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Molecular Fingerprinting and Autocrine Growth Regulation of Endothelial Cells in a Murine Model of Hepatocellular Carcinoma

Eduard Ryschich,1 Paulius Lizdenis,1 Carina Ittrich,2 Axel Benner,2 Simone Stahl,3 Alf Hamann,4 Jan Schmidt,1 Percy Knolle,5 Bernd Arnold,3 Gu¨nter J. Ha¨mmerling,3 and Ruth Ganss3

1Department of Surgery, University of Heidelberg; 2Central Unit Biostatistics and 3Department of Molecular Immunology, German Cancer Research Center, Heidelberg, Germany; 4Experimental Rheumatology, Charite University, Berlin, Germany; and 5Institute for Molecular Medicine and Experimental Immunology, University of Bonn, Bonn, Germany

Abstract vessel remodeling, the tumor vasculature develops distinct and In a mouse model of hepatocellular carcinogenesis, highly stage-specific morphologic features compared with their normal vascularized tumors develop through two distinct morpho- counterparts (3, 4). Molecular events that direct angiogenesis have logic phases of neovascularization. We show that increased been extensively studied in vitro. For instance, human umbilical vascular caliber occurs first, followed by extensive vessel vein endothelial cells (HUVEC) incubated with growth factors, such sprouting in late-stage carcinomas. To define molecular as vascular endothelial growth factor (VEGF) and basic fibroblast pathways in tumor neovascularization, endothelial cells were growth factor, are classically used to recapitulate tumor angiogen- directly purified from normal liver and advanced tumors. esis (5–9). However, much as these in vitro systems have contributed to our understanding of vessel proliferation, they are Gene expression profiling experiments were then designed to identify genes enriched in the vascular compartment. We selective for single growth factors and poorly mimic the report that Cathepsin S is the major protease specifically three-dimensional stromal architecture. Furthermore, they do not overexpressed during vessel sprouting. We also show that account for organ specificities of microvessels. Indeed, endothelial the CC chemokines CCL2 and CCL3 are secreted by neo- cells are morphologically and functionally heterogeneous popula- vessels and stimulate proliferation through their cognate tions that are highly adapted to their microenvironment (10–12). receptors in an autocrine fashion. This suggests that Importantly, there is increasing evidence that the tumor micro- chemokine signaling represents the most prominent signal- milieu, like its normal counterpart, shapes and imprints unique ing pathway in tumor-associated endothelial cells and features onto the neovasculature. In vivo phage-display profiling of directly regulates vessel remodeling. Furthermore, high murine vessels, for instance, revealed profound molecular differ- angiogenic activity is associated with attenuated lymphocyte ences among endothelial cells of normal organs (13, 14) and extravasation and correlates with expression of the vascular beds of different tumors (15, 16). Therefore, the molecular immunomodulatory cytokine interleukin 10. This is the first anatomy of tumor vessels differs between tumor stages as well as tumor types (15). comprehensive study addressing liver-specific vascular changes in a murine autochthonous tumor model. These Comprehensive molecular analyses of endothelial cells purified novel insights into liver angiogenesis infer an environmental from normal mouse organs are limited (12), and even less is known control of neovascularization and have important implica- about isolated tumor endothelial cells (17). We have established tions for the design of antiangiogenic therapies. (Cancer Res a murine model for hepatocellular carcinoma with the goal of 2006; 66(1): 198-211) characterizing angiogenic changes during tumorigenesis. Trans- genic mice expressing the oncogene SV40 large T antigen (Tag) under the control of the albumin promoter/enhancer (Alb; ref. 18) Introduction sequentially develop highly vascularized liver cancers. A major Carcinogenesis is primarily a consequence of nuclear events advantage of this model over transplanted tumors is that Tag- within transformed cell clones but equally requires stromal induced tumors spontaneously develop within the liver, enabling interactions to cause cancer progression (1). Tumor-associated analysis of the neovasculature in its native architecture and stroma provides essential components that promote tumor cell location. To reveal molecular alterations induced in the vasculature proliferation and formation of a new vascular network. Angiogen- of AlbTag tumors, we purified endothelial cells from normal esis is indeed a discrete and rate-limiting step that enables small, liver and late-stage tumors and did comprehensive gene profiling avascular tumors to develop into hypervascular, rapidly growing experiments. Here, we present evidence for novel mechanisms cancers (2). During neovascularization, endothelial cells, which intrinsic to liver tumor endothelial cells (LTEC) that regulate vessel form the lining of blood vessels, proliferate, invade into surround- sprouting, migration, and growth. ing stroma, and finally form new vascular sprouts. Due to constant Materials and Methods Mice and cell lines. The 3.8 kb Alb promoter/enhancer (18) was cloned Note: E. Ryschich and P. Lizdenis contributed equally to this work. upstream of a 2.7 kb fragment coding for the early region of Tag (19) and Requests for reprints: Ruth Ganss, Department of Molecular Immunology, injected into (C57BL/6ÂDBA/2)F1 oocytes. AlbTag mice were backcrossed German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, into the C3HeBFe background for 20 generations. Tag-expressing murine Germany. Phone: 49-6221-423754; Fax: 49-6221-401629; E-mail: [email protected]. + I2006 American Association for Cancer Research. hepatocellular carcinoma cells (Tag-HCC) were established ex vivo from Tag doi:10.1158/0008-5472.CAN-05-1636 tumors by enzymatic digestion, in vitro culture and in vivo passages through

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Figure 1. Vessel transformation during AlbTag carcinogenesis. A, intravital microscopy of normal mouse liver displaying a homogeneous network of liver sinusoids (arrow) and a postsinusoidal venule (arrowhead). B, a similar vessel microarchitecture is apparent in nodular carcinoma from AlbTag mice with dilated sinusoids (arrow) and postsinusoidal venules (arrowheads). C, a representative intravital picture from AlbTag carcinoma displaying a heterogeneous vasculature with broad vessels adjacent to small vessels. D, comparative analysis of the size range of sinusoids from normal liver and nodular carcinoma of AlbTag mice. E, size distribution of postsinusoidal venules in normal liver and nodular carcinoma. F, vessel diameters as measured in AlbTag carcinoma of different size. The data summarize the analyses of 10 normal livers, 10 nodular carcinoma, and 17 carcinoma. Columns, mean; bars, SD.

