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Human and mouse VEGFA-amplified hepatocellular carcinomas are highly
sensitive to sorafenib treatment
Elad Horwitz1, Ilan Stein1,2, Mariacarla Andreozzi3, Julia Nemeth4, Avivit Shoham1, Orit
Pappo2, Nora Schweitzer5, Luigi Tornillo3, Naama Kanarek1, Luca Quagliata3, Farid
Zreik2, Rinnat M. Porat2, Rutie Finkelstein1,2, Hendrik Reuter6, Ronald Koschny11, Tom
Ganten11, Carolin Mogler7, Oren Shibolet8, Jochen Hess4,9,10, Kai Breuhahn7, Myriam
Grunewald12, Peter Schirmacher7, Arndt Vogel5, Luigi Terracciano3, Peter Angel4, Yinon
Ben-Neriah1 & Eli Pikarsky1,2
1 - The Lautenberg Center for Immunology, IMRIC, Hebrew University - Hadassah
Medical School, Jerusalem, Israel
2 - Department of Pathology, Hadassah-Hebrew University Medical Center, Jerusalem,
Israel
3 - Institute of Pathology, University Hospital Basel, Basel, Switzerland
4 - Division of Signal Transduction and Growth Control (A100), German Cancer
Research Center (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany
5 - Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical
School, Hannover, Germany
6 - Division of Molecular Genetics (B060) German Cancer Research Center (DKFZ),
Heidelberg, Germany
7 - Institute of Pathology, University Hospital Heidelberg, Germany
8 - Liver Unit, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel
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9 - Junior Group Molecular Mechanisms of Head and Neck Tumors (A102), German
Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany
10 - Department of Otolaryngology, Head and Neck Surgery, University Hospital
Heidelberg, Germany
11 - Department of Internal Medicine, University Hospital Heidelberg, Germany
12 - Department of Developmental Biology and Cancer Research, IMRIC, Hebrew
University - Hadassah Medical School, Jerusalem, Israel
Correspondence should be addressed to Yinon Ben-Neriah ([email protected]) and Eli
Pikarsky ([email protected])
We declare no conflict of interest
Keywords: HCC, Sorafenib, VEGFA, HGF, copy number variation, macrophages
Abbreviations: HCC - Hepatocellular Carcinoma, VEGFR - VEGFA Receptor, HGF - Hepatocyte Growth Factor, Mdr2-/- - Multiple drug resistance variant 2 deficient mice, aCGH - array Comparative Genomic Hybridization, qPCR - quantitative PCR, CISH - Chromogenic In Situ Hybridization, Mbp - Mega base pairs, FISH - Fluorescent In Situ Hybridization, Amppos - VEGFA amplification positive tumor, Ampneg - VEGFA amplification negative tumor, TAMs - Tumor Associated Macrophages, AST - Aspartate Aminotransferase, pHH3 - phosphorylated Histone H3.
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.P. and 294390 to Y.B-N., the Dr.
Miriam and Sheldon G. Adelson Medical Research Foundation (AMRF), the German
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Research Foundation (DFG, SFB-TR77), the Cooperation Program in Cancer Research
of the Deutsches Krebsforschungszentrum (DKFZ) and Israeli's Ministry of Science,
Culture and Sport (MOST) to E.P. & Y.B-N., the Israel Science Foundation (ISF)
Centers of Excellence to E.P. & Y.B-N. and ISF project Grant to I.S. & O.P.
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Abstract
Death rates from hepatocellular carcinoma (HCC) are steadily increasing yet
therapeutic options for advanced HCC are limited. We identify a subset of mouse and
human HCCs harboring VEGFA genomic amplification, displaying distinct biological
characteristics. Unlike common tumor amplifications, this one appears to work via
heterotypic paracrine interactions: stromal VEGF receptors (VEGFRs), responding to
tumor VEGF-A, produce hepatoycte 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 sensitive 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.
Statement of significance
Using a mouse model of inflammation driven cancer, we identified a subclass of HCC
carrying VEGFA amplification, which is particularly sensitive to VEGF-A inhibition. We
found that a similar amplification in human HCC identifies patients who favorably
responded to sorafenib – the frontline treatment of advanced HCC, which has an overall
moderate therapeutic efficacy.
