVERY JUNE 2014 www.aacrjournals.org
RESEARCH ARTICLE
Human and Mouse VEGFA-Amplified
Hepatocellular Carcinomas Are Highly
Sensitive to Sorafenib Treatment
Elad Horwitz1
, Ilan Stein1,3
, Mariacarla Andreozzi5
, Julia Nemeth6
, Avivit Shoham1
, Orit Pappo3
,
Nora Schweitzer12
, Luigi Tornillo5
, Naama Kanarek1
, Luca Quagliata5
, Farid Zreik3
, Rinnat M. Porat3
,
Rutie Finkelstein3
, Hendrik Reuter7
, Ronald Koschny11
, Tom Ganten11
, Carolin Mogler9
,
Oren Shibolet4
, Jochen Hess6,8,10
, Kai Breuhahn9
, Myriam Grunewald2
, Peter Schirmacher9
,
Arndt Vogel12
, Luigi Terracciano5
, Peter Angel6
, Yinon Ben-Neriah1
, and Eli Pikarsky1,3
ABSTRACT Death rates from hepatocellular carcinoma (HCC) are steadily increasing, yet thera-
peutic options for advanced HCC are limited. We identify a subset of mouse and
human HCCs harboring VEGFA genomic amplification, displaying distinct biologic characteristics.
Unlike common tumor amplifications, this one seems to work via heterotypic paracrine interactions;
stromal VEGF receptors (VEGFR), responding to tumor VEGF-A, produce hepatocyte growth factor
(HGF) that reciprocally affects tumor cells. VEGF-A inhibition results in HGF downregulation and
reduced proliferation, specifically in amplicon-positive mouse HCCs. Sorafenib—the first-line drug
in advanced HCC—targets multiple kinases, including VEGFRs, but has only an overall mild beneficial
effect. We found that VEGFA amplification specifies mouse and human HCCs that are distinctly sensi-
tive to sorafenib. FISH analysis of a retrospective patient cohort showed markedly improved survival
of sorafenib-treated patients with VEGFA-amplified HCCs, suggesting that VEGFA amplification is a
potential biomarker for HCC response to VEGF-A–blocking drugs.
SIGNIFICANCE: Using a mouse model of inflammation-driven cancer, we identified a subclass of HCC
carrying VEGFA amplification, which is particularly sensitive toVEGF-A inhibition.We found that a simi-
lar amplification in human HCC identifies patients who favorably responded to sorafenib—the first-line
treatment of advanced HCC—which has an overall moderate therapeutic efficacy. Cancer Discov; 4(6);
730–43. ©2014 AACR.
See related commentary by Luo and Feng, p. 640.
Authors’ Affiliations: 1
The Lautenberg Center for Immunology; 2
Depart-
ment of Developmental Biology and Cancer Research, IMRIC, Hadassah
Medical School, Hebrew University; 3
Department of Pathology, Hadassah-
Hebrew University Medical Center, Jerusalem; 4
Liver Unit, Tel Aviv Sour-
asky Medical Center, Tel Aviv, Israel; 5
Institute of Pathology, University
Hospital Basel, Basel, Switzerland; 6
Division of Signal Transduction and
Growth Control (A100), 7
Division of Molecular Genetics (B060), and 8
Jun-
ior Group Molecular Mechanisms of Head and Neck Tumors (A102), Ger-
man Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance; 9
Institute of
Pathology, University Hospital Heidelberg; Departments of 10
Otolaryngol-
ogy, Head and Neck Surgery and 11
Internal Medicine, University Hospital
Heidelberg, Heidelberg; and 12
Department of Gastroenterology, Hepatology
and Endocrinology, Hannover Medical School, Hannover, Germany
Note: Supplementary data for this article are available at Cancer Discovery
Online (http://cancerdiscovery.aacrjournals.org/).
Corresponding Authors: Eli Pikarsky, The Lautenberg Center for Immunol-
ogy, IMRIC, Hadassah Medical School, Hebrew University, Kiryat Hadas-
sah, P.O.B. 12272, Jerusalem 91120, Israel. Phone: 972-2-675-8202; Fax:
972-2-642-6268; E-mail: peli@hadassah.org.il; and Yinon Ben-Neriah,
The Lautenberg Center for Immunology, IMRIC, Hadassah Medical School,
Hebrew University, Kiryat Hadassah, P.O.B. 12272, Jerusalem 91120,
Israel. Phone: 972-2-675-8718; E-mail: yinonb@ekmd.huji.ac.il
doi: 10.1158/2159-8290.CD-13-0782
©2014 American Association for Cancer Research.
on March 27, 2016. © 2014 American Association for Cancer Research.cancerdiscovery.aacrjournals.orgDownloaded from
Published OnlineFirst March 31, 2014; DOI: 10.1158/2159-8290.CD-13-0782

JUNE 2014 CANCER DISCOVERY | 731
INTRODUCTION
Hepatocellular carcinoma (HCC) is the third leading cause of
cancer-related mortality worldwide, with the highest increase
rate in North America (1, 2). Sorafenib is the mainstay of
therapy for advanced HCC and the only systemic drug that has
shown any survival advantage in HCC so far (3, 4). However,
patients’ response is modest, and sorafenib treatment is asso-
ciated with side effects (3–8). Thus, several studies looked for
predictive markers for sorafenib response (9–11), yet no such
biomarkers have entered the clinical setting. Predictive biomar-
kers, identifying patient subsets to guide treatment choices, are
usually based on distinct pathogenetic mechanisms, and are
the cornerstone of personalized medicine (12, 13). Prominent
examples include ERBB2 amplification and KRAS mutations
that serve as key determinants of treatment with trastuzumab
or cetuximab, respectively (14, 15).
Sorafenib is a multikinase inhibitor, the targets of which
include BRAF, CRAF, PDGFR2, c-KIT, and the VEGFA recep-
tors (VEGFR; refs. 10, 16). Although VEGFRs were postulated
as mediators of sorafenib response in HCC, testing for VEGF-A
serum levels was not found to be predictive of sorafenib
treatment success (10). Moreover, bevacizumab, an antibody
against VEGF-A, only shows minimal responses in HCC
(17–20). A possible explanation for this is that the mecha-
nism of action of sorafenib is predominantly independent of
VEGF-A inhibition. Alternatively, a small subset of patients
who can respond to VEGF-A blockers may exist that was
underrepresented in bevacizumab studies.
VEGF-A is a master regulator of angiogenesis whose role
in tumor vessel recruitment is well established (21). However,
additional roles for VEGF-A in tumorigenesis are emerging.
VEGF-A was shown to promote tumor cell growth in an
autocrine manner, in skin and lung cancer cells expressing
VEGFRs (22, 23). Moreover, VEGF-A also has nonangiogenic
functions in normal physiology. In liver tissue, VEGF-A elicits
hepatocyte proliferation by elevating expression of hepato-
cyte mitogens in liver sinusoidal endothelial cells (24, 25).
HCC is most commonly the outcome of chronic injury and
inflammation, resulting in hepatocyte regeneration and dys-
regulated growth factor signaling (1, 26). It has become clear
that inflammatory signaling pathways can support survival,
growth, and progression of cancer. Accordingly, secreted
cytokines and effector molecules, which are abundant in the
tumor microenvironment, could constitute suitable targets
for treatment and primary prevention of HCC (26). Here, we
used Mdr2-deficient mice (Mdr2−/−
), which develop chronic
liver inflammation, eventually leading to inflammation-
induced liver tumors similar to human HCC (27, 28), to look
for candidate treatment targets modulating the tumor micro-
environment that are relevant to human HCC.