Rag-1À/À C3HebFe mice.6 To collect conditioned medium, Tag-HCC cells FITC (rat IgG2a, 30 Ag/mL; ref. 21). Propidium iodide (1 Ag/mL) was were grown in DMEM containing 4.5 g/L glucose, 10% FCS, 2 mmol/L gluta- added to exclude dead cells. Cells were sorted using a FACSVantage SE mine, 100 units/mL penicillin, and 100 Ag/mL streptomycin for 3 to 5 days. flow cytomer (Becton Dickinson, Heidelberg, Germany). For in vitro Intravital microscopy. The procedure has been described previously (3) studies, LSECs and LTECs were separated by magnetic cell sorting using and modified for liver by placing liver lobes on a rubber stage superfused biotinylated anti-CD31 antibodies and streptavidin-conjugated magnetic with Ringer’s solution (37jC). Statistical analysis was done by using SPSS beads (Miltenyi Biotech, Bergisch Gladbach, Germany). Cells were seeded software (version 11.5.1, SPSS, Inc., Chicago, IL). Fisher’s exact test was used on collagen (Sigma)–coated flasks and grown in DMEM containing 4.5 g/L to compare vessel diameters. The differences of adherent leukocytes glucose, 10% FCS, 2 mmol/L glutamine, 100 units/mL penicillin, and 100 between groups were compared using Mann-Whitney U test. Ag/mL streptomycin. Endothelial cell purity was assessed by incubation Isolation of primary cell populations and fluorescence-activated with 200 Ag/mL acetylated low-density lipoprotein (LDL), conjugated with cell sorting. Isolation of murine liver sinusoidal endothelial cells (LSEC) the fluorochrome 1,1V-dioctadecyl-3,3,3V,3V-tetramethylindo-carbocyanine has been described (20). For the isolation of LTECs, AlbTag mice were perchlorate (DiI, Paesel&Lorei, Hanau, Germany) for 4 hours. Tissue- injected i.p. with 200 units heparin (Sigma, Taufkirchen, Germany) and infiltrating lymphocytes (TIL) were prepared as described for endothelial perfused via the inferior vena cava with Spinner’s modified Eagle’s cells but separated on a Percoll gradient. Lymphocytes were washed in medium containing 100 mmol/L EGTA/50 units heparin, followed by PBS and cultured in RPMI 1640, 10% FCS, 2 mmol/L glutamine, 100 units/ 0.025% collagenase A (Roche, Mannheim, Germany) in Williams’ medium mL penicillin and 100 Ag/mL streptomycin, and 0.05 mmol/L E. Tumor nodules were digested in 0.025% collagenase A for 30 minutes in 2-mercaptoethanol. Surface markers were analyzed on a FACScan (BD a rotary water bath at 37jC. LTECs were filtered, washed, and separated PharMingen) with the following antibodies purchased from BD PharMin- in a Histodenz (nonionic density gradient medium, Sigma) gradient. Cells gen and used at 10 Ag/mL: anti-CD3 (hamster IgG), anti-CD4 (rat IgG2b), were further incubated with 0.01% dispase (Roche) for 30 minutes at 37jC anti-CD8 (rat IgG2a), anti-CD45R/B220 (rat IgG2a), anti-DX5 (rat IgM), in a rotary water bath, washed, and concentrated via a second density and anti-CD11b (rat IgG2b). gradient centrifugation. For fluorescence-activated cell sorting (FACS) RNA preparation and microarray analysis. Total RNA from 2 Â 105 to staining, cells were incubated with Fc block (CD16/CD32, 2.4G2, 2.5 Ag/AL; 3 Â 105 purified LSECs or LTECs was prepared using the Absolutely RNA BD PharMingen, Heidelberg, Germany) and specifically labeled with anti- Microprep kit (Stratagene, Amsterdam, the Netherlands). Total RNA from CD31-phycoerythrin (rat IgG2a, 4 Ag/mL; BD PharMingen) and ME-9F1- whole organs was isolated with the RNeasy mini kit (Qiagen, Hilden, Germany). RNA quality was analyzed using RNA 6000 Nano Assays and the Bioanalyzer 2100 Lab-on-a-Chip system (Agilent Technologies, Palo Alto, CA). For all probe syntheses, 250 ng RNA were used. RNA was amplified 6 S. Stahl, unpublished results. according to Baugh et al. (22) and modified by Kenzelmann et al. (23). Five www.aacrjournals.org 199 Cancer Res 2006; 66: (1). January 1, 2006

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Cancer Research micrograms of biotinylated cRNA were used for hybridization on Affymetrix (R = ratio of expression in the endothelial cells to expression in whole murine U74Av2 arrays following the instructions of the manufacturer. Three tissue), (b) difference in fluorescence intensity between the two cell types independent probes for LSECs, LTECs, normal liver, and liver tumor were z50 units, and (c) unadjusted P value for the corresponding difference V synthesized and three hybridizations per group were done. To collect the 0.05. P values for the comparison LTEC versus LSEC were adjusted material for individual probes, organs from three mice were pooled. Public according to Benjamini and Hochberg (28) to control the false discovery database references for the U74Av2 gene chip are available on the rate. A threshold of 0.05 for the adjusted P values and probe sets with a fold Affymetrix NetAffx Analysis Center (www.affymetrix.com). change of z3 and enrichment in endothelial cells of either normal or tumor Statistical data evaluation. Statistical analysis was done using the tissue were used. software package R, version 1.9.1 (24), together with libraries gcrma and Quantitative reverse transcription-PCR analysis. Quantitative reverse limma of the Bioconductor Project, version 1.4 (25). Data preprocessing transcription-PCR (RT-PCR) was done using real-time PCR TaqMan steps, background adjustment, normalization, and computation of GCRMA technology (Applied Biosystems, Weiterstadt, Germany) as described (29). gene expression measures were done according to Wu et al. (26). For The mouse hypoxanthine phosphoribosyltransferase (Hprt) gene served as an statistical analysis, empirical Bayes inference for linear models with factors internal control. Primer sequences are available on request. tissue type [normal (normal liver, LSEC) or malignant (liver tumor, LTEC)] Immunohistochemistry. The procedure was described previously (29). and sample type (whole tissue or purified endothelial cells) and their The following reagents were used at 10 Ag/mL: anti-CD31 and ME-9F1, interaction was used (27). Moderated t statistics based on shrinkage of the biotinylated anti-CD3, and biotinylated anti-B220. The concentration of estimated sample variance towards a pooled estimate and corresponding CCR2B (goat polyclonal IgG, C-20, Santa Cruz Biotechnology, Heidelberg, P values were calculated for comparisons, LSEC versus normal liver, LTEC Germany) and CCR5 (goat polyclonal IgG, M-20, Santa Cruz Biotechnology) versus liver tumor tissue, and LTEC versus LSEC. According to Favre et al. was 4 Ag/mL. All secondary reagents were used at 3 Ag/mL: cyanin-3 or (12), candidates for genes/expressed sequence tags (EST) enriched in the FITC-conjugated IgG F(abV)2 fragment goat anti-rat (Dianova, Hamburg, endothelial cells were chosen by three criteria: (a) enrichment ratio R z 1.5 Germany), cyanin-3-conjugated donkey anti-goat IgG (Dianova), and streptavidin-phycoerythrin or streptavidin-FITC (BD PharMingen). Histology was analyzed using the Axioplan 2 microscope (Carl Zeiss, Hallbergmoos, Germany) equipped with Plan-Neofluar objective lenses 10Â/0.30, 25Â/0.08 oil, and 40Â/1.30 oil. AxioCAM camera and AxioVision 3.1 (Carl Zeiss) were used for image recording. Images were processed using Adobe Photoshop software (San Jose, CA). ELISA. Tag-HCC cells, TILs, LSECs, and LTECs were seeded on six-well plates at a density of 4 Â 105/cm2. Supernatants were collected for 3 days. ELISAs were done according to the instructions of the manufacturer for mouse MCP-1/CCL2 (BD OptEIA set, BD Biosciences, Heidelberg, Germany) and mouse MIP-1a/CCL3 (DuoSet, R&D Systems, Wiesbaden, Germany) and measured by Multiskan Ascent (Labsystems, Helsinki, Finland). LSEC proliferation. LSECs were seeded on collagen-coated 12-well plates at a density of 2 Â 105/cm2 and treated for 36 hours with reagents from R&D Systems: 5 ng/mL recombinant mouse CCL2, 5 ng/mL recombinant CCL3, antimouse CCL2 antibody (rat IgG2a, clone 123616, 1 Ag/mL), and antimouse CCL3 antibody (goat polyclonal IgG, 1.0 Ag/mL). For the last 12 hours, 1 ACi/mL [3H]thymidine (Amersham, Freiburg, Germany) was added to the culture. Following incubation with [3H]thymidine, cells were washed in PBS and treated with 10% trichloroacetic acid for 15 minutes. Cells were washed thrice with 90% ethanol, lysed in 0.3 mol/L sodium hydroxide, and collected on nitrocellulose filters (Schleicher & Schu¨ll, Dassel, Germany). Samples were measured in scintillation fluid (Fisher Chemicals, Heidelberg, Germany) in a liquid scintillation counter (Wallac, Turku, Finland).

