Human and mouse VEGFA-amplified hepatocellular ......2014/03/29 · Author Manuscript Published...

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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

Research. on August 26, 2021. © 2014 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 31, 2014; DOI: 10.1158/2159-8290.CD-13-0782

http://cancerdiscovery.aacrjournals.org/

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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




24

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




25

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




26

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.).




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




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,




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.




30

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34

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




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.




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




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




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.




A BMurine HCCs

Tumor #1 Tumor #2ne

g

t

Murine Amppos HCCs43.3

43.8

amplified

non-amplified

minimal amplified region

Figure 1

Tumor #3 Tumor #4

Am

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44.4

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46.15-46.17Vegfa

45.82-45.84Mrpl1445.78-45.82Tmem63b

45.77-45.79Capn11

46.39-46.41Tjap146.33-46.37Xpo5

45.71-45.71Hsp90ab1

46.24-46.26Mrps18a

45.69-45.70Nfkbie

46.43-46.44EGFL9

45.64-45.66Aars2

44.36-44.41Cdc5L

C

normal polysomic amplified

Human HCCs Mbp

48.5

46.63-46.68Parc46.86-46.87Gnmt

47.4

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n.s.

WT Ampneg AmpposWT A A pos WT A A pos

** ***

D E

4

5

6

7

/ pg/

ug p

rote

in

VEGFa mRNA (qPCR)

VEGFa Protein (ELISA)

uctio

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)

normal polysomic amplified

WT liver

Amp g

TumorAmpp

Tumor

***

WT liver

Ampneg

TumorAmppos

TumorWT liver

Ampneg

TumorAmppos

Tumor

WT liver

Ampneg

TumorAmppos

TumorWT liver

Ampneg

TumorAmppos

TumorWT liver

Ampneg

TumorAmppos

Tumor

*** ***

Cul9

0

1

2

3

1 2 1 2 3 4 1 2 3 4

fold

indu

ctio

n /

fold

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WT liver

Ampneg Amppos

Mdr2-/- tumors

FHoechst E-cadherin VEGFA Merge

sTu

mor

Am

ppos




BAmpneg Tumor Amppos Tumor

15

20

nucl

ei **BrdU

WT liver

Ampneg Tumor

A

Figure 2

Brd

U

0

5

10

vWF

perc

ent p

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ve n

Amppos Tumor

10

12

14

e ar

ea

vWF*

vWF

0

2

4

6

8

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vWF

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ent p

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t pos

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a

�

n.s.n.s. n.s.enic

WT liver

Ampneg

TumorAmppos

Tumor

***** **

WT liver

Ampneg

TumorAmppos

TumorWT liver

Ampneg

TumorAmppos

Tumor

Pro

-tum

orig

em

arke

rs

WT liver

Ampneg

TumorAmppos

TumorWT liver

Ampneg

TumorAmppos

TumorWT liver

Ampneg

TumorAmppos

Tumor

Ant

i-tum

orig

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arke

rs

D ED

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1

Ampneg Tumor Amppos TumorE

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BAmppos TumorAmpneg Tumor

**

A

Figure 3

C

HG

F

WT liver

Ampneg

TumorAmppos

Tumor

vWF F4/80 HGF Merge

or

D

Am

ppos

Tum

o

D

FEA A

H&

E

Ampneg

TumorAmppos

Tumor

KD

R

**

***6

8

10

12

14

16

18

lativ

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pres

sion

Hepatocytes

Macrophages**

****

***

0

2

4

FLT1 KDR Nrp1 Nrp2 c-Met

re

KDR FLT1FLT1G H

HGF

WT liver

Ampneg

TumorAmppos

Tumor

***

WT liver

Ampneg

TumorAmppos

Tumor

***

Msr1 CD105

*

***

WT liver

Ampneg

TumorAmppos

TumorWT liver

Ampneg

TumorAmppos

Tumor

*** peritoneal macrophages




B

4

5

Ampneg

GFPAmpneg GFPAmpneg sFLTAmppos GFP

BrdU

e nu

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GFPAmpneg

sFLTAmppos

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A

Figure 4

C ***

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GFPAmppos

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GFPAmpneg

sFLTAmppos

GFPAmppos

sFLT

Glut1 PGK1

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EAmpneg

GFPAmpneg

sFLTAmppos

GFPAmppos

sFLTAmpneg

GFPAmpneg

sFLTAmppos

GFPAmppos

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D

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Figure 5

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Figure 6

Ampneg Amppos Ampneg Amppos Ampneg Amppos Ampneg Amppos Ampneg Amppos

Angiopoietin 1 Angiopoietin 2 Angptl 2 FGF 1 FGF 2

n.s.

n.s.n.s.n.s.

*****

n.s.

****

n.s.n.s.* *

n.s.*

n.s.

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Ampneg Amppos Ampneg Amppos Ampneg Amppos Ampneg Amppos Ampneg Amppos

PDGF-A PDGF-B PDGF-C PLGF VEGF-B

n.s.n.s.




B

1.82 *

Amppos

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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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