RESULTS
Array CGH Reveals Recurrent Gains in the VEGFA
Locus Identifying a Molecularly Distinct Tumor
Subpopulation
In search of microenvironment-affecting factors whose
amplification or deletion plays a role in inflammation-induced
HCC development, we applied array-based comparative
genomic hybridization (aCGH) to 10 HCCs obtained from
16-month-old Mdr2−/−
mice (Supplementary Fig. S1A). We
detected several amplifications and deletions, including an
amplification in the qB3 band of murine chromosome 17
(Chr17qB3; Supplementary Table S1), encoding among oth-
ers the gene for VEGF-A. Genomic amplification of Vegfa was
of interest as it is a cytokine gene that can modulate several
components of the tumor microenvironment and induce liver
cell growth (24, 25). To determine the frequency of this ampli-
fication, we tested a larger cohort of Mdr2−/−
tumors by quan-
titative PCR (qPCR) of tumor DNA (Supplementary Fig. S1B)
and chromogenic in situ hybridization (CISH; Fig. 1A); 13 of
93 (∼14%) HCCs harbored this amplicon. To map the minimal
amplified region of this amplicon, we used DNA qPCR directed
at several loci along murine chromosome 17 in a cohort of
tumors bearing this amplification (Fig. 1B). We found that
the common proximal border lies between 43.3 and 48.5 mega
base pairs (Mbp) from the chromosome 17 start. This minimal
amplified region spanned 53 genes (Supplementary Table S2).
Amplification of human Chr6p21 (the region syntenic for
murine Chr17qB3) and whole chromosome gains were previ-
ously reported in several whole-genome analyses of human
HCC with a frequency ranging between 7% and 40% (29–33).
Accordingly, through FISH, we found VEGFA amplifications
and chromosome 6 polysomies in 11% of human HCCs we
tested (21 of 187; Fig. 1C; Supplementary Table S3).
To elucidate the tumor relevance of these chromosomal
gains, we analyzed the expression of mouse Chr17qB3 ampli-
con genes in amplicon-positive (herein Amppos
) and amplicon-
negative (Ampneg
) tumors. Matched increases in mRNA and
DNA levels were found for Vegfa, Tjap1, and Xpo5 (Fig. 1D
and Supplementary Fig. S2A). We further found a correlation
between Chr17qB3 amplification and VEGF-A protein levels
in both tumor extracts and serum (Fig. 1E and Supplemen-
tary Fig. S2B). Furthermore, double immunostaining for
VEGF-A and E-cadherin in Amppos
tumors showed that the
tumor cells are indeed the main origin of VEGF-A in these
tumors (Fig. 1F). Thus, amplification of murine Chr17qB3 is
a recurrent event in HCC associated with an elevated expres-
sion of several resident genes, including Vegfa.
Amppos
Tumors Display Distinct
Histologic Features
Tumor cell proliferation is a strong and consistent marker
of poor prognosis in human HCC (34). Bromodeoxyuridine
(BrdUrd) immunostaining revealed that Amppos
mouse HCCs
displayed a 2-fold higher proliferation index compared with
Ampneg
tumors (Fig. 2A and B, top). No differences were
noted in apoptosis (Supplementary Fig. S2C) or in the pres-
ence of neutrophils or fibroblasts (data not shown). Other
histologic features, differing between Amppos
and Ampneg
mouse HCCs, include a 6-fold higher vessel density (Fig.
2A and B, middle) and 4-fold higher macrophage content
(Fig. 2A and B, bottom). Moreover, several signature genes
of tumor-associated macrophages (TAM), including those
encoding Arginase 1, TGFβ, and YM1 (35, 36), were elevated
in the Amppos
group (Fig. 2C), whereas markers of classically

Horwitz et al.RESEARCH ARTICLE
A B
n.s.
WT
liver
Amp
neg
tumor
Amppos
tumor
***
** ***
*** ***
Cul9
D E
0
1
2
3
4
5
6
7
1 2 1 2 3 4 1 2 3 4
Vegfa mRNA (qPCR)
VEGF-A protein (ELISA)
Foldinduction(qPCR)pg/µgprotein(ELISA)
Mdr2–/–
tumors
C
Normal Polysomic Amplified
Murine HCCs
Tumor #1 Tumor #2
Tumor #3
HumanHCCs
Tumor #4
Ampneg
Amppos
Mbpfromchromosome17start
Murine Amppos
HCCs
43.3
43.8
44.4
45.0
45.7
45.9
46.16
46.4
46.9
48.5
46.15–46.17Vegfa
45.82–45.84Mrpl14
45.78–45.82Tmem63b
45.77–45.79Capn11
46.39–46.41Tjap1
46.33–46.37Xpo5
45.71–45.71Hsp90ab1
46.24–46.26Mrps18a
45.69–45.70Nfkbie
46.43–46.44Egfl9
45.64–45.66Aars2
44.36–44.41Cdc5L
46.63–46.68Parc
46.86–46.87Gnmt
47.4
47.9
Amplified
Nonamplified
Minimal amplified region
F
Hoechst E-cadherin VEGF-A Merge
Amppostumor
10
8
6
4
2
0
5
4
3
2
1
0
Cdc5L
Tjap1 Xpo5
Hsp90ab1 Vegfa
6
4
2
0
Relativeexpression
40
30
20
10
0
8
6
4
2
0
6
4
2
0
Relativeexpression
Relativeexpression
Relativeexpression
Relativeexpression
Relativeexpression
WT
liver
Amp
neg
tumor
Amppos
tumor
WT
liver
Amp
neg
tumor
Amppos
tumor
WT
liver
Amp
neg
tumor
Amp
pos
tumor
WT
liver
Amp
neg
tumor
Amp
pos
tumor
WT
liver
Amp
neg
tumor
Amp
pos
tumor
WT
liver
Ampneg
Amppos
Figure 1. A recurrent gain in the VEGFA locus identifying a molecularly distinct tumor subpopulation. A, representative photomicrographs of CISH
using probes specific for the murine Chr17qB3. B, DNA qPCR analysis using primers specific for different loci on the qB3 arm of chromosome 17. Each
vertical line represents a single Amppos
tumor. The thin line represents nonamplified regions, and the thick line represents amplified regions. The list
includes several of the residing genes (a full list is available as Supplementary Data). C, representative photomicrographs of FISH of human HCC using
Chr6p12 probe (red) and chromosome 6 centromere probe (green). D, qPCR analysis of the mRNA levels of murine genes encoded on the amplified region.
Each dot represents a different tumor. The cross line signifies geometric mean (n.s., not significant; **, P < 0.01; ***, P < 0.0001). E, qPCR and ELISA
analyses of VEGF-A performed on extracts of wild-type (WT) livers, Ampneg
, and Amppos
Mdr2−/−
tumors in matching pairs show a correlation between
the increase in mRNA and protein levels. F, confocal microscopy images of an Mdr2−/−
Amppos
tumor immunostained for E-cadherin and VEGF-A. Hoechst
33342 marks nuclei. Scale bar, 20 μm.

VEGFA Amplification in HCC RESEARCH ARTICLE
Figure 2. Amppos
tumors are a distinct tumor subpopulation. A, representative IHC photomicrographs for BrdUrd, vWF, and F4/80. Scale bar, 50
μm (BrdUrd) and 100 μm (vWF and F4/80). B, IHCs were quantified using automated image analysis (n ≥ 7; *, P < 0.01; **, P < 0.05). C, qPCR analysis of
tumor-associated (protumorigenic) and classically activated (antitumorigenic) macrophage markers. Each dot represents a different tumor. The cross line
signifies geometric mean (n.s., not significant; **, P < 0.01; ***, P < 0.001). D, immunofluorescent stain for MRC1 on Ampneg
and Amppos
tumors. Scale bar,
40 μm. E, quantification of MRC1 immunofluorescence. Bars represent geometric mean; **, P < 0.01.
B
C
n.s.n.s.
n.s.