Results Vascular transformation occurs late during liver carcinogenesis. Liver carcinogenesis in AlbTag mice is multistep, prog- ressing from preneoplastic foci to nodular adenoma and, finally, carcinoma. Hepatocellular carcinoma eventually arises in all Figure 2. Isolation of endothelial cells from normal liver and AlbTag tumors. AlbTag mice, which live an average of 16 weeks. Angiogenesis in A, staining of sinusoids in normal mouse liver as seen with anti-CD31 these mice is central to liver tumor progression, similar to the antibodies on frozen sections. B, corresponding staining with ME-9F1. C, immunohistochemistry on AlbTag late-stage tumors (14-16 weeks) using the hypervascularity in poorly differentiated human hepatocellular anti-CD31 antibody. D, tumor vessel morphology as displayed by staining with carcinoma (30). We used intravital microscopy to compare trans- the ME-9F1 antibody (A-D, original magnification, Â10). E, ex vivo purified forming, tumorigenic vasculature to normal vessels (Fig. 1A-F). endothelial cells from normal liver (LSEC) were stained with CD31-phycoerythrin (CD31-PE) and ME-9F1-FITC. Approximately 35% of double-labeled cells with The liver has a unique microvascular system consisting of afferent high fluorescence were sorted by FACS. Some sorted cells were cultured on arterioportal and efferent venular blood vessels. Figure 1A shows collagen and purity of endothelial cells was assessed by the uptake of DiI-lableled, acetylated LDL (inset). F, FACS analysis showing a typical staining the typical microarchitecture of normal liver with an intercon- of freshly purified liver tumor-associated endothelial cells (LTEC) with CD31 and necting network of sinusoids and postsinusoidal venules. Sinus- ME-9F1. Approximately 5% of double-labeled, highly fluorescent cells were oids in the mouse liver are small, usually with a diameter of 5 to FACS sorted. Inset, endothelial cell-specific uptake of acetylated LDL. Dead cells were excluded from the sort (E and F, insets, original 10 Am (Fig. 1D), whereas postsinusoidal venules range from 20 to magnification, Â40). 30 Am (Fig. 1E). Surprisingly, even in macroscopically detectable

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Table 1. Genes up-regulated in tumor-derived endothelial cells

Public ID Gene title Gene Fold Adjusted P Function symbol change

X70058 Chemokine (C-C motif) ligand 7 Ccl7 82 0.007094 Chemotaxis AJ223208 Cathepsin S Ctss 74 0.002918 Proteolysis and peptidolysis AB023418 Chemokine (C-C motif) ligand 8 Ccl8 66 0.033688 Chemotaxis J04491 Chemokine (C-C motif) ligand 3 Ccl3 64 0.000088 Chemotaxis M90388 Protein tyrosine phosphatase, Ptpn8 47 0.000003 Protein amino nonreceptor type 8 acid dephosphorylation X98471 Epithelial membrane protein 1 Emp1 34 0.000134 Cell growth X16834 Lectin, galactose binding, soluble 3 Lgals3 33 0.002719 — M80778 Selectin, endothelial cell Sele 28 0.000222 Cell adhesion Z80112 Chemokine (C-X-C motif) receptor 4 Cxcr4 27 0.000426 Chemotaxis U83148 Nuclear factor, interleukin 3, regulated Nfil3 25 0.000747 Regulation of transcription U35330 Histocompatibility 2, class II, H2-DMb1 25 0.000036 Antigen presentation locus Mb1 M37897 Interleukin 10 Il10 25 0.002238 Immune response L00039 Myelocytomatosis oncogene Myc 25 0.000406 Regulation of cell cycle M94584 Chitinase 3-like 3 Chi3l3 24 0.000270 Inflammatory response D13458 Prostaglandin E receptor 4 (subtype EP4) Ptger4 24 0.000527 G-protein signaling X62502 Chemokine (C-C motif) ligand 4 Ccl4 23 0.000888 Chemotaxis AW124113 Brain abundant, membrane Basp1 21 0.001284 Regulation of transcription attached signal protein 1 M55181 Preproenkephalin 1 Penk1 19 0.000057 Neuropeptide signaling, angiogenesis M19681 Chemokine (C-C motif) ligand 2 Ccl2 18 0.005244 Chemotaxis AI120844 Pleckstrin homology, Sec7 and Pscdbp 18 0.001328 Regulation of cell adhesion coiled-coil domains, binding protein AB006361 Prostaglandin D2 synthase (brain) Ptgds 15 0.013120 Prostaglandin biosynthesis L24118 Tumor necrosis factor, a-induced protein 2 Tnfaip2 14 0.006839 Angiogenesis AI120844 Pleckstrin homology, Sec7 and coiled-coil Pscdbp 13 0.001900 Regulation of cell adhesion domains, binding protein X53526 CD48 antigen Cd48 12 0.000180 — AF030185 chemokine (C-C motif) receptor-like 2 Ccrl2 12 0.001361 G protein–coupled receptor protein signaling Y17860 Ganglioside-induced Gdap10 12 0.003327 — differentiation-associated-protein 10 X87128 Tumor necrosis factor receptor Tnfr2 12 0.001153 Inflammatory response superfamily, member 1b U39827 G protein–coupled receptor 65 Gpr65 12 0.001125 G protein–coupled receptor protein signaling Y12657 Cytochrome P450, family 26, Cyp26a1 12 0.001221 Electron transport subfamily a, polypeptide 1 AV223216 Interleukin 1 receptor, type II Il1r2 11 0.009835 Transport Y11666 Hexokinase 2 Hk2 11 0.009072 Glycolysis X62700 Urokinase plasminogen uPAR 11 0.000118 Angiogenesis activator receptor AF000236 Chemokine orphan receptor 1 Cmkor1 11 0.005225 Chemotaxis AF065947 Chemokine (C-C motif) ligand 5 Ccl5 11 0.007047 Chemotaxis U23778 B-cell leukemia/lymphoma 2 Bcl2a1d 11 0.001365 — related protein A1d M34896 Ecotropic viral integration site 2a Evi2a 10 0.000412 Cell growth and/or maintenance M58004 Chemokine (C-C motif) ligand 6 Ccl6 10 0.027468 Chemotaxis U56819 Chemokine (C-C motif) receptor 2 Ccr2 9 0.000773 Chemotaxis AW122039 Coronin, actin binding protein 1A Coro1a 9 0.000308 — U87948 Epithelial membrane protein 3 Emp3 9 0.000852 Cell growth M16238 Fibrinogen-like protein 2 Fgl2 9 0.001188 Cytolysis X53798 Chemokine (C-X-C motif) ligand 2 Cxcl2 9 0.003481 Chemotaxis

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Table 1. Genes up-regulated in tumor-derived endothelial cells (Cont’d)