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Introduction
Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality
worldwide, with the highest increase rate in northern 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, patient’s response is modest, and
sorafenib treatment is associated with side effects (3-8). Thus, several studies looked
for predictive markers for sorafenib response (9-11), yet none such biomarkers have
entered the clinical setting. Predictive biomarkers, 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 K-RAS 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: B-Raf, c-Raf, PDGFR2,
c-Kit, and the VEGFR receptors (VEGFRs) (10, 16). While 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’s 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 mechanism of action of sorafenib is predominantly
independent of VEGF-A inhibition. Alternatively, a small subset of patients that can
respond to VEGF-A blockers may exist, that was underrepresented in bevacizumab
studies.
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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 the VEGFRs (22, 23). Moreover, VEGF-A also
has non-angiogenic functions in normal physiology. In liver tissue, VEGF-A elicits
hepatocyte proliferation by elevating expression of hepatocyte mitogens in liver
sinusoidal endothelial cells (24, 25).
HCC is most commonly the outcome of chronic injury and inflammation, resulting in
hepatocyte regeneration and dysregulated 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 utilized 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 microenvironment that are relevant to human HCC.
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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 comparative genomic
hybridization (aCGH) to 10 HCCs obtained from 16 months old Mdr2-/- mice
(Supplementary Fig. 1A). We detected several amplifications and deletions, including an
amplification in the qB3 band of murine chromosome 17 (Chr17qB3, Supplementary
Table 1) encoding among others 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 amplification we tested a larger cohort of Mdr2-/- tumors by quantitative
PCR (qPCR) of tumor DNA (Supplementary Fig. 1B) and chromogenic in situ
hybridization (CISH, Fig. 1A); 13 out 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 to
48.5 mega base pairs (Mbp) from the chromosome 17 start. This minimal amplified
region spanned 53 genes (Supplementary Table 2).
Amplification of human Chr6p21 (the region syntenic for murine Chr17qB3) and whole
chromosome gains were previously reported in several whole genome analyses of
human HCC with a frequency ranging between 7-40% (29-33). Accordingly, through
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fluorescent in situ hybridization (FISH) we found VEGFA amplifications and
chromosome 6 polysomies in 11% of human HCCs we tested (21/187, Fig. 1C and
Supplementary Table 3).
To elucidate the tumor relevance of these chromosomal gains we analyzed the
expression of mouse Chr17qB3 amplicon genes in amplicon positive (herein Amppos)
and negative (Ampneg) tumors. Matched increases in mRNA and DNA levels were found
for VEGFA, Tjap1 and Exportin 5 (Fig. 1D and Supplementary Fig. 2A). We further
found a correlation between Chr17qB3 amplification and VEGF-A protein levels in both
tumor extracts and serum (Fig. 1E and Supplementary Fig. 2B). 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 expression of several resident genes including VEGFA.
Amppos tumors display distinct histological features
Tumor cell proliferation is a strong and consistent marker of poor prognosis in human
HCC (34). BrdU immunostaining revealed that Amppos mouse HCCs displayed a 2-fold
higher proliferation index compared to Ampneg tumors (Fig. 2A and B, upper panels). No
differences were noted in apoptosis (Supplementary Fig. 2C) or in the presence of
neutrophils or fibroblasts (data not shown). Other histological features, differing between
Amppos and Ampneg mouse HCCs, include a 6-fold higher vessel density (Fig. 2A and B,
middle panels) and 4-fold higher macrophage content (Fig. 2A and B, lower panels).
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Moreover, several signature genes of tumor associated macrophages (TAMs) including
Arginase 1, TGF�, and Ym1 (35, 36), were elevated in the Amppos group (Fig. 2C), while
markers of classically activated macrophages, including TNF�, iNOS, CXCL10, and
IL12p40 were similar in both tumor groups (Fig. 2C and Supplementary Fig. 3A). Along
with these, immunofluorescent stain for the pro-tumorigenic macrophage marker Mrc1,
revealed a higher presence 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.