Antitumorigenic
markers
***** **
Protumorigenic
markers
Ampneg tumor Amppos tumor
Amppos
tumor
Ampneg
tumor
WT liver
Ampneg tumor Amppos tumor
BrdUrd
0
5
10
15
20
vWF
Percentagepositivenuclei
**
BrdUrd
vWF
0
2
4
6
8
10
12
14
Percentagepositivearea
vWF
*
F4/80
0
2
4
6
8
10
12
14
*
F4/80
A
D
MRC1
E MRC1
**
1.5
Arg1
Nos2 Cxcl10
Tgfb Ym1
Tnfa
8 40
30
20
10
0
6
4
2
0
1.0
0.5
RelativeexpressionRelativeexpression
Relativeexpression
Relativeexpression
Relativeexpression
Relativeexpression
0.0
600 50
40
30
20
10
0
15
10
5
0
60
40
20
0
400
200
15
10
5
0
WT
liver
Amp
neg
tumor
Amp
pos
tumor
WT
liver
Amp
neg
tumor
Amppos
tumor
WT
liver
Ampneg
tumor
Amppos
tumor
WT
liver
Amp
neg
tumor
Amp
pos
tumor
WT
liver
Amp
neg
tumor
Amp
pos
tumor
Amp
neg
Amp
pos
WT
liver
Amp
neg
tumor
Amp
pos
tumor

activated macrophages, including TNFα, inducible nitric
oxide synthase (iNOS), CXCL10, and IL12p40, were similar
in both tumor groups (Fig. 2C and Supplementary Fig. S3A).
Along with these, immunofluorescent staining for the protu-
morigenic macrophage marker MRC1 revealed a higher pres-
ence of MRC1-expressing cells in Amppos
tumors (Fig. 2D and
E). Of note, VEGF-A was shown to act as a chemoattractant
for naïve myeloid cells, which facilitate the generation of new
blood vessels (37). Together, these data signify Amppos
tumors
as a distinct subgroup of HCCs characterized by enhanced
presence of specific microenvironmental components and
increased proliferation rate.
Histologic analysis of human HCCs revealed several char-
acteristic traits of HCCs harboring VEGFA gains. A higher
incidence of vascular invasion was found in the Amppos
group (9 of 20 Amppos
tumors vs. 1 of 20 Ampneg
tumors;
P < 0.01; Supplementary Table S4). A lower incidence of fibrosis
within tumor tissue was found in the Amppos
group as well. Few
additional traits nearly reaching statistical significance were
identified (Supplementary Table S4). We found no differences
in several clinical characteristics of HCC including underlying
disease, gender, or tumor size (Supplementary Fig. S4A–S4C).
Altogether, these results indicate that in both murine Mdr2−/−
and human HCCs, tumors that harbor genomic gains in the
VEGFA locus are distinct from those that do not.
Macrophage–Tumor Cell Cross-talk
in Amppos
Tumors
Previous studies have delineated hepatocyte–endothelial
cross-talk taking place in non-neoplastic liver, wherein VEGF-A
stimulates endothelial cells to secrete several mitogens includ-
ing hepatocyte growth factor (HGF; refs. 24, 25). We hypothe-
sized that Amppos
HCCs exploit this interaction for promoting
tumor cell proliferation. Following this notion, we detected a
3-fold elevation of Hgf mRNA levels in Amppos
versus Ampneg
mouse tumors (Fig. 3A). We did not find significant changes
in other angiocrine-produced molecules (24, 25)—Wnt2, IL6,
and heparin binding EGF-like growth factor (HB-EGF; data
not shown). Immunostaining detected HGF expression only
in Amppos
tumors, exclusively in the non-neoplastic stromal
cells (Fig. 3B). Immunofluorescent staining for von Willebrand
Factor (vWF), F4/80, and HGF suggested that macrophages are
the major cell type expressing HGF (Fig. 3C).
To understand the VEGF-A–HGF relationship, we isolated
hepatocytes and macrophages from Mdr2−/−
livers at the age
of 8 months (Fig. 3D and Supplementary Fig. S3B), a time
point signified by marked dysplasia, yet no overt HCC for-
mation (27). mRNA profiling of these fractions showed that
the genes encoding VEGFRs (FLT1 and KDR) and corecep-
tors [Neuropilin (Nrp)1 and 2] were higher in macrophages
whereas the HGF receptor (c-MET) was more abundant in
hepatocytes (Fig. 3E). This aligns with previous work showing
that hepatocytes are inert to direct activation with VEGF-A
(24). Immunostaining for KDR in Amppos
tumors demon-
strated that its expression in these tumors was restricted to
non-neoplastic cells (Fig. 3F). This correlated with a modest
increase in mRNA levels of both Kdr and Flt1 in total tumor
lysates, which was comparable with the increase in mRNA
levels of recruited macrophage and endothelial markers Msr1
and Cd105, respectively (Fig. 3G). Recapitulating this interac-
tion in vitro, we treated peritoneal macrophages with recom-
binant VEGF-A and detected an increase in Hgf mRNA levels
(Fig. 3H). This raises the possibility that VEGF-A in Amppos
tumors does not provide autocrine signals to hepatocytes,
but rather acts through manipulation of the microenviron-
ment to induce HGF secretion.
VEGF-A Increases Cellular Proliferation in HCC
To prove the functional role of VEGFA in this genomic ampli-
fication, we set out to inhibit VEGF-A in Mdr2−/−
tumors. To
this end, we injected intravenously adenoviral vectors encoding
GFP alone or GFP and the soluble VEGFR1 (sFLT), a potent
inhibitor of VEGF-A (38), into 56 tumor-harboring Mdr2−/−
mice of ages 14 to 18 months and sacrificed them 10 days fol-
lowing injection (Supplementary Fig. S5A). Vegfa amplification
status was determined after sacrifice by both DNA qPCR and
Vegfa mRNA expression (Fig. 4C and data not shown). Liver
damage, measured through plasma aspartate aminotransferase
(AST) activity, was similar in all groups (Supplementary Fig.
S5B). Immunostaining for BrdUrd, Ki67, and phosphorylated
histone H3 (pHH3) revealed that blocking VEGF-A markedly
inhibited tumor cell proliferation in Amppos
tumors, but not
in Ampneg
ones (Fig. 4A and B and Supplementary Fig. S5C
and S5D). This decrease in proliferation was accompanied by
reduced Hgf mRNA levels (Fig. 4C). Treatment with adenovi-
rus encoding GFP alone did not induce any change in either
group. Macrophage infiltration and protumorigenic macro-
phage expression profile did not decrease following the sFLT
treatment (Supplementary Fig. S5C and S5D and data not
shown). Macroscopic and histologic analyses revealed multiple
foci of coagulative necrosis only in sFLT-treated Amppos
tumors
(3 of 6 vs. 0 of 10 in Ampneg
; P < 0.05; Fig. 4D). In these specific
tumors, we also found an elevation of the hypoxia-inducible
factor-1α (HIF1α) target genes Glut1 and Pgk1 (Fig. 4C), indica-
tive of tissue hypoxia. Immunostaining for vWF revealed a
trend of decrease in vasculature only in VEGF-A–blocked
Amppos
tumors, particularly in the hypoxic tumors (Supple-
mentary Fig. S5C and S5D). These data denote Amppos
tumors
as hypersensitive to direct inhibition of VEGF-A.
Overexpression of VEGF-A in HCC Cells Increases
Proliferation Only In Vivo
To further substantiate the tumor–stroma relationship with
respect to VEGF amplification, in particular hepatocyte VEGF-
A eliciting a macrophage HGF–heterotypic circuit in tumor
growth, we injected human Hep3B HCC cells transduced with
a lentivector overexpressing human VEGF-A into immuno-
deficient mice (Supplementary Fig. S6A). The in vivo growth
rate of VEGF-A–overexpressing cells was higher than control
vector–transduced cells (Fig. 5A and Supplementary Fig. S6B).
This correlated with higher proliferation rate and increased
HGF expression (Fig. 5B–D and Supplementary Fig. S6C and
S6D), phenocopying the Amppos
Mdr2−/−
HCCs. Supporting
the non–cell-autonomous role, VEGF-A overexpression did
not affect the in vitro growth rate of Hep3B cells (Fig. 5E). We
also found no difference in expression of the hypoxia markers
prolyl hydroxylase domain 3 (PHD3) and lactate dehydroge-
nase A (LDHA) in these xenografts, lending further support to
a nonangiogenic role for VEGF-A in HCC (Supplementary Fig.