Public ID Gene title Gene Fold Adjusted P Function symbol change

U34277 Phospholipase A2, group VII Pla2g7 9 0.023772 Inflammatory response (platelet-activating factor acetylhydrolase) M12330 Ornithine decarboxylase, structural 1 Odc1 9 0.000056 Sonic hedgehog signaling M72332 Selectin, platelet Selp 9 0.004675 Inflammatory response AF009246 RAS, dexamethasone-induced 1 Rasd1 9 0.000075 Small GTPase-mediated signal transduction U10551 GTP-binding protein Gem 8 0.000497 Small GTPase-mediated (gene overexpressed in skeletal muscle) signal transduction M15131 Interleukin 1b Il1b 8 0.007405 Immune response U12884 Vascular cell adhesion molecule 1 Vcam1 8 0.004675 Cell adhesion U22262 Apolipoprotein B editing complex 1 Apobec1 8 0.006328 mRNA editing AF099973 Schlafen 2 Slfn2 7 0.004604 Negative regulation of cell proliferation C79248 Expressed sequence C79248 — 7 0.022702 — U12919 Adenylate cyclase 7 Adcy7 7 0.000340 Cyclic AMP biosynthesis AV152244 IFN, a-inducible protein G1p2 7 0.004984 Chemotaxis AV370035 Chemokine (C-C motif) receptor 5 Ccr5 7 0.000655 Chemotaxis AI839109 Tumor necrosis factor, a-induced protein 8 Tnfaip8 7 0.006148 — U94828 Regulator of G-protein signaling 16 Rgs16 7 0.007984 G protein–coupled receptor protein signaling AW230891 Leucine-rich a-2-glycoprotein 1 Lrg1 7 0.000344 — X89749 TG interacting factor Tgif 7 0.000758 TGF-h receptor signaling pathway L19932 Transforming growth factor, b induced Tgfbi 6 0.005967 Cell adhesion X82786 Antigen identified by monoclonal Mki67 6 0.009691 Cell proliferation antibody Ki 67 D16432 Cd63 antigen Cd63 6 0.006801 — Y07836 Basic helix-loop-helix domain Bhlhb2 6 0.002148 Regulation of transcription containing, class B2 M32490 Cysteine rich protein 61 Cyr61 5 0.008365 Regulation of cell growth, angiogenesis U67187 Regulator of G-protein signaling 2 Rgs2 5 0.014020 G protein–coupled receptor protein signaling M61007 CAAT/enhancer binding protein, b Cebpb 5 0.036020 Regulation of transcription AF020313 Amyloid b(A4) precursor protein-binding, Apbb1ip 5 0.037024 Neuropeptide signaling pathway family B, interacting protein U00937 Growth arrest and DNA-damage- Gadd45a 5 0.011075 Regulation of cell cycle inducible 45 a AW061318 CUG triplet repeat, RNA binding protein 2 Cugbp2 5 0.002034 mRNA splice site selection AA204579 RIKEN cDNA 2510004L01 gene — 5 0.000899 — AB011812 Protein kinase C, d Prkcd 5 0.001361 Protein amino acid phosphorylation AW120868 TCDD-inducible poly(ADP-ribose) polymerase Tiparp 5 0.000682 Protein amino acid ADP-ribosylation AF053959 Ras association (RalGDS/AF-6) Rassf5 5 0.006784 Neuropeptide signaling pathway domain family 5 AI835159 PTK2 protein tyrosine kinase 2 b Ptk2b 4 0.016812 Protein amino acid phosphorylation M24377 Early growth response 2 Egr2 4 0.004604 Regulation of transcription X16995 Nuclear receptor subfamily 4, group A, member 1 Nr4a1 4 0.027893 Regulation of transcription U74683 Cathepsin C Ctsc 4 0.009244 Proteolysis and peptidolysis U25685 Spleen tyrosine kinase Syk 4 0.019266 G protein–coupled receptor protein signaling Z16410 B-cell translocation gene 1, Btg1 4 0.001908 Cell growth and/or maintenance antiproliferative X66032 Cyclin B2 Ccnb2 4 0.001295 Cell cycle, sonic hedgehog signaling J04596 Chemokine (C-X-C motif) ligand 1 Cxcl1 3 0.006109 Regulation of cell cycle U44088 Pleckstrin homology-like domain, Phlda1 3 0.014372 FasL biosynthesis family A, member 1 AW049031 Core promoter element binding protein Copeb 3 0.000183 — U88327 Suppressor of cytokine signaling 2 Socs2 3 0.027363 Regulation of cell growth

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Table 1. Genes up-regulated in tumor-derived endothelial cells (Cont’d)

Public ID Gene title Gene Fold Adjusted P Function symbol change

M97590 Protein tyrosine phosphatase, Ptpn1 3 0.000218 Protein amino acid nonreceptor type 1 dephosphorylation AB031292 Proteolipid protein 2 Plp2 3 0.000852 — M82831 Matrix metalloproteinase 12 Mmp12 3 0.012457 Proteolysis and peptidolysis AV218205 Cystatin C Cst3 3 0.015164 — U57524 Nuclear factor of jlight chain gene Nfkbia 3 0.001739 Regulation of cell proliferation enhancer in B-cells inhibitor, a U19118 Activating transcription factor 3 Atf3 3 0.037702 Regulation of transcription AJ001418 Pyruvate dehydrogenase Pdk4 3 0.021345 Signal transduction annotation kinase, isoenzyme 4 X57437 Histidine decarboxylase Hdc 3 0.006871 — K02236 Metallothionein 2 Mt2 3 0.010883 Zinc ion homeostasis AA163960 RAS p21 protein activator 4 Rasa4 3 0.039682 Intracellular signaling cascade U59864 TRAF family member-associated Tank 3 0.011823 I-nB kinase/NF-nB cascade Nf-jB activator AB013137 Glutaredoxin 1 (thioltransferase) Glrx1 3 0.002616 Electron transport AI851255 Cathepsin B Ctsb 3 0.042611 Proteolysis and peptidolysis AJ242663 Cathepsin Z Ctsz 3 0.024982 Proteolysis and peptidolysis AI846289 Casein kinase, d Csnk1d 3 0.000875 Wnt receptor signaling pathway

NOTE: List of 100 from 156 genes found to be a) enriched in LTEC compared with whole liver tumors and b) up-regulated compared with LSEC (adjusted P value z0.05).

nodular carcinoma, the principal vascular architecture is not cells from normal liver (Fig. 2E) and cancers (Fig. 2F) were disturbed (Fig. 1B). Instead, sinusoidal diameter was significantly isolated by enzymatic tissue dissociation, enriched by gradient increased to >10 Am (Fig. 1D) and postsinusoidal venules to >30 centrifugation, and labeled with CD31 and ME-9F1 antibodies. Am (Fig. 1E). Thus, the earliest vessel transformation in AlbTag Cells with the highest fluorescence for both markers were sorted. liver carcinogenesis is an increase in vessel caliber. In contrast, To confirm identity and viability of endothelial cells, uptake of loss of vessel hierarchy is only evident in more advanced LDL modified by acetylation and conjugated with the fluoro- hepatocellular carcinomas, when tumor nodules range from 2 chrome DiI was measured on sorted cells plated on collagen to 10 mm (Fig. 1C and F). Here, sinusoids and postsinusoidal (Fig. 2E and F, insets). Isolated endothelial cells were at least venules cannot be structurally discriminated. Instead, advanced 95% pure. tumors display a chaotic vascular distribution with marked Genes involved in angiogenesis during hepatocellular variability in vessel diameter, generally between 20 and 100 Am, carcinogenesis. To gain insight into molecular mechanisms indicative of high angiogenic activity and the formation of a true underlying vessel remodeling, we first identified genes enriched in tumor-specific vasculature. endothelial cells (12) by comparing gene expression between Ex vivo isolation of normal and tumor-derived liver purified endothelial cells and their tissue of origin (normal liver endothelial cells. Having identified profound morphologic or tumor). RNA from normal liver, isolated LSECs, liver tumor, changes in the tumor vasculature during neovascularization, and isolated LTECs was probed on Affymetrix oligonucleotide we aimed to characterize molecular differences between LTECs microarrays encompassing 12,000 genes and ESTs. Comparing and the corresponding normal LSECs. We isolated these normal liver and its corresponding purified endothelial cells endothelial cells by FACS using specific antibodies. CD31 is an (LTEC), 1,521 genes/ESTs (12.7%) were enriched in LSECs. When endothelial-specific antibody reacting with normal liver sinusoids whole liver tumors were compared with the corresponding (Fig. 2A) and heterogeneous vessels of AlbTag tumors (Fig. 2C) purified LTECs, 892 genes/ESTs (7.4%) were enriched in LTEC. but is unsuitable as a single FACS marker due to cross-reactivity Among genes enriched in both LSECs and LTECs are known with a subset of immune cells. We therefore used a second endothelial markers, such as vascular endothelial cadherin; endothelial-specific antibody, purified from the hybridoma ME- claudin 5; von Willebrand factor; VEGF receptors (VEGFR) 9F1 (21). Although cross-reactive with DX5-positive natural killer VEGFR1, VEGFR2, and VEGFR3; angiopoietin 2 (Ang2); and the (NK) cells,7 ME-9F1 is highly endothelial specific in histology, tyrosine kinase receptor 1 (Tie1), which are not differentially staining normal liver sinusoids as well as tumor vessels in a regulated (data not shown; ref. 31). The accumulation of well- pattern indistinguishable from CD31 (Fig. 2B and D). Endothelial known endothelial-specific genes in the enriched gene fractions confirms the validity of our approach. Differential analysis of endothelial-enriched genes from normal and tumor tissue was subsequently done to show tumor-specific 7 A. Hamann, personal communication. endothelial alterations. We identified 156 genes/ESTs being www.aacrjournals.org 203 Cancer Res 2006; 66: (1). January 1, 2006