Histological analysis of human HCCs revealed several characteristic traits of HCCs
harboring VEGFA gains. A higher incidence of vascular invasion was found in the
Amppos group (9/20 Amppos tumors vs. 1/20 Ampneg tumors, p<0.01, Supplementary
Table 4). 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 4). We found no differences in several clinical characteristics of
HCC including underlying disease, gender or tumor size (Supplementary Fig. 4A-C).
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.
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Macrophage-tumor cell crosstalk in Amppos tumors
Previous studies have delineated a hepatocyte-endothelial crosstalk taking place in
non-neoplastic liver, wherein VEGF-A stimulates endothelial cells to secrete several
mitogens including HGF (24, 25). We hypothesized 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 vs. Ampneg mouse tumors (Fig. 3A). We
did not find significant changes in other angiocrine-produced molecules (24, 25) – Wnt2,
IL-6 and 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 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. 3B), a time point signified by marked dysplasia, yet no overt HCC formation (27).
mRNA profiling of these fractions portrayed that VEGFRs (FLT1 and KDR) and
coreceptors [Neuropilin (Nrp)1 and 2] were higher in macrophages while 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 demonstrated 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 to the increase in mRNA levels of recruited macrophage and endothelial
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markers Msr1 and CD105, respectively (Fig. 3G). Recapitulating this interaction in vitro,
we treated peritoneal macrophages with recombinant 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 microenvironment to induce HGF secretion.
VEGF-A increases cellular proliferation in HCC
In order to prove the functional role of VEGFA in this genomic amplification, we set 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 aged 14 to 18 months and
sacrificed them ten days following injection (Supplementary Fig. 5A). VEGFA
amplification status was determined after sacrifice by both DNA qPCR and VEGF-A
mRNA expression (Fig. 4C and data not shown). Liver damage, measured through
plasma Aspartate Aminotransferase (AST) activity, was similar in all groups
(Supplementary Fig. 5B). Immunostaining for BrdU, Ki67, and phospho-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. 5C and
D). This decrease in proliferation was accompanied by reduced HGF mRNA levels (Fig.
4C). Treatment with adenovirus encoding GFP alone did not induce any change in
either group. Macrophage infiltration and protumorigenic macrophage expression profile
did not decrease following the sFLT treatment (Supplementary Fig. 5C and D and data
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not shown). Macroscopic and histological analyses revealed multiple foci of coagulative
necrosis only in sFLT treated Amppos tumors (3/6 vs. 0/10 in Ampneg, p<0.05 Fig. 4D). In
these specific tumors we also found an elevation of the HIF1� target genes Glut1 and
PGK1 (Fig. 4C), indicative 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 (Supplementary Fig. 5C and D). 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 macrophage HGF - heterotypic
circuit in tumor growth, we injected human Hep3B HCC cells transduced with a
lentivector overexpressing human VEGF-A into immune-deficient mice (Supplementary
Fig. 6A). The in vivo growth rate of VEGF-A overexpressing cells was higher than
control vector transduced cells (Fig. 5A and Supplementary Fig. 6B). This correlated
with higher proliferation rate and increased HGF expression (Fig. 5B-D and
Supplementary Fig. 6C and D), 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 PHD3 and LDHA in these xenografts, lending further support to a non-
angiogenic role for VEGF-A in HCC (Supplementary Fig. 6E). Immunofluorescence
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confirmed VEGF-A expression in transduced tumor cells and revealed HGF expression
in macrophages (Supplementary Fig. 7A).
Next, we generated single cell suspensions from VEGF-A overexpressing or control
Hep3B ZsGreen labeled xenografts and isolated macrophages (ZsGreen-CD45+F4/80+)
and endothelial cells (ZsGreen-CD45-Meca32+) using FACS sorting. qPCR analysis of
the macrophage and endothelium specific genes, Msr1 and CD105 respectively,
confirmed successful separation of cell populations (Supplementary Fig. 7B). While
VEGFA and control tumor groups did not show significant differences in the tumor
associated macrophage genes TGF�, Ym1, TNF� and iNOS (Supplementary Fig. 7C),
we found a twofold increase in HGF expression in macrophages, but not endothelial
cells, isolated from VEGF-A overexpressing tumors (Fig. 5F). Thus, VEGF-A
overexpression by HCC cells, is sufficient to induce upregulation of HGF, mostly in
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 Angiopoietin 1
and 2, Angiopoietin like 2, FGF 1 and 2, PDGF-A, -B and -C, PLGF and VEGF-B
revealed that several of these factors were overexpressed in Mdr2-/- HCCs compared
with normal livers, irrespectively of the amplicon status. Notably, PDGF-C 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 angiogenesis in a VEGF-
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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 eligible for local therapies and is a frontline treatment for
these patients (3). As sorafenib inhibits VEGFRs and B-Raf - 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 aged 14
to 18 months with sorafenib or vehicle alone for three days after which they were
sacrificed and tumor tissue was analyzed (experimental design depicted in
Supplementary Fig. 5A). Amplification status was assessed after sacrifice by DNA
qPCR and verified by VEGF-A mRNA expression (Fig. 7C and data not shown).