S6E). Immunofluorescence confirmed VEGF-A expression in

Figure 3. A macrophage-
tumor cell cross-talk within
Amppos
tumors. A, qPCR
analysis of Hgf mRNA in WT
livers, Ampneg
, and Amppos
tumors. The cross line signi-
fies geometric mean (**,
P < 0.001). B, representative
photomicrographs of IHC for
HGF on Ampneg
and Amppos
tumors. Scale bar, 100 μm.
C, representative confocal
microscopy images of Amppos
tumor immunostained for
vWF, F4/80, and HGF. Hoechst
33342 marks nuclei. Scale
bar, 40 μm. D, representative
cytospin preparations of
macrophage and hepatocyte
fractions from Mdr2−/−
liv-
ers. E, qPCR analysis of
hepatocyte and macrophage
fractions (n = 11 different
mice), isolated from livers of
Mdr2−/−
mice. Bars represent
geometric mean (**, P < 0.001;
***, P < 0.0001). F, representa-
tive photomicrograph of IHC
for the VEGFR KDR in Ampneg
and Amppos
tumors. Note
that the expression of KDR is
confined to endothelial and
stromal cells. Scale bar, 100 μm.
G, mRNA qPCR analysis
for the VEGFRs Kdr and Flt1
and the macrophage and
endothelium markers Msr1 and
Cd105 on tissue lysates from
the indicated groups. Each dot
represents a different tumor.
The cross line represents geo-
metric mean (***, P < 0.0001).
H, peritoneal macrophages
were cultured under serum-free
conditions followed by 8 hours
of exposure to recombinant
murine VEGF-A (100 ng/mL).
Hgf expression was measured
by qPCR. Bars represent
geometric mean; n ≥ 3;
*, P < 0.05.
C
B
HGF
**
D
FE
KDR
**
***
0
2
4
6
8
10
12
14
16
18
Flt1 Kdr Nrp1 Nrp2 Met
Relativeexpression
Hepatocytes
Macrophages
**
**
**
***
Kdr
***
Flt1
***
Msr1
***
Cd105
A
G
H&E
H
*
Hgf
Peritoneal
macrophages
vWF F4/80 HGF
Macrophages Hepatocytes
Merge
Ampneg tumor
Ampneg
Amppos tumor
Amppos
Amppostumor
WT
Hgf
6
4
2
Relativeexpression
0
liver
Amp
neg
tumor
Amppos
tumor
3 5
2.5
2.0
1.5
1.0
0.5
0.0
NT 8h rVEGF-A
4
3
2
1
0
2
1
Relativeexpression
0
10 3
2
1
0
8
6
4
2
0
WT
liver
Amp
neg
tumor
Amp
pos
tumor
WT
liver
Amp
neg
tumor
Amp
pos
tumor
WT
liver
Ampneg
tumor
Amp
pos
tumor
WT
liver
Amp
neg
tumor
Amp
pos
tumor
transduced tumor cells and revealed HGF expression in mac-
rophages (Supplementary Fig. S7A).
Next, we generated single-cell suspensions from VEGF-A–
overexpressingorcontrolHep3BZsGreen-labeledxenografts
andisolatedmacrophages(ZsGreen−
CD45+
F4/80+
)andendo-
thelial cells (ZsGreen−
CD45−
Meca32+
) using FACS sorting.
qPCR analysis of the macrophage-specific and endothelium-
specific genes, Msr1 and Cd105, respectively, confirmed
successful separation of cell populations (Supplementary
Fig. S7B). Although VEGF-A and control tumor groups
did not show significant differences in the TAM genes Tgfb,
Ym1, Tnfa, and Nos2 (Supplementary Fig. S7C), we found
a 2-fold increase in Hgf expression in macrophages, but
not endothelial cells, isolated from VEGF-A–overexpress-
ing tumors (Fig. 5F). Thus, VEGF-A overexpression by HCC
cells is sufficient to induce upregulation of HGF, mostly in

Figure 4. VEGF-A inhibition impedes proliferation in Amppos
tumors. Mdr2−/−
mice were treated with adenovectors expressing either GFP alone or GFP
and sFLT for 10 days. A, representative photomicrographs of IHC for BrdUrd. Tumor-infiltrating cells remain proliferative. Scale bar, 100 μm. B, BrdUrd
immunostaining was quantified using automated image analysis (n ≥ 6; *, P < 0.05). C, mRNA qPCR analysis of the indicated genes in Ampneg
and Amppos
tumors treated with the indicated adenovectors. Each dot represents a different tumor. The cross line signifies geometric mean (***, P < 0.0001). D, left,
histologic section stained with H&E depicting necrosis, representing three out of the six sFLT-treated Amppos
tumors. Scale bar, 500 μm. Right, a macro-
scopic picture of a tumor with hemorrhagic necrosis. Scale bar, 0.5 cm.
C D
B
***
0
1
2
3
4
5
BrdUrd
Ampneg
GFP
Ampneg
sFLT
Amppos
GFP
Amp
pos
sFLT
BrdUrd
*
A
8
30
20
10
0
5
4
3
2
1
0
6
4
2
0
Relativeexpression
8
6
4
2
0
Relativeexpression
Relativeexpression
Relativeexpression
Amp
neg
sFLT
Amp
neg
GFP
Amppos
GFP
Amppos
sFLT
Ampneg
sFLT
Amp
neg
GFP
Amp
pos
GFP
Amp
pos
sFLT
Amp
neg
sFLT
Ampneg
GFP
Amppos
GFP
Amppos
sFLT
Amp
neg
sFLT
Ampneg
GFP
Amppos
GFP
Amppos
sFLT
Vegfa
Glut1 Pgk1
Hgf
Ampneg GFP Ampneg sFLT Amppos GFP Amppos sFLT
H&E
Amppos sFLT
Figure 5. VEGF-A overexpression enhances tumor cell proliferation. Immunodeficient mice were subcutaneously injected with Hep3B cells transduced
with control vector or with a human VEGF-A vector. A, growth curve of xenografts transduced with control vector (black line) or with VEGF-A lentivec-
tor (gray line). Tumor volumes were measured topically with a caliper (*, P < 0.05; **, P < 0.01). B, representative photomicrographs of IHC for BrdUrd in
control or VEGF-A lentivector-transduced xenografts. Scale bar, 100 μm. C, BrdUrd immunostaining was quantified using automated image analysis (n ≥
3; *, P < 0.05; a representative experiment of two performed is shown). D, mRNA qPCR analysis in control or VEGF-A lentivector-transduced xenografts.
Bars represent geometric mean (n ≥ 4; *, P < 0.05; **, P < 0.01). E, XTT in vitro proliferation assay of the indicated lentivector-transduced cultured cells.
F, expression of Hgf in macrophage and endothelial fractions from tumors overexpressing VEGF-A and controls determined by qPCR. Bars represent
geometric mean; n ≥ 3; *, P < 0.05; ND, not detected—amplification did not occur in any of the sample’s wells.
D
B
E
A C
F
*
ND ND
Mac. End. Mac. End.
Control
tumor
VEGF-A
tumor
Control VEGF-A
BrdUrd
*
Control
VEGF-A
Control
VEGF-A
2,000
1,800
1,600
1,400
1,200
1,000
800
600
400
200
0
6 1.9
Control
VEGF-A
BrdUrd
35
30
25
20
15
10
5
0
Hgf
2.5
2.0
1.5
1.0
0.5
0.0
1.7
1.5
1.3
1.1
0.9
0.7
0.5
0 1 2 3
Days in culture
4
Control
VEGF-A
5 6
Tumorvolume(mm3
)
Relativeexpression
Absorbance(450nm)
Relativeexpression
5
4
3
2
1
0
0 10
**
*
**
*
*
**
Days
hVEGFA mHgf
20 30

macrophages, and leads to increased proliferation of tumor
cells.