Table 2. Genes down-regulated in tumor-derived endothelial cells

Public ID Gene title Gene Fold Adjusted P Function symbol change

M34476 Retinoic acid receptor, c Rarg 25 0.000036 Regulation of transcription AW049768 Lipocalin 7 Lcn7 22 0.000913 Proteolysis AB010833 Patched homologue 2 Ptch2 21 0.000097 Sonic hedgehog signaling AA879929 DNA segment, Chr 17, — 12 0.003040 — ERATO Doi 288, expressed AF035645 Protein tyrosine Ptp4a3 11 0.001042 Protein amino acid phosphatase 4a3 dephosphorylation AI850533 Bone morphogenic protein 6 Bmp6 9 0.003748 TGF-h receptor signaling pathway AA838868 Latent transforming growth Ltbp4 9 0.000210 TGF-h receptor signaling pathway factor bbinding protein 4 AI846549 RIKEN cDNA 2610019F03 gene — 9 0.030927 — AI841235 Transcription factor 3 Tcf3 8 0.000031 Wnt receptor signaling pathway U22399 Cyclin-dependent kinase Cdkn1c 8 0.000036 Cell cycle inhibitor 1C (P57) D49473 SRY-box containing gene 17 Sox17 8 0.000110 Wnt receptor signaling pathway U12620 Dipeptidylpeptidase 4 Dpp4 8 0.000009 Proteolysis and peptidolysis AI846896 RIKEN cDNA 2310004I03 gene — 8 0.000392 — AI837497 Disabled homologue 2 (Drosophila) Dab2ip 8 0.000150 — interacting protein U30292 Procollagen, type XIII, a1 Col13a1 7 0.000240 Cell adhesion X51905 Lactate dehydrogenase 2, B chain Ldh2 7 0.000937 Glycolysis AI595313 BTB (POZ) domain containing 4 Btbd4 7 0.001006 Regulation of transcription AI846678 cDNA sequence BC060632 — 7 0.000046 — M21019 Harvey rat sarcoma Rras 6 0.001311 Small GTPase-mediated oncogene, subgroup R signal transduction D11091 Protein kinase C, u Prkcq 6 0.000000 Protein amino acid phosphorylation AF047355 DNase1-like 3 Dnase1l3 6 0.013120 DNA catabolism AB012886 Insulin-like growth factor Igfbp7 6 0.002071 Regulation of cell growth binding protein 7 AF010133 MAD homologue 6 (Drosophila) Smad6 6 0.000100 TGF-h receptor signaling pathway Y08027 ADP-ribosyltransferase 3 Art3 6 0.002299 Protein amino acid ADP ribosylation U33629 Myeloid ecotropic viral Meis1 6 0.002722 Regulation of transcription integration site 1 Z31664 Activin A receptor Alk1 6 0.001299 TGF-h receptor signaling pathway L35032 SRY-box containing gene 18 Sox18 6 0.000312 Regulation of transcription U43541 Laminin, b2 Lamb2 6 0.018968 Electron transport AJ131021 Ribosomal protein Rps6ka2 5 0.000488 Protein amino acid S6 kinase, polypeptide 2 phosphorylation L25602 Bone morphogenetic protein 2 Bmp2 5 0.010433 TGF-h receptor signaling pathway X98207 Histone deacetylase 1 Hdac1 5 0.000118 Regulation of transcription AA648027 cDNA sequence BC029719 — 5 0.001553 — AF006492 Zinc finger protein, Zfpm1 5 0.000464 Negative regulation multitype 1 of transcription AW048347 Tripartite motif protein 47 Trim47 5 0.000218 — AJ000059 Hyaluronidase 2 Hyal2 5 0.000709 Carbohydrate metabolism AI854522 Protocadherin c subfamily A Pcdhga 5 0.001361 Cell adhesion AW047228 Histone deacetylase 7A Hdac7a 5 0.000031 Negative regulation of transcription AI849048 Vesicle-associated membrane Vapb 5 0.000374 — protein, associated protein B, C AI851258 RIKEN cDNA E030006K04 gene — 5 0.001231 —

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Table 2. Genes down-regulated in tumor-derived endothelial cells (Cont’d)

Public ID Gene title Gene Fold Adjusted P Function symbol change

AF016697 Duffy blood group Dfy 5 0.000135 G protein–coupled receptor protein signaling AF017111 Interleukin 16 Il16 5 0.000118 Immune cell chemotaxis L22482 Transforming growth factor Tgfb1i1 5 0.000225 — b1-induced transcript 1 AA709672 Kinesin 2 Kns2 5 0.002176 — X74134 Nuclear receptor subfamily 2, Nr2f1 5 0.000488 Regulation of cell cycle group F, member 1 D30779 Phospholipase A2, group IB, Pla2g1br 5 0.000823 — pancreas, receptor AF004106 Dimethylarginine Ddah2 5 0.006736 Arginine catabolism dimethylaminohydrolase 2 AI841022 Thyroid hormone receptor a Thra 4 0.000240 Regulation of cell cycle AI843845 RIKEN cDNA 2310061B02 gene — 4 0.000720 — U37500 Polymerase (RNA) II Polr2a 4 0.000432 Transcription (DNA directed) polypeptide A AA209597 RIKEN cDNA C530030I18 gene — 4 0.043795 — Y08026 Immunity-associated protein Imap38 4 0.004264 — AI845581 RIKEN cDNA 4931426K16 gene — 4 0.013572 — U05245 T-cell lymphoma invasion Tiam1 4 0.011354 Small GTPase-mediated and metastasis 1 signal transduction AI839175 Serum deprivation response Sdpr 4 0.012973 — AI841654 G protein–coupled receptor 56 Gpr56 4 0.010435 G protein–coupled receptor protein signaling AW121826 Inositol 1,4,5-trisphosphate 3-kinase B Itpkb 4 0.000758 — U89489 LIM domain binding 2 Ldb2 4 0.013882 Development D45903 Syntaxin binding protein 1 Stxbp1 4 0.000913 Protein transport D10475 Epimorphin Epim 4 0.007119 Intracellular protein transport AI835373 RIKEN cDNA F830020C16 gene — 4 0.007777 Regulation of transcription U29055 Guanine nucleotide Gnb1 4 0.016943 G protein–coupled receptor binding protein, b1 protein signaling AI157060 RIKEN cDNA D030068L24 gene — 4 0.000020 — AA397054 Adenylate cyclase 4 Adcy4 4 0.000895 Cyclic AMP biosynthesis X71426 Endothelial-specific receptor Tek 4 0.001938 Phosphorylation, tyrosine kinase angiogenesis AI854794 Tensin like C1 domain-containing Tenc1 4 0.008350 Wnt receptor phosphatase signaling pathway AJ250490 Receptor (calcitonin) activity Ramp2 4 0.002071 Adrenomedullin signaling, modifying protein 2 angiogenesis L33726 Fascin homologue 1, actin bundling Fscn1 4 0.000688 — protein (Strongylocentrotus) L31958 Phosphoprotein enriched Pea15 4 0.000758 Intracellular in astrocytes 15 signaling cascade AB000096 GATA binding protein 2 Gata2 4 0.000118 Regulation of transcription AW048884 DNA segment, Chr 8, ERATO D8Ertd319e 4 0.016682 Metabolism Doi 319, expressed AI853996 RAB11a, member RAS Rab11a 4 0.012143 Small GTPase-mediated oncogene family signal transduction AW124853 cDNA sequence BC030183 — 4 0.000692 — X57971 Gap junction membrane Gja4 4 0.017023 Blood vessel development channel protein a4 X61452 Septin 4 Septin4 4 0.015164 Cell cycle AI841629 Guanine nucleotide binding Gnai2 4 0.000338 G protein–coupled receptor protein, a inhibiting 2 protein signaling AA763950 Zinc and ring finger 1 Znrf1 4 0.000325 — AW061228 ATPase, Ca2+-sequestering Atp2c1 4 0.005184 Cation transport

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Table 2. Genes down-regulated in tumor-derived endothelial cells (Cont’d)

