tumor cell plasticity in ewing sarcoma, an …...tumor cell plasticity in ewing sarcoma, an...

Tumor Cell Plasticity in Ewing Sarcoma, an Alternative Circulatory

System Stimulated by Hypoxia

Daisy W.J. van der Schaft,1Femke Hillen,

1Patrick Pauwels,

1Dawn A. Kirschmann,

3

Karolien Castermans,1Mirjam G.A. oude Egbrink,

2Maxine G.B. Tran,

4Rafael Sciot,

5

Esther Hauben,6Pancras C.W. Hogendoorn,

7Olivier