Profiling Proangiogenic-Factor
Expression in Mdr2−/−
HCC
As Ampneg
tumors did not respond to sFLT treatment, we
profiled other proangiogenic factors that can support tumor
vascularization. mRNA qPCR analysis of the genes encod-
ing angiopoietin 1 and 2, angiopoietin like 2, FGF 1 and 2,
platelet-derived growth factor (PDGF)-A, -B, and -C, placental
growth factor (PLGF), and VEGF-B revealed that several of
these factors were overexpressed in Mdr2−/−
HCCs compared
with normal livers, irrespective of the amplicon status. Nota-
bly, Pdgfc levels were significantly higher (2.4-fold) in Ampneg
compared with Amppos
tumors (Fig. 6). This is in line with
a previous report showing that PDGF-C can promote ang-
iogenesis in a VEGF-A–independent manner (39) and could
provide a plausible explanation for the lack of effect on vessel
density in Ampneg
tumors in response to sFLT.
Murine Amppos
Tumors Are Uniquely
Sensitive to Sorafenib
Sorafenib is the only systemic drug showing a clinical
advantage in patients with advanced HCC who are not eligi-
ble for local therapies and is a first-line treatment for these
patients (3). As sorafenib inhibits VEGFRs and BRAF—a
downstream effector of both VEGFRs and the HGF receptor
c-MET (21, 40, 41)—we tested whether sorafenib may have a
selective advantage in Amppos
tumors. We treated 58 Mdr2−/−
mice ages 14 to 18 months with sorafenib or vehicle alone
for 3 days, after which they were sacrificed and tumor tissue
was analyzed (experimental design depicted in Supplemen-
tary Fig. S5A). Amplification status was assessed after sac-
rifice by DNA qPCR and verified by Vegfa mRNA expression
(Fig. 7C and data not shown). Immunostaining for BrdUrd
and pHH3 demonstrated decreased proliferation in soraf-
enib-treated mice in Amppos
but not Ampneg
HCCs (Fig. 7A
and B and Supplementary Fig. S8A and S8B). Similar to
VEGF-A inhibition, Hgf levels were decreased only in soraf-
enib-treated Amppos
tumors (Fig. 7C). Although no effects
were observed on tumor macrophage density (Supplemen-
tary Fig. S8A and S8B), expression of the TAM marker TGFβ
was decreased in sorafenib-treated Amppos
tumors (Sup-
plementary Fig. S8C). Blood vessel density changes or signs
of hypoxia were absent (Supplementary Fig. S8A–S8C),
possibly due to the short treatment duration, implying
that the early inhibitory effect of sorafenib in VEGF-A–
amplified tumors may be independent of angiogenesis.
Figure 6. Angiogenic factors elevated in murine Mdr2−/−
HCC. mRNA qPCR analysis of Mdr2−/−
tumors for the indicated angiogenesis regulators. Values
shown are fold over normal livers’ average. Each dot represents a different tumor. The cross line signifies geometric mean, significance marks immedi-
ately above each group refer to comparison with normal livers. Topmost significance marks represent the comparison between Ampneg
and Amppos
tumors
(n = 4 normal liver; n ≥ 6 in tumor groups; n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001). PDGF, platelet-derived growth factor.
Angpt1 Angpt2 Angptl2 Fgf1 Fgf2
n.s.
50
40
30
20
10
5
RelativetonormalliverRelativetonormalliver
4
3
2
1
0
n.s.n.s.
*
*n.s.
n.s.
n.s.n.s.
n.s.
n.s.n.s.
n.s.
*****
Pdgfa Pdgfb Pdgfc Pgf Vegfb
n.s.
****
n.s.
n.s.* *
n.s.
*
n.s.
n.s.n.s.
n.s.
n.s.*
Ampneg Amppos
Ampneg Amppos
Ampneg Amppos
Ampneg Amppos
Ampneg Amppos
Ampneg Amppos
Ampneg Amppos
Ampneg Amppos
Ampneg Amppos
Ampneg Amppos
80
70
60
50
40
30
20
10
5
4
3
2
1
0

Figure 7. Amppos
tumors are uniquely sensitive to sorafenib. A, representative BrdUrd immunostains. Scale bar, 100 μm. B, BrdUrd immunostaining was
quantified using automated image analysis (n ≥ 6; *, P < 0.05). C, qPCR analysis of Ampneg
and Amppos
tumors treated as indicated. Each dot represents a
different tumor. The cross line signifies geometric mean (n.s., not significant; *, P < 0.01). D, growth curves of xenografts transduced with control (dashed
lines) or VEGF-A (solid lines) lentivectors, treated daily with sorafenib (gray lines) or nontreated (NT, black lines). Tumor volumes were measured with
a caliper (n ≥ 6; *, P < 0.05). E, Kaplan–Meier curves showing survival of resected HCC patients negative (n = 96) or positive (n = 14) for VEGFA gain
(p log-rank > 0.05). F, Kaplan–Meier curves showing survival of patients with HCC treated with sorafenib, negative (n = 47, 10 months median) or positive
for VEGFA gain (n = 7; median undefined; plog-rank = 0.029). G and H, genomic gains in VEGFA promote tumorigenesis through the microenvironment.
G, an increase in VEGFA gene copy number in liver tumor cells leads to elevated VEGF-A secretion. VEGF-A modulates the tumor microenvironment in
favor of tumor cell growth through several modes: (i) recruitment of TAMs expressing the mitogen HGF and (ii) activating the liver endothelium to secrete
angiocrine factors and enhance the tumor blood supply. H, inhibition of VEGF-A through soluble receptor or sorafenib results in decreased HGF signaling
and blood supply, impeding tumor growth.
Resected patients treated with sorafenib
B
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2 *
BrdUrd Ampneg
vehicle
Ampneg
sorafenib
Amppos
vehicle
Amppos
sorafenib
BrdUrd
Amp
neg
Veh
Amp
neg
Sor
Amp
pos
Veh
Amp
pos
Sor
C
*
Hgf
0
1
2
3
4
Relativeexpression
Vegfa
0
2
4
6
8
10
Relativeexpression
n.s.
A
Resected HCC patients
Percentagesurvival
VEGFA gains
Normal VEGFA
VEGFA gains
Normal VEGFA
FE
HG
Daily treatment period
Tumorvolume(mm2
)
D 1,500
Control - NT
Control - Sorafenib
VEGFA - NT
VEGFA - Sorafenib
1,000
500
0
0
100
50
0
0 1,000 2,000
Days
3,000 4,000 5,000 0
0
Percentagesurvival
100
50
Plogrank = 0.029
500 1,000
Days
1,500 2,000
5 10
Days
15 20
*
*
Amp
neg
vehicle
Amp
neg
sorafenib
Amp
pos
vehicle
Amp
pos
sorafenib
Amp
neg
vehicle
Amp
neg
sorafenib
Amp
pos
vehicle
Amp
pos
sorafenib
VEGFA–amplified
malignant hepatocyte
Protumorigenic
macrophages
Angiocrine
factors
HGF
VEGF-A
Activated
endothelial
cells
VEGF-A
VEGF receptors
VEGF genomic region
HGF
C-Met
VEGFA–amplified
malignant hepatocyte
Decrease in mitogensAngiocrine
factors
HGF
VEGF-A
Decreased
vascularity
Soluble VEGF receptor
Sorafenib

Ampneg
tumors did not show any measurable response to
sorafenib.
In addition, we treated mice bearing Hep3B xenografts,
with or without VEGF overexpression, with sorafenib for 10
days. Similar to the Mdr2−/−
HCCs, this treatment markedly
reduced growth, proliferation, and HGF expression in VEGF-
A–overexpressing HCCs but did not affect control HCCs (Fig.
7D and Supplementary Fig. S9A–S9D). Despite the longer
duration of treatment, we still could not detect changes in
macrophage or blood vessel densities (Supplementary Fig.
S10A–S10C). Of note, a differential response to sorafenib was
not evident in vitro, emphasizing the importance of microen-
vironmental factors (Supplementary Fig. S10D).