Public ID Gene title Gene Fold Adjusted P Function symbol change

X58250 H2.0-like homeobox 1 Hlx1 4 0.002244 Regulation of transcription (Drosophila) AW121844 Adducin 1 (a) Add1 4 0.000021 — AI154249 RIKEN cDNA 6330406L22 gene — 4 0.000622 Intracellular signaling cascade AF031380 Riboflavin kinase Rfk 4 0.001382 Vitamin B2 biosynthesis AF055665 Kinesin 2 Kns2 4 0.003299 — AV333272 Bone morphogenetic protein 2 Bmp2 4 0.016525 TGF-h receptor signaling pathway AW122407 Catenin src Catns 4 0.001808 Cell adhesion AF015260 MAD homologue 7 (Drosophila) Smad7 4 0.002249 TGF-h receptor signaling pathway AI851984 Protein phosphatase 1, regulatory Ppp1r2 4 0.001006 Glycogen metabolism (inhibitor) subunit 2 AW124340 Solute carrier family 39 Slc39a8 4 0.002779 Metal ion transport (metal ion transporter), member 8 U44389 Hydroxyprostaglandin Hpgd 4 0.029232 Prostaglandin dehydrogenase 15 (NAD) metabolism AF057286 Epsin 2 Epn2 4 0.002026 Endocytosis AW046775 RIKEN cDNA 2610319K07 gene 2610319K07 4 0.037007 — AF110520 DNA segment, Chr 17, ERATO — 4 0.003264 — Doi 288, expressed AF014010 Polycystic kidney disease 2 Pkd2 4 0.021826 Transport AW120767 RIKEN cDNA 5830406C15 gene — 4 0.001965 — AI851750 Arrestin, b1 Arrb1 3 0.006148 G protein–coupled receptor protein AI509617 Tumor necrosis Tnfaip1 3 0.007525 Potassium ion transport factor, a-induced protein 1 (endothelial) D78643 Ornithine decarboxylase Oaz2 3 0.001564 Sonic hedgehog signaling antizyme 2 U70674 Numb gene Numb 3 0.010883 Notch signaling pathway homologue(Drosophila) Y07919 Adaptor protein Ap1b1 3 0.000705 Notch signaling pathway complex AP1, b 1 subunit AW121446 Casein kinase 1, c2 Csnk1g2 3 0.003987 Protein amino acid phosphorylation AI851163 Tryptophanyl-tRNA synthetase Wars 3 0.011609 Protein biosynthesis, angiogenesis

NOTE: List of 100 genes from 142 genes found to be enriched in LTECs compared with whole liver tumors and down-regulated compared with LSECs (adjusted P z 0.05).

up-regulated (z3-fold, adjusted P V 0.05) and 142 genes/ESTs being the transforming growth factor-h (TGF-h) signaling pathway (35), down-regulated (z3-fold, adjusted P V 0.05) in LTECs compared which are essential for normal vascular development, are with LSECs. Tables 1 and 2 list 100 genes that are up-regulated or significantly altered in angiogenic vessels [TG interacting factor down-regulated, respectively. A considerable number of differen- (Tgif ), bone morphogenetic protein 2 and 6 (Bmp2/Bmp6; ref. 36), tially expressed genes cluster in three major signaling pathways, latent TGF-b-binding protein 4 (Ltbp4; ref. 37), MAD homologue6 namely, notch [numb gene homologue (Numb) and adaptor and 7 (Smad6/Smad7), and activin receptor kinase 1 (Alk1; protein complex (Ap1b1); ref. 32], sonic hedgehog [ornithine ref. 38)]. Other genes that have previously been implicated in decarboxylase (Odc), cyclin B (Ccnb2), patched homologue2 neovascularization and are up-regulated in LTECs include myelo- (Ptch2), and ornithine decarboxylase antizyme 2 (Oaz2); ref. 33), cytomas oncogene (myc) and B-cell translocation gene 1 (btg1), both and wnt (transcription factor 3 (Tcf3 ); casein kinase 1, c2 regulators of cell growth and angiogenesis (39, 40). Preproenkepha- (Csnk1g2) and casein kinase, d (Csnk1d); Syr-box containing gene lin (Penk1) encodes opioid growth factor, which is important for 17 (Sox17); and tensin-like C1 domain-containing phosphatase embryonic vessel development (41), as is spleen tyrosine kinase (Syk; (Tenc1); ref. 34), which have crucial roles in cell proliferation, ref. 42). The urokinase plasminogen activator system is represented survival, and endothelial differentiation. Moreover, members of by its receptor (uPar) and is strongly induced in migrating

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Endothelial Cells in Hepatocellular Carcinoma endothelial cells (43). Inflammation promotes angiogenesis, which tumors (size range, 2-10 mm). The numbers of loosely (Fig. 4A) is reflected by up-regulation of interleukin-1b (IL-1b; ref. 44) and and firmly (Fig. 4B) adherent leukocytes were evaluated after tumor necrosis factor receptor 2 (Tnfr2), a mediator of the tumor intravascular labeling of leukocytes. Surprisingly, despite the up- necrosis factor–induced angiogenic pathway (45). The helix-loop- regulation of some adhesion molecules, low- and high-affinity helix transcription factor Dec1 (Bhlhb2) is up-regulated in tumor leukocyte-endothelium interactions were not enhanced in tumor endothelial cells under hypoxia and may regulate cell death (46). vessels. Next, TILs from solid tumors and lymphocytes from In contrast, aminoacyl-tRNA synthetase (TrpRS or Wars)isan normal liver were isolated and quantified using FACS (Fig. 4C). antiangiogenic molecule and is down-modulated in LTECs (47). Normal liver contains large numbers of CD4+ and CD8+ T cells, Thus, the molecular profile of neovessels in hepatocellular B cells, monocytes/macrophages, and also NK and NKT cells carcinoma shows considerable parallels to previously documented (Fig. 4C). These cells do not normally infiltrate the parenchyma pathways regulating vascular development as well as endothelial (50). In contrast, AlbTag tumors contain significantly increased cell proliferation and migration. numbers of B cells, NK, and NKT cells, while CD4+ and CD8+ T Prominent features of liver angiogenesis. Vessel remodeling cells and monocytes/macrophage numbers remain comparable. during tumor growth is a multistep process requiring endothelial However, surprisingly few immune cells attach to tumor vessels or cell proliferation and tissue invasion through degradation of infiltrate into tumor parenchyma (Fig. 4D and E), which sharply extracellular matrix. Proteases, in particular matrix metallopro- contrasts the massive leukocyte recruitment into inflamed liver teases (MMP), participate in invasive growth (48). Our differential (51). Thus, despite enhanced leukocyte recruitment, liver tumors analysis revealed, however, that whereas only a single MMP family do not display features of an enhanced inflammatory response. member, MMP12, is up-regulated in the tumor vasculature, several Interestingly, attenuation of lymphocyte migration into tumors members of a family of cysteine proteases, the so-called cathepsins, are highly enriched in LTECs. This suggests a crucial role for cathepsins during liver angiogenesis. Cathepsins are also expressed in immune and tumor cells and may, therefore, play a more general role in tumor-stromal interactions (49). To evaluate the relative importance of cathepsins B, C, S, and Z and MMP12 for vascular sprouting, expression levels in different tumor constituents, including tumor cells (Tag-HCC), LTECs, and TILs, were analyzed by quantitative RT-PCR (Fig. 3A). Consistent with the microarray analysis, all proteases are up-regulated in LTECs. However, cathepsin S is most dramatically increased and significantly higher in LTECs than in TILs, suggesting an important function during liver neovascularization. Surprisingly, VEGFR1 and VEGFR2 as well as neuropilin and Tie1 are expressed at similar levels in normal liver and tumor endothelium and not elevated in the liver tumor vasculature. In contrast, endothelial-specific receptor tyrosine kinase (Tie2), a receptor for angiopoietins, is significantly down-regulated in LTECs compared with LSECs (Fig. 3B). This is an intriguing finding because VEGF and angiopoietins are important growth- promoting and vessel-stabilizing factors during angiogenesis (31). Another unexpected but important finding of our microarray analysis is the abundance of chemokines and G protein–coupled chemokine receptors in LTECs. Most strikingly, a variety of CC chemokines (CCL2, CCL3, CCL4, CCL5, CCL7, and CCL8) and their cognate receptors (CCR2 and CCR5) are up-regulated in tumor endothelium, a finding confirmed by quantitative RT-PCR (Fig. 3C). Thus, enhanced CC chemokine signaling represents one of the most significant molecular changes in neovessels in AlbTag tumors and is indicative of an inflammatory response of the vessel wall. Vascular inflammation does not promote leukocyte-tumor endothelium interactions. Chemokines classically recruit leu- Figure 3. Differential gene expression profiles of LSECs and LTECs. A, relative kocytes to inflamed tissue in a paracrine manner. Moreover, mRNA expression levels of the metalloprotease MMP12 and various members of LTECs overexpress vascular cellular adhesion molecule 1 as well the cathepsin cysteine protease family. Expression profiles were assessed for whole normal liver tissue and whole liver tumors from 14- to 16-week-old AlbTag as endothelial and platelet selectins (Table 1), which are thought mice as well as for freshly purified tumor constituent cell types, such as TILs, to facilitate leukocyte adhesion. These findings motivated us LSECs, and LTECs. B, comparative RT-PCR analysis for tyrosine kinase receptors of the VEGF (VEGFR1,VEGFR2, and neutropilin-1) and angiopoietin to analyze leukocyte-endothelial interactions and extravasation (Tie1 and Tie2) signaling in LTECs versus LSECs. C, differential gene of immune cells into liver tumors. We used intravital micros- expression of CC chemokines and cognate receptors. Numbers above columns, copy to monitor adherence of nonspecific leukocytes to vessels fold of induction in LTECs compared with LSECs as assessed by quantitative RT-PCR. Data are from three independently isolated cell populations for TILs, of normal liver and tumors. Postsinusoidal venules of normal LSECs, and LTECs, and five different liver and tumor samples. Expression levels liver were compared with vessels of similar diameter in AlbTag were normalized to the housekeeping gene Hprt. Columns, mean; bars, SD. www.aacrjournals.org 207 Cancer Res 2006; 66: (1). January 1, 2006