Immunostaining for BrdU and pHH3 demonstrated decreased proliferation in sorafenib
treated mice in Amppos but not Ampneg HCCs (Fig. 7A and B and Supplementary Fig. 8A
and B). Similarly to VEGF-A inhibition, HGF levels were decreased only in sorafenib-
treated Amppos tumors (Fig. 7C). While no effects were observed on tumor macrophage
density (Supplementary Fig. 8A and B), expression of the tumor associated
macrophage marker TGF� was decreased in sorafenib-treated Amppos tumors
(Supplementary Fig. 8C). Blood vessel density changes or signs of hypoxia were absent
(Supplementary Fig. 8A-C), possibly due to the short treatment duration, implying that
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the early inhibitory effect of sorafenib in VEGFA amplified tumors may be independent
of angiogenesis. 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. Similarly 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. 9A-D). Despite
the longer duration of treatment, we still could not detect changes in macrophage or
blood vessel densities (Supplementary Fig. 10A-C). Of note, a differential response to
sorafenib was not evident in vitro, emphasizing the importance of microenvironmental
factors (Supplementary Fig. 10D).
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 HCC patients that underwent tumor resection. This retrospective cohort
was collected from 3 different centers. To assess the correlation between VEGFA gain
and survival, we analyzed human tumor samples by FISH (Fig. 1C). Survival of HCC
patients that did not receive sorafenib was independent of the VEGFA status (Fig. 7E).
However, a markedly improved survival was seen in the VEGFA-gain group compared
to the non-gain group in sorafenib treated patients (indefinable median survival from
sorafenib treatment start and 11 months, respectively, pLog-Rank=0.029, Fig. 7F).
Taken together, our mouse data and retrospective analysis of a human cohort imply that
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VEGFA gains correlate with a particularly beneficial response to sorafenib, and possibly
other VEGF-A inhibitors, in HCC.
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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 histological appearance, rate of
proliferation and microenvironmental content. We delineated a cytokine-based
heterotypic crosstalk between malignant Amppos hepatocytes and tumor stromal cells.
Importantly, we show 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. Therefore, 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-
microenvironment 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). While hepatocytes are inert to VEGF-A,
liver sinusoidal cells respond to VEGF-A with counter secretion of HGF (24).
Furthermore, in regenerating 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.
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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 phenotype, 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 findings therefore raise the interesting possibility that
the VEGFA amplicon contributes to the inflammatory microenvironment, 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 proliferation
via stroma-derived HGF secretion (and possibly other cytokines as well); iii. Enhancing
tumor angiogenesis. Interestingly, all these modes entail heterotypic cellular
interactions, distinguishing this particular genomic gain from other studied amplicons
(43). We maintain that the unique sensitivity of tumors with VEGFA gains to VEGF-A
blockade stems from these multiple pro-tumorigenic functions of VEGF-A (Fig. 7G and
H).
An amplicon spanning VEGFA was noted in several different human cancers (29-31,
33, 43-51). A linear correlation was found between mRNA levels of VEGF-A and extent
of amplification in human HCC (29). Amplifications in 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 vascular invasion and
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shorter survival (46, 51). Cumulative analysis of these reported human studies shows
that gains and amplifications of VEGFA are found in 7-30% 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 sorafenib. Nevertheless, we cannot completely
exclude contribution of other amplicon genes to tumorigenesis.