Beneficial Effect of Sorafenib Treatment in Human
Patients with HCCs Bearing VEGFA Gains
Noting the predictive potential of Vegfa gains in the mouse
model, we analyzed samples from patients with HCC who
underwent tumor resection. This retrospective cohort was col-
lected from three different centers. To assess the correlation
between VEGFA gain and survival, we analyzed human tumor
samples by FISH (Fig. 1C). Survival of patients with HCC who
did not receive sorafenib was independent of VEGFA status
(Fig. 7E). However, markedly improved survival was seen in
the VEGFA-gain group compared with the non-gain group
in sorafenib-treated patients (indefinable median survival
from sorafenib treatment start and 11 months, respectively;
p log-rank = 0.029; Fig. 7F). Taken together, our mouse data
and retrospective analysis of a human cohort imply that
VEGFA gains correlate with a particularly beneficial response
to sorafenib, and possibly other VEGF-A inhibitors, in HCC.
DISCUSSION
Using a mouse model of inflammation-induced HCC,
we identified and characterized a unique group of HCC in
humans and mice. These tumors are defined by genomic gains
of a region encompassing VEGFA, and are distinct in histologic
appearance, rate of proliferation, and microenvironmental
content. We delineated cytokine-based heterotypic cross-talk
between malignant Amppos
hepatocytes and tumor stromal
cells. Importantly, we showed that mouse Amppos
tumors are
uniquely sensitive to VEGF-A inhibition and to sorafenib. A
retrospective analysis of human HCCs indicates that genomic
gains of VEGFA can predict response to sorafenib.
The Mdr2−/−
model of inflammation-induced HCC yields
primary tumors each holding specific genetic changes. There-
fore, we denote it as a sound platform to test the pro-
tumorigenic effects of recurring changes in an unbiased
manner. Moreover, the inflammatory background in this
model is particularly relevant for studying tumor–microenvi-
ronment interactions in clinically relevant settings. Previous
work showed that systemic elevation of VEGF-A induces
proliferation of normal hepatocytes through factors secreted
from endothelial cells (24). Although hepatocytes are inert
to VEGF-A, liver sinusoidal cells respond to VEGF-A with
counter-secretion of HGF (24). Furthermore, in regenerat-
ing livers, hepatocyte proliferation was shown to depend on
VEGF-A–induced expression of HGF from endothelial cells
(25). Here, we show that recurring genomic gains in VEGFA,
detected in a subset of HCCs, can promote tumor growth
through a similar cell–cell interaction module.
TAMs are key players in tumor progression and are known
to modulate invasion, angiogenesis, immune response, and
metastasis (36). TAMs are characterized by a specific phe-
notype, distinguished by a unique expression signature.
Previous studies have shown that VEGF-A recruits myeloid
cells that play an active part in vessel growth processes (37,
42). In agreement, we observed that Amppos
tumors harbor
higher numbers of TAMs compared with Ampneg
tumors and
detected features of protumorigenic macrophages. Our find-
ings therefore raise the interesting possibility that the VEGFA
amplicon contributes to the inflammatory microenviron-
ment, which supports developing tumors.
Our data suggest that VEGFA genomic gains facilitate
tumor development by several different modes: (i) providing
a microenvironment rich in TAMs; (ii) promoting prolif-
eration via stroma-derived HGF secretion (and possibly other
cytokines as well); and (iii) enhancing tumor angiogenesis.
Interestingly, all these modes entail heterotypic cellular inter-
actions, distinguishing this particular genomic gain from
other studied amplicons (43). We maintain that the unique
sensitivity of tumors with VEGFA gains to VEGF-A block-
ade stems from these multiple protumorigenic functions of
VEGF-A (Fig. 7G and H).
An amplicon spanning VEGFA was noted in several differ-
ent human cancers (29–31, 33, 43–51). A linear correlation
was found between mRNA levels of VEGFA and extent of
amplification in human HCC (29). Amplifications in the
VEGFA locus and juxtaposed regions were associated with
advanced-stage HCC (30). In colorectal carcinoma and breast
cancer, this amplification was found in correlation with vas-
cular invasion and shorter survival (46, 51). Cumulative anal-
ysis of these reported human studies shows that gains and
amplifications of VEGFA are found in 7% to 30% of human
HCCs (29–33). Our interventional studies in the Mdr2−/−
model indicate that Vegfa is a major driver of this amplicon,
which minimally harbors 53 genes in Mdr2−/−
murine tumors
and 11 genes in humans (32). Xenograft experiments reveal
that overexpression of VEGF-A in a human HCC cell line is
sufficient to upregulate HGF expression and increase tumor
cell proliferation in vivo and that the tumor growth advantage
gained through this overexpression can be negated by sora-
fenib. Nevertheless, we cannot completely exclude the contri-
bution of other amplicon genes to tumorigenesis.
The first line of treatment for advanced HCC is the multi-
kinase inhibitor sorafenib, which prolongs median survival
by 10 to 12 weeks (3, 4). The response to sorafenib seems
to be variable, and treatment is associated with significant
side effects (3–8). Importantly, there are no clinically applied
biomarkers for predicting sorafenib response in HCC (10).
Among the multiple targets of sorafenib (16) are VEGFRs
and BRAF—a downstream effector of both VEGFRs and the
HGF receptor c-MET (21, 40). Indeed, in line with our mouse
results, sorafenib treatment in patients led to a decrease in
serum HGF (10). Unlike most tumors, advanced HCC is
usually diagnosed and treated without obtaining tumor tis-
sue, making it difficult to establish tissue-based predictive
biomarkers. On the basis of our small-scale retrospective
study, we show that VEGFA gains may predict response to

sorafenib in HCC, thus enabling the tailoring of treatment
to only those patients who may benefit from this side effect–
prone therapy. Notably, the same amplification was also
found in lung, colorectal, bone, and breast cancers (44–48);
thus, it is plausible that similar considerations could be
applied to other tumors harboring VEGFA gains.
METHODS
Human Tissue Samples and Tissue Microarray
Human HCC tissues were obtained from resected patients from
the institutes of Basel University Hospital (Basel, Switzerland), Han-
nover Medical School Hospital (Hannover, Germany), and Heidel-
berg University Hospital (Heidelberg, Germany). Clinical information
included age at diagnosis, tumor diameter, gender, and survival time
information. Examination of tumor hematoxylin and eosin (H&E)
sections was performed by an expert liver pathologist (O. Pappo).
Samples from Heidelberg were also reviewed by Heidelberg patholo-
gists (C. Mogler and P. Schirmacher). The study was approved by
each of the institutions’ ethics committees—numbered 206/05
(Heidelberg), 660-2010 (Hannover), and EKBB20 (Basel). Required
cohort size was computed by power analysis to yield a power of at
least 90% with an α value of 0.05. Construction of the tissue micro-
array (TMA) was performed as follows: tissue samples were fixed
in buffered 10% formaldehyde and embedded in paraffin. H&E-
stained sections were made from each selected primary block (named
donor blocks) to define representative tissue regions. Tissue cylinders
(0.6 mm in diameter) were then punched from the region of the
donor block with the use of a custom-made precision instrument
(Beecher Instruments). Afterward, tissue cylinders were transferred to
a 25 mm × 35 mm paraffin block to produce the TMAs. The resulting
TMA block was cut into 3-μm sections that were transferred to glass
slides by use of the Paraffin Sectioning Aid System (Instrumedics).
Sections from the TMA blocks were used for FISH analysis.
Mice
Male and female Mdr2−/−
mice on FVB background were held in
specific pathogen-free conditions. Experiments on Mdr2−/−
mice were
performed in cohorts of 10 to 20 mice each time; results show the
combined data from at least four different experiments. Wild-type
(WT) controls were age-matched FVB mice. Mouse body weights dur-
ing the experiments were 30 to 45 g. Sorafenib (Xingcheng Chemp-
harm Co., Ltd.) was administered daily (50 mg/kg) by oral gavage.
Cremophor EL (Sigma)/ethanol/water (1:1:6) was used as a vehicle.