Figure 4. Leukocyte-endothelial interactions and infiltration of AlbTag tumors. Loose leukocyte (rollers/mm2;A) and firm leukocyte (stickers/mm2;B) adherence in vessels of normal liver (n = 10) and liver tumors of different size range (n = 17) were quantified intravitally. RBC velocity was unchanged in tumors compared with normal liver (data not shown). C, normal and malignant livers were enzymatically dissected and the frequency of released CD3+CD4+ (T-helper T cells), CD3+CD8+ (cytotoxic T cells), B220+ (B cells), CD3+DX5+ (NKT cells), DX5+ (NK cells), and CD11b+ (monocytes/macrophages) populations were quantified by FACS. D, representative histology showing tumor vessels (CD31+, red) and infiltration of AlbTag carcinoma by CD3+cells (green). E, infiltration by B220+ (green) cells. Few immune cells interact with the vessel wall (arrows). Some leukocytes diffusely infiltrate tumor parenchyma (original magnification, Â25). The immunosuppressive cytokine IL-10 is strongly expressed in LTECs and 17 fold up-regulated compared with levels in LSECs. Representative quantitative RT-PCR analyses of three independent probes for LTECs and LSECs normalized against Hprt. Columns, mean; bars, SD.

coincides with expression of the immune-suppressive cytokine response was measured. Incubation of LSECs with tumor- IL-10 (Fig. 4F) exclusively by tumor endothelial cells. conditioned medium alone enhances proliferation, which is in part Chemokine signaling promotes vessel proliferation in an mediated by VEGF (800 pg/mL in 5-day Tag-HCC-conditioned autocrine loop. Chemokines, in particular CXC chemokines, are medium). Addition of recombinant CCL2 or CCL3 induced multifunctional and control leukocyte recruitment and angiogen- proliferation by 3- to 4-fold, which was abolished in the presence esis (52). The majority of chemokines up-regulated in AlbTag of neutralizing antibodies. Proliferation was further increased tumor vessels belongs to the CC family. To assess the role of CCL2 (5-fold) when LSECs were incubated with CCL2 and CCL3 and CCL3 as angiogenic growth regulators, protein secretion by simultaneously (Fig. 5G). Therefore, expression of chemokines and cultured tumor cells, TILs, LSECs, and LTECs was analyzed using their cognate receptors on tumor vessels in vivo are part of a ELISA. Release of CCL2 (Fig. 5A) and CCL3 (Fig. 5B) in short-term synergistic autocrine growth loop, which induces endothelial cell cultured LTECs is f6-fold increased compared with LSECs. proliferation in vitro and may be crucial for vessel remodeling during Moreover, these chemokines are exclusively produced by endothe- liver angiogenesis. lial cells (Fig. 5A and B). CCR2 and CCR5, the cognate receptors for CCL2 and CCL3, respectively, are both detectable in the vasculature of AlbTag tumors by immunohistochemistry (Fig. 5C and E). Vessel Discussion staining of CCR2 and CCR5 is noncontinuous, indicating that only Neovascularization is essential for solid tumor progression subsets of endothelial cells express high levels of these chemokine and is an integral element of tumorigenesis. Although different receptors. Furthermore, mRNA expression of both receptors can be tumors frequently share common biological principles, each induced in ex vivo purified LSECs upon stimulation with liver tumor possesses a unique environment that shapes the pheno- tumor-conditioned medium and increases over time (Fig. 5D and F). type of resident cells, including the vasculature. Indeed, the Simultaneous expression of chemokines and their cognate receptors present study reveals novel aspects of tumor vessel remodeling on LTECs implies a potential autocrine growth regulation of that have not been apparent by studying in vitro models of endothelial cells within the tumor bed. To assess this further, LSECs angiogenesis. were induced by conditioned medium to express CCR2 and CCR5, We show with intravital microscopy of AlbTag tumors that then stimulated with purified CCL2 and CCL3, and the proliferative increased vessel caliber was the first detectable abnormality,