The first line of treatment for advanced HCC is the multikinase inhibitor sorafenib, which
prolongs median survival by 10-12 weeks (3, 4). The response to sorafenib appears 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 B-Raf - 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 tissue, making it difficult to establish tissue-based predictive biomarkers. Based
on our small-scale retrospective study, we show that VEGFA gains may predict
response to sorafenib in HCC, thus enabling to tailor treatment only to those patients
who may benefit from this side effects-prone therapy. Notably, the same amplification
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was found also 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.
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Methods
Human tissue samples and tissue microarray
Human HCC tissues were obtained from resected patients from the institutes of Basel
University Hospital, Switzerland; Hannover Medical School Hospital and Heidelberg
University Hospital, Germany. Clinical information included age at diagnosis, tumor
diameter, gender, and survival time information. Examination of tumor H&E sections
was performed by an expert liver pathologist (O.P.). Samples from Heidelberg were also
reviewed by Heidelberg pathologists (C.M., P.S.). The study was approved by each of
the institutions ethics committee – 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 � value of 0.05. Construction of tissue
microarray (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, Silver
Spring, USA). Afterwards, tissue cylinders were transferred to a 25x35 mm paraffin
block to produce the TMAs. The resulting TMA block was cut into 3-�m sections that
were transferred to glass slides by use of the Paraffin Sectioning Aid System
(Instrumedics, Hackensack, USA). Sections from the TMA blocks were used for FISH
analysis.
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Mice
Male and female Mdr2-/- mice on FVB background were held in specific pathogen free
conditions. Experiments performed on Mdr2-/- mice were performed in cohorts of 10-20
mice each time, results show the combined data from at least 4 different experiments.
WT controls were aged matched FVB mice. Mouse body weights during the
experiments were 30-45 grams. Sorafenib (Xingcheng Chempharm Co., Ltd Taizhou,
China) was administered daily (50 mg/kg) by oral gavage. Cremophor EL
(Sigma)/ethanol/water; (1:1:6) was used as vehicle. Two hours prior to sacrifice, mice
were injected with 10 �l BrdU (Cell Proliferation labeling reagent, Amersham) per 1
gram body weight. Mice were anesthesized 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 subjected to standard histological
procedures. Xenografts experiments were performed by subcutaneous injection of
transduced Hep3B cells (2.5×106) suspended in 100 �l PBS and 100 �l Matrigel
(Becton Dickinson) into NOD/SCID mice. Tumor volumes were assessed by external
measurement with caliper. 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) and Yosef Haviv (Hadassah Hospital, Israel) were
prepared in GH354 cells using standard procedures. A titer of 109 transducing units was
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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 NK cells, a kind gift from Ofer Mandelboim, Hebrew University, Jerusalem) into
pSC-B plasmid using the StrataClone 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, Boston) 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 DMEM (10% fetal bovine serum). Cells
were tested free of mycoplasma prior to 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, Beit
Haemek) using the manufacturer’s protocol. Murine recombinant VEGF-A (R&D
systems) was used at the concentration of 100ng/ml.
Immunohistochemistry, immunofluorescence and ELISA
Antibodies used for tissue immunostaining throughout the work were - vWF (dilution
1:300, Dako), phosphorylated Histone H3 (pHH3, 1:800, Upstate), cleaved Caspase 3
(1:200, Cell Signaling), F4/80 (1:300, Seroteq), HGF (1:100, R&D), BrdU (1:200,
NeoMarkers), KDR (1:400, Cell Signaling), Ki67 (1:100, NeoMarkers), e-cadherin
(1:100, Cell Signaling) and VEGF (1X as supplied, Spring). IHC was performed on 5 μm
paraffin sections. Antigen retrieval was performed in a decloaking chamber (Biocare
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Medical) in citrate buffer for all antibodies except vWF and F4/80 for which retrieval was
performed with Pronase (Sigma). HRP conjugated secondary antibodies for all
immunohistochemistry 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 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 (distinguished by morphology) was designated as either
positive or negative by these parameters. The fraction of positive cells was calculated
from counting at least 5 randomly selected fields in each tumor.
Immunofluoresence was performed on snap frozen tissue embedded in OCT gel
(Sakura Finetek) and sectioned to 8 μm slices. Slides were incubated at 370C and fixed
with both acetone and 4% paraformaldehyde sequentially. Fluorophore conjugated
secondary antibodies used were Donkey anti-Goat Alexa 647 (Invitrogen), Donkey anti
Rabbit Cy2/Cy5, Donkey anti mouse Cy3 and Goat anti-Rat Cy3 (Jackson
Laboratories). Hoechst 33342 was used as a nuclei marker (Invitrogen). Antibodies
used for flow FACS sorting 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
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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 sonicated 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 in 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, Berlin, Germany), 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) according to the instructions of the manufacturer.
Isolated BAC-DNA (1 �g) was digested with AluI restriction enzyme (Invitrogen) and
labelled with Cy3-dUTP (GE Healthcare) using the BioPrime Array CGH Kit (Invitrogen).
Labeling reaction was assessed by Nanodrop (Nanodrop, Wilmington, DE, USA).
Labeled DNA was purified with the FISH Tag DNA Kit (Invitrogen). Tissue microarrays
and whole tissue sections were deparaffinized in xylene for 20 minutes and
subsequently 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. Slides were
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then boiled in pretreatment buffer (70% formamide, 2x 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 immersion of slides in 70%, 96%, and
100% ethanol (2 minutes each step). Slides were then air dried at 75°C for 3 minutes.
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
DAPI I solution (1000 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, amplified - VEGFA/Chr6
ratio higher than 2.2, as proposed by the ASCO/CAP guidelines for HER2 amplification
in breast cancer. High polysomy was defined as >3.75 copies of the CEP6 probe,
Low polysomy was defined as cases displaying between 2.26-3.75 copies of the CEP6
probe(52, 53). All cases displaying either amplification or polysomy were collectively
defined as VEGFA gain. FISH quantification and classification were done by an expert
molecular pathologist that had no access to the clinical data (L.T.).
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27
Array-based comparative genomic hybridization and qPCR
Genomic DNA was isolated using the QIAGEN DNAeasy Tissue kit. Samples were
hybridized to mouse CGH 60-mer oligonucleotides microarrays (Agilent Technologies),
washed and scanned according to Agilent Technologies instructions. Data was
analyzed 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 maximum speed. cDNA was prepared with 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 Supplementary Table 5. In
the Xenografts experiment, murine HGF mRNA levels were assessed with Taqman
probe (Life Sciences). Human HPRT and UBC were used as reference genes in the
xenograft experiment. HPRT and PPIA combined were used as reference genes in all
murine analyses except for the Hepatocyte vs. Macrophage comparison in which UBC,
�2M and TBP were additionally applied. Primers detecting the murine chromosome 17
pericentromeric 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 Collagenase (Sigma) by in-situ perfusion. Hepatocytes were isolated
by centrifugation at 50xg for 2 minutes, and after 3 washes were frozen immediately in
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28
liquid nitrogen for RNA preparation. Non parenchymal cells were pelleted by
centrifugation at 150xg 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
(Beckman). Liver macrophages were recovered from the interface between 8% and
12% Larcoll, washed 3 times and immediately frozen in liquid nitrogen. Purity of
hepatocytes and macrophage fractions was determined by Hematoxylin & Eosin
staining of cytospin preparations and always exceeded 90%. Dissociation of cells from
tumor xenografts was performed using the gentleMACS dissociator (Miltenyi Biotech)
according to manufacturer’s protocol.
Statistics
Data was analyzed using a paired two tails Student’s T-Test at p<0.05. Histological
differences were analyzed using Pearson’s �2 test at p<0.05. Data was 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 standard error of the mean (SEM).
Acknowledgments
We thank Rivka Ben-Sasson, Reba Condioti, Shaffika El-Kawasmi, Etti Avraham,
Mohamad Juma' and Drs. Hila Giladi, Gustavo Mostoslavsky, David Curiel, Yosef Haviv,
Shemuel Ben-Sasson, and Sharon Elizur for providing expertise and reagents. We
thank Drs. Jacob Hannah, Hidekazu Tsukamoto, Ofer Mandelboim, Noam Stern-
Ginosar, Noa Stanietsky, Rachel Yamin, Seth Salpeter, Temima Schnitzer-Perlman,
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29
Dan Lehmann, Rachel Horwitz and Chamutal Gur for expert advice and kind
assistance. We are indebted to Dr. Daniel Goldenberg for supplying aged Mdr2-/- mice.
We are grateful to Drs. Christoffer Gebhardt, Robert Goldstein, Moshe Biton, Zvika
Granot and Tzachi Hagai for fruitful discussions.
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49. Andreozzi, M., Quagliata, L., Gsponer, J.R., Ruiz, C., Vuaroqueaux, V., Eppenberger-Castori, S., et al. VEGFA gene locus analysis across 80 human tumour types reveals gene amplification in several neoplastic entities. Angiogenesis 2013.
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Figure legends
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.
Thin line represents non-amplified regions, 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. 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 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 bars:
20 μm.
Figure 2. Amppos
tumors are a distinct tumor subpopulation. (A) Representative IHC
photomicrographs for BrdU, vWF, and F4/80. Scale bars: 50 μm (BrdU) 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 (pro-tumorigenic) and
classically activated (anti-tumorigenic) macrophage markers. Each dot represents a
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35
different tumor. 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
bars: 40 μm. (E) Quantification of Mrc1 immunofluorescence. Bars represent geometric
mean, **p<0.01.
Figure 3. A macrophage-tumor cell cross talk within Amppos tumors. (A) qPCR analysis
of HGF mRNA in WT livers, Ampneg and Amppos tumors. Cross line signifies geometric
mean (**p<0.001). (B) Representative photomicrographs of IHC for HGF on Ampneg and
Amppos tumors. Scale bars: 100 �m. (C) Representative confocal microscopy images of
Amppos tumor immunostained for vWF, F4/80 and HGF. Hoechst 33342 marks nuclei.
Scale bars: 40 μm. (D) Representative cytospin preparations of macrophage and
hepatocyte fractions from Mdr2-/- livers. (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) Representative
photomicrograph of IHC for the VEGF receptor KDR in Ampneg and Amppos tumors. Note
that expression of KDR is confined to endothelial andh stromal cells. Scale bars: 100
�m. (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. Cross line represents geometric mean
(***p<0.0001). (H) Peritoneal macrophages were cultured under serum free conditions
followed by 8h exposure to recombinant murine VEGF-A (100ng/ml). HGF expression
was measured by qPCR. Bars represent geometric mean, n�3, *p<0.05.
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36
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 BrdU. Tumor infiltrating cells remain
proliferative. Scale bars: 100 μm. (B) BrdU 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. Cross line signifies geometric mean (***p<0.0001). (D) Left
panel - histological section stained with H&E depicting necrosis, representing three out
of the six sFLT treated Amppos tumors. Scale bar: 500 μm. Right panel - a macroscopic
picture of a tumor with hemorrhagic necrosis. Scale bar: 0.5 cm.
Figure 5. VEGF-A overexpression enhances tumor cell proliferation. Immune deficient
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 lentivector (gray line). Tumor volumes were
measured topically with a caliper (*p<0.05, **p<0.01). (B) Representative
photomicrographs of IHC for BrdU in control or VEGF-A lentivector-transduced
xenografts. Scale bars: 100 μm. (C) BrdU 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
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37
of HGF in macrophage and endothelial fractions from tumors overexpressing VEGFA
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.
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. Cross line signifies
geometric mean, significance marks immediately above each group refer to comparison
with normal livers. Top most 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).
Figure 7. Amppos tumors are uniquely sensitive to sorafenib. (A) Representative BrdU
immunostains. Scale bars: 100 μm. (B) BrdU 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. 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 non-treated (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 (pLog-
Rank>0.05). (F) Kaplan-Meier curves showing survival of HCC patients treated with
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38
sorafenib, negative (n=47, 10 months median) or positive for VEGFA gain (n=7, median
undefined, pLog-Rank=0.029). (G & 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 tumor associated macrophages expressing the mitogen HGF (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 with
decreased HGF signaling and blood supply, impeding tumor growth.
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A BMurine HCCs
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Published OnlineFirst March 31, 2014.Cancer Discovery Elad Horwitz, Ilan Stein, Mariacarla Andreozzi, et al. are highly sensitive to sorafenib treatmentHuman and mouse VEGFA-amplified hepatocellular carcinomas
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