Two hours before sacrifice, mice were injected with 10 μL BrdUrd
(Cell Proliferation labeling reagent; Amersham) per 1 g body weight.
Mice were anesthetized with ketamine and xylazine and the liver was
perfused via the heart with PBS–Heparin solution followed by 4%
formaldehyde. Following perfusion, livers were removed and sub-
jected to standard histologic procedures. Xenograft experiments were
performed by subcutaneous injection of transduced Hep3B cells (2.5
× 106
) suspended in 100 μL PBS and 100 μL Matrigel (Becton Dickin-
son) into NOD/SCID mice. Tumor volumes were assessed by external
measurement with calipers. All animal experiments were performed
in accordance with the guidelines of the institutional committee for
the use of animals for research. In all mouse experiments, the different
groups were housed together in the same cages.
Viral Vectors and Cultured Cells
Adenoviral vectors encoding GFP or GFP and sFLT—a kind gift
from David Curiel (Washington University, St. Louis, MO) and Yosef
Haviv (Hadassah Hospital, Jerusalem, Israel)—were prepared in GH354
cells using standard procedures. A titer of 109
transducing units was
injected into mice tail veins. Mice whose livers did not yield minimal
60% adenovector transduction efficiency (by tissue staining) were
excluded. Lentiviral-based vectors were prepared by subcloning the
PCR products of the human VEGFA165 gene [from cDNA of decidual
natural killer (NK) cells; a kind gift from Ofer Mandelboim, Hebrew
University, Jerusalem, Israel] into the pSC-B plasmid using the Strata-
Clone Kit (Stratagene), subsequently digested with BamHI and NotI
and subcloned into the self-inactivating lentiviral vector pHAGE (gift
of Gustavo Mostoslavsky, Boston University School of Medicine, Bos-
ton, MA) digested with BamHI and NotI. Lentivectors were produced
by cotransfection of the backbone vector plasmid with the gag-pol
and pMD.G plasmids and using standard procedures. Hep3B cells
(obtained from the ATCC) were grown in Dulbecco’s Modified Eagle
Medium (DMEM; 10% FBS). Cells were tested free of Mycoplasma
before transduction and injection. Lentivector transduction efficiency
was assessed by fluorescent microscopy and was estimated as 80%.
In vitro proliferation was determined through XTT assay (Biological
Industries) using the manufacturer’s protocol. Murine recombinant
VEGF-A (R&D Systems) was used at the concentration of 100 ng/mL.
IHC, Immunofluorescence, and ELISA
Antibodies used for tissue immunostaining throughout the
work were—vWF (dilution at 1:300; Dako), pHH3 (1:800; Upstate),
cleaved caspase-3 (1:200; Cell Signaling Technology), F4/80 (1:300;
Seroteq), HGF (1:100; R&D Systems), BrdUrd (1:200; NeoMarkers),
KDR (1:400; Cell Signaling Technology), Ki67 (1:100; NeoMarkers),
E-cadherin (1:100; Cell Signaling Technology), and VEGF (1× as
supplied; Spring). IHC was performed on 5-μm paraffin sections.
Antigen retrieval was performed in a decloaking chamber (Biocare
Medical) in citrate buffer for all antibodies except vWF and F4/80,
for which retrieval was performed with pronase (Sigma). Horserad-
ish peroxidase (HRP)–conjugated secondary antibodies for all IHC
antibodies used were Histofine (Nichirei Biosciences), except for anti-
mouse–derived antibodies that were detected with Envison (Dako).
3,3′-Diaminobenzidine (DAB; Lab Vision) was used as a chromogen.
Immunohistochemical stainings were quantitated when indicated
using an Ariol SL-50 system (Applied Imaging). For quantification
of nuclear immunostaining, the ki-sight module of the Ariol-SL50
robotic image analysis system was applied. This system designates
classifiers for positive (red-brown) and negative (azure) nuclei defined
by color intensity, size, and shape. Each tumor cell nucleus (distin-
guished by morphology) was designated as either positive or negative
by these parameters. The fraction of positive cells was calculated
from counting at least five randomly selected fields in each tumor.
Immunofluoresence was performed on snap-frozen tissue embed-
ded in OCT gel (Sakura Finetek) and sectioned to 8-μm slices.
Slides were incubated at 37°C and fixed with both acetone and 4%
paraformaldehyde sequentially. Fluorophore-conjugated secondary
antibodies used were donkey anti-goat Alexa Fluor 647 (Invitro-
gen), donkey anti-rabbit Cy2/Cy5, donkey anti-mouse Cy3, and goat
anti-rat Cy3 (The Jackson Laboratory). Hoechst 33342 was used as
a nuclear marker (Invitrogen). Antibodies used for flow FACS sort-
ing were CD45-pacific blue, F4/80-PE (both 1:50; BioLegend), and
Meca32-biotin (1:50; BioLegend) used with streptavidin APC-Cy7
(BD Biosciences). Flow cytometry–based cell sorting was performed
in a FACSAria III cell sorter (BD Biosciences). VEGF-A ELISA was
performed using Quantikine mouse ELISA kit (R&D Systems).
DNA In Situ Hybridizations
Probes for CISH analysis of mouse tumors were prepared from the
BAC clones RP24-215A3 for the murine Chromosome 17 (BACPAC
resources center). BAC clones were labeled with DIG using Nick-
Translation mix (Roche). Mouse Cot-1 DNA (Invitrogen) and soni-
cated murine genomic DNA were added to the probe for background
block. Tissues were prepared by boiling in pretreatment buffer and
digestion with pepsin (Zymed). Hybridization was performed at 37°C

overnight after denaturation at 95°C for 5 minutes. The SPoT-Light
Detection Kit (Invitrogen) was used for anti-DIG antibody and HRP-
conjugated secondary antibody.
FISH analysis for human HCCs was performed as follows. The
genomic BAC clone RPCIB753M0921Q (imaGENES), which covers the
human VEGFA gene region, was used for preparation of the FISH probe.
BAC-DNA was isolated using the Large-Construct Kit (Qiagen) accord-
ing to the instructions of the manufacturer. Isolated BAC-DNA (1 μg)
was digested with AluI restriction enzyme (Invitrogen) and labeled with
Cy3-dUTP (GE Healthcare) using the BioPrime Array CGH Kit (Invit-
rogen). The labeling reaction was assessed by NanoDrop. Labeled DNA
was purified with the FISH Tag DNA Kit (Invitrogen). TMAs and whole-
tissue sections were deparaffinized in xylene for 20 minutes and subse-
quently washed with 100%, 96%, and 70% ethanol followed by a wash
with tap water (2 minutes each step). Slides were air dried at 75°C for 3
minutes.Slideswerethenboiledinpretreatmentbuffer[70%formamide,
2× saline-sodium citrate buffer (SSC)] at 100°C for 15 minutes followed
by a wash with tap water. Tissue was then subjected to Proteinase K
(Sigma) treatment at 37°C for 70 minutes followed by a wash in tap
water (2 minutes). Dehydration of slides was performed by serial immer-
sion of slides in 70%, 96%, and 100% ethanol (2 minutes each step). Slides
were then air dried at 75°C for 3 minutes. The FISH probe was applied
and slides were sealed with rubber cement. Following a denaturation
step (10 minutes at 75°C), slides were incubated overnight at 37°C.
Slides were washed in Wash Buffer (2× SSC, 0.3% NP40, pH 7–7.5) and
counterstained with 4′,6-diamidino-2-phenylindole (DAPI) I solution
(1,000 ng/mL; Vysis Abbott Molecular). As reference, a Spectrum Green-
labeled chromosome 6 centromeric probe (Vysis Abbott Molecular) was
used. Images were obtained with a Zeiss fluorescence microscope using
a 63× objective (Zeiss) and the Axiovision software (Zeiss).
FISH results were evaluated according to (i) absolute VEGFA gene
copy number and chromosome 6 copy number and (ii) VEGFA gene/
chromosome 6 copy number ratio. The following classification was
used: not amplified—VEGFA/Chr6 ratio of less than 1.8; equivocal/
borderline—VEGFA/Chr6 ratio between 1.8 and 2.2; and amplified—
VEGFA/Chr6 ratio higher than 2.2, as proposed by the ASCO/CAP
(American Society of Clinical Oncology/College of American Pathol-
ogists) guidelines for HER2 amplification in breast cancer. High poly-
somy was defined as >3.75 copies of the CEP6 probe, low polysomy
was defined as cases displaying between 2.26 and 3.75 copies of the
CEP6 probe (52, 53). All cases displaying either amplification or poly-
somy were collectively defined as VEGFA gain. FISH quantification
and classification were done by an expert molecular pathologist who
had no access to the clinical data (L. Tornillo).
aCGH and qPCR
Genomic DNA was isolated using the QIAGEN DNAeasy Tissue
Kit. Samples were hybridized to mouse CGH 60-mer oligonucle-
otides microarrays (Agilent Technologies), washed, and scanned
according to the Agilent Technologies’ instructions. Data were ana-
lyzed using Feature Extraction software V8.1 (Agilent), GeneSpring
GX V7.3.1, and CGH Analytics V3.4.27 (Agilent) software. RNA
was extracted from tissues by mechanical grinding in TriReagent
(Sigma) with a Polytron tissue homogenizer (Kinematica) at maxi-
mum speed. cDNA was prepared with Moloney murine leukemia
virus (MMLV) reverse transcriptase (Invitrogen). qPCR analyses were
carried out with SYBR green (Invitrogen) in 7900HT Fast Real-Time
PCR System (Applied Biosystems). Results were analyzed using the
qBase v1.3.5 software. Primer sequences are available in Supplemen-
tary Table S5. In the xenografts experiment, murine Hgf mRNA levels
were assessed with TaqMan probe (Life Sciences). Human hypox-
anthine phosphoribosyltransferase (HPRT) and ubiquitin C (UBC)
were used as reference genes in the xenograft experiment. Hprt and
peptidylprolyl isomerase A (Ppia) combined were used as reference
genes in all murine analyses except for the hepatocyte versus mac-
rophage comparison in which Ubc, B2m, and Tbp were additionally
applied. Primers detecting the murine chromosome 17 pericentro-
meric region were used as references in DNA qPCR analyses.
Cell Separation
Hepatocytes and macrophages were isolated from Mdr2−/−
mice
livers essentially as described by Kamimura and Tsukamoto (54).
Briefly, livers were digested enzymatically with pronase and colla-
genase (Sigma) by in situ perfusion. Hepatocytes were isolated by cen-
trifugation at 50 × g for 2 minutes, and after three washes were frozen
immediately in liquid nitrogen for RNA preparation. Nonparenchy-
mal cells were pelleted by centrifugation at 150 × g for 8 minutes, laid
on top of a four-density gradient of Larcoll (Sigma) and centrifuged
at 20,000 rpm at 25°C for 30 minutes using a SW41Ti rotor (Beck-
man). Liver macrophages were recovered from the interface between
8% and 12% Larcoll, washed three times and immediately frozen in
liquid nitrogen. Purity of hepatocytes and macrophage fractions was
determined by H&E staining of cytospin preparations and always
exceeded 90%. Dissociation of cells from tumor xenografts was per-
formed using the gentleMACS dissociator (Miltenyi Biotec) accord-
ing to the manufacturer’s protocol.
Statistical Analysis
Data were analyzed using a paired two-tailed Student t test at P <
0.05. Histologic differences were analyzed using the Pearson χ2
test
at P < 0.05. Data were processed using Microsoft Excel 2007. Graphs
were generated using either GraphPad Prism 5.0 or Excel software.
Kaplan–Meier calculations and graphs were performed in GraphPad
Prism 5.0. Log-rank (Mantel–Cox) was used to determine survival P
value. Throughout the work, error bars represent 1 SEM.
Disclosure of Potential Conflicts of Interest
R. Koschny has received travel grants from Bayer Company.
E. Pikarsky has filed for a provisional patent application based on
this article. No potential conflicts of interest were disclosed by the
other authors.
Authors’ Contributions
Conception and design: E. Horwitz, I. Stein, J. Hess, P. Angel,
Y. Ben-Neriah, E. Pikarsky
Development of methodology: E. Horwitz, I. Stein, M. Andreozzi,
R.M. Porat, M. Grunewald
Acquisition of data (provided animals, acquired and managed
patients, provided facilities, etc.): E. Horwitz, I. Stein, J. Nemeth,
A.Shoham,O.Pappo,N.Schweitzer,L.Tornillo,N.Kanarek,L.Quagliata,
F. Zreik, R.M. Porat, R. Finkelstein, R. Koschny, T. Ganten, C. Mogler,
O. Shibolet, K. Breuhahn, M. Grunewald, P. Schirmacher, A. Vogel,
L. Terracciano
Analysis and interpretation of data (e.g., statistical analysis,
biostatistics, computational analysis): E. Horwitz, A. Shoham,
O. Pappo, N. Schweitzer, L. Quagliata, H. Reuter, C. Mogler, O. Shibolet,
J. Hess, A. Vogel, L. Terracciano, P. Angel, Y. Ben-Neriah, E. Pikarsky
Writing, review, and/or revision of the manuscript: E. Horwitz,
I. Stein, N. Schweitzer, R. Koschny, T. Ganten, C. Mogler, O. Shibolet,
J. Hess, P. Schirmacher, P. Angel, Y. Ben-Neriah, E. Pikarsky
Administrative, technical, or material support (i.e., reporting or
organizing data, constructing databases): E. Horwitz, A. Shoham,
N. Kanarek, L. Quagliata, T. Ganten, J. Hess, P. Schirmacher, A. Vogel,
L. Terracciano
Study supervision: Y. Ben-Neriah, E. Pikarsky
Acknowledgments
The authors thank Rivka Ben-Sasson, Reba Condioti, Shaffika El-
Kawasmi, Etti Avraham, Mohamad Juma’, and Drs. Hila Giladi, Gus-
tavo Mostoslavsky, David Curiel, Yosef Haviv, Shemuel Ben-Sasson,

and Sharon Elizur for providing expertise and reagents. The authors
also thank Drs. Jacob Hannah, Hidekazu Tsukamoto, Ofer Man-
delboim, Noam Stern-Ginosar, Noa Stanietsky, Rachel Yamin, Seth
Salpeter, Temima Schnitzer-Perlman, Dan Lehmann, Rachel Horwitz,
and Chamutal Gur for expert advice and kind assistance. The authors
are indebted to Dr. Daniel Goldenberg for supplying aged Mdr2−/−
mice and are grateful to Drs. Christoffer Gebhardt, Robert Goldstein,
Moshe Biton, Zvika Granot, and Tzachi Hagai for fruitful discussions.
Grant Support
The research leading to these results has received funding from
the European Research Council under the European Union’s Seventh
Framework Programme (FP/2007-2013)/ERC Grant Agreements
281738 to E. Pikarsky and 294390 to Y. Ben-Neriah; the Dr. Miriam
and Sheldon G. Adelson Medical Research Foundation (AMRF), the
German Research Foundation (DFG, SFB-TR77), the Cooperation
Program in Cancer Research of the Deutsches Krebsforschungszen-
trum (DKFZ), and Israel’s Ministry of Science, Culture and Sport
(MOST) to E. Pikarsky, Y. Ben-Neriah, and P. Angel; the Israel Sci-
ence Foundation (ISF) Centers of Excellence to E. Pikarsky and
Y. Ben-Neriah; and an ISF Project Grant to I. Stein and O. Pappo.
Received October 21, 2013; revised March 26, 2014; accepted
March 26, 2014; published OnlineFirst March 31, 2014.
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2014;4:730-743. Published OnlineFirst March 31, 2014.Cancer Discovery
Elad Horwitz, Ilan Stein, Mariacarla Andreozzi, et al.
Are Highly Sensitive to Sorafenib Treatment
-Amplified Hepatocellular CarcinomasVEGFAHuman and Mouse
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