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Endothelial Cells in Hepatocellular Carcinoma followed by vessel sprouting. Notably, increasing vessel heteroge- are marked by an increase in vessel diameter rather than vessel neity and chaotic vessel organization were only observed late in numbers. Thus, expansion of the endothelial surface area is the first tumorigenesis, when the carcinoma had progressed beyond the adaptive process of the vasculature during tumorigenesis. nodular stage. These findings are similar to those in our earlier To focus on molecular changes intrinsic to the vasculature, studies, using a mouse model of insulinoma (3) and rat we first identified genes enriched in purified endothelial cells and hepatocellular cancer (4), where early phases of angiogenesis then compared expression profiles between purified endothelial cells of normal liver and liver tumors, to identify tumor-specific changes. There are currently limited numbers of molecular studies on normal (10, 12) or tumor vessels (17) because of technical difficulties of ex vivo purification of endothelial cells. However, our microarray studies identified a considerable number of genes shown by gene targeting experiments to be critical, thus confirming the effectiveness of our strategy. These genes include the tyrosine kinase receptors of the VEGF and angiopoietin signaling pathways, hedgehog signaling, and ‘‘neuronal’’ genes, such as neuropilin, ephrins, and members of the notch and delta families. Typically, both up- and down-regulation of these molecules results in vascular defects (53, 54) and, indeed, various members of these families are differentially regulated during liver neovascularization (Tables 1 and 2). Interestingly, some genes that we have now identified as modulators of liver angiogenesis are reported to impair placental or cardiac development. Cysteine-rich protein 61 (Cyr61), for instance, is an extracellular matrix– associated angiogenic inducer strongly expressed in liver neovessels as well as in proliferating endothelial cells in vitro (5, 7) and plays an essential role in placental development (55). Ltbp4 and bmp2, both molecules of the TGF-h signaling pathway, are essential for heart development (36, 37) and are shown to be down-regulated in LTECs. Similarly, the transcriptional cofactor friend of gata-1 (Fog1 or zinc finger protein multitype 1, Zfpm1; ref. 56) and polycystic kidney disease-2 (Pkd2), mutations of which also cause autosomal dominant polycystic kidney disease (57), are both necessary for normal cardiac function and are significantly down-regulated in LTECs. Thus, a constellation of genes with known effects on embryonic, placental, and myocardial vascularization is likely to also have overlapping functions in liver angiogenesis. The Tie2 receptor, which mediates signaling of Ang1, and its antagonist Ang2, is also decreased in AlbTag tumor vessels. Because interaction of Ang1 and Tie2 strengthens cell junctions (58, 59), impaired signaling may prevent vessel stabilization during neovascularization. Surprisingly, however, receptors for VEGF, such as VEGFR1, VEGFR2, and neuropilin, prototypic mediators of angiogenic signaling, are expressed at comparable levels in LSECs and LTECs. This provocative finding implies that VEGF signaling is as important for tumor growth as for normal Figure 5. Autocrine growth stimulation of LTECs. A, secretion of CCL2 by a endothelial homeostasis. Indeed, VEGF has been implicated in murine hepatocellular carcinoma cell line (Tag-HCC), ex vivo purified AlbTag the homeostasis of lung endothelium (12) as well as in neuro- TILs, and freshly isolated normal and tumor-derived endothelial cells, LSECs and protection (60). Alternatively, VEGF might be involved in the LTECs, respectively. Protein levels were assessed in 2-day cultures by ELISA. B, corresponding analysis for the chemokine CCL3. Protein concentrations early phases of angiogenesis, whereas late-stage tumors rely on were measured in two independent experiments. C, immunohistochemistry on a different growth factors to promote neovascularization. The liver tumor of a 14-week-old AlbTag mouse using a CCR2-specific antibody. D, freshly purified endothelial cells from mouse liver were plated on collagen fact that liver tumor angiogenesis progresses through at least and incubated in growth medium (control) or 5-day Tag-HCC tumor two different morphologic phases (Fig. 1) supports this view. cell-conditioned medium (Tag-HCC) for 1, 2, and 3 days. Expression of CCR2 Additional evidence that different mechanisms might operate was analyzed by quantitative real-time RT-PCR. E, vessel-specific expression of CCR5 in AlbTag tumors. F, induction of CCR5 mRNA in LSECs cultured with in the early versus late stages of angiogenesis comes from the normal growth medium (control) or tumor-conditioned medium (Tag-HCC). finding in pancreatic insulinomas that MMP-9 is particularly G, LSECs were cultured in vitro in the presence or absence of tumor-conditioned important in mobilizing VEGF during the ‘‘switch’’ from vascular Tag-HCC medium, 5 ng/mL recombinant CCL2 or CCL3, and/or neutralizing antibodies for CCL2 (aCCL2 ab,1Ag/mL) and CCL3 (aCCL3 ab,1Ag/mL) for 36 quiescence to angiogenesis (61). Moreover, in this model of islet hours. The cells were radiolabeled with [3H]thymidine for the last 12 hours and carcinogenesis, metalloproteases and proteases of the cathepsin the fold increase in [3H]thymidine incorporation to LSECs cultured in normal growth medium is displayed. Columns, mean (n = 3); bars, SD. Representative family have nonoverlapping functions at different stages of tumor of two experiments. development (49). Intriguingly, we have found that cathepsins, www.aacrjournals.org 209 Cancer Res 2006; 66: (1). January 1, 2006

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Cancer Research and not MMPs, are the dominant proteases in endothelial cells not considered to be angiogenic, there is precedence for CCR2 of late-stage hepatocellular carcinoma. In particular, cathepsin S expression in some vascular cells and recombinant CCL2 induces is dramatically increased in LTECs compared with LSECs and migration of HUVEC in a dose-dependent manner (68, 69). CCR5 may, therefore, play a major role in the control of endothelial cell is expressed on human brain microvessels (70) but growth invasion. Although cathepsin S deficiency impairs wound repair stimulation by CCL3 has not been assessed. Here, we show (62), this is the first report of cathepsin S being critically involved functional CCR2 and CCR5 receptor expression on AlbTag tumor in tumor angiogenesis. vessels and show that this angiogenic effect can be mimicked Increased expression of chemokines and chemokine receptors is in vitro by incubating LSECs with tumor-conditioned medium. also prominent in liver tumor angiogenesis. The CXC chemokine Subsequent ligand engagement then leads to enhanced endothe- family plays a pivotal role in angiogenesis and, indeed, the CXCR4 lial proliferation. Thus, simultaneous expression of CCL2 and receptor is highly overexpressed in LTECs, consistent with results CCL3, together with their cognate receptors on LTECs, critically from in vitro angiogenesis assays (6, 9) and gene-deficient mice controls vessel remodeling in AlbTag tumors in an autocrine (63). Chemokines attract different leukocyte populations that, in manner. turn, may induce angiogenesis. Indeed, increased numbers of NK, Our data also show that chemokines may play a broader role in NKT, and B cells are recruited into AlbTag tumors. Surprisingly, angiogenesis than previously appreciated. In this context, it is of however, leukocyte adherence in situ is not significantly different particular interest that late-stage AlbTag tumors incorporate bone between normal and tumor tissue. Moreover, leukocyte extrava- marrow–derived endothelial cell precursors into neovessels, a sation into tumor parenchyma seems attenuated and liver tumors process that involves chemokine signaling and can be blocked by are only diffusely infiltrated. These findings are similar to those in interfering with G protein–coupled receptor signaling.8 Recruit- a rat hepatoma model, where chemoattractant-superfused tumors ment of progenitors into tumor lesions is a feature of late lack leukocyte-endothelium interactions (64), and in human angiogenesis and is consistent with our intravital microscopy hepatocellular carcinoma, where decreased numbers of infiltrating findings and the notion of multistage angiogenesis. Thus, our novel CD8+ T cells were observed in tumors (65). Impaired tumor findings in liver-associated angiogenesis emphasize the importance infiltration by immune effector cells may indeed contribute to of tumor-specific as well as stage-specific mechanisms for the immune evasion of tumor and an attractive mechanism for neovascularization and will confer critical insights for therapeutic the impaired infiltration is the secretion of inhibitory factors. interference. Interestingly, endothelial cells possess immunomodulatory prop- erties and LSECs, as organ-resident antigen-presenting cells, Acknowledgments can induce tolerance (11, 20). Intriguingly, LTECs secrete IL-10, Received 5/12/2005; revised 9/26/2005; accepted 10/18/2005. which has been shown to be immunosuppressive for circulating Grant support: Deutsche Forschungsgemeinschaft grant SFB405 and Cooperation dendritic cells in patients with hepatocellular carcinoma (66). Program in Cancer Research of the Deutsches Krebsforschungszentrum and Israel Ministry of Science. Furthermore, IL-10 secretion defines a prototype of regulatory, The costs of publication of this article were defrayed in part by the payment of page tissue-resident antigen-presenting cells that are able to induce charges. This article must therefore be hereby marked advertisement in accordance IL-10-producing regulatory T cells with the capacity to suppress with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Ludmila Umansky and Christine Schmitt for excellent technical immune responses (67). assistance, Klaus Hexel and Manuel Scheuermann for operating the FACSVantage Although chemokines recruit leukocytes, lack of dense infil- SE flow cytometer, Mark Kenzelmann for advice on Affymetrix probe synthesis, and trates in AlbTag tumors indicates an additional role for chemo- Manfred Hergenhahn for Affymetrix chip hybridizations. kine signaling during liver carcinogenesis. Of particular interest is the finding that CCL2 and CCL3 are coexpressed on ex vivo 8 H. Spring, T. Schu¨ler, B. Arnold, G. J. Ha¨mmerling, and R. Ganss. Chemokines purified neovessels together with their cognate receptors CCR2 direct endothelial progenitors into tumor neovessels. Proc Natl Acad Sci U S A, in and CCR5, respectively. Although CC chemokines are classically press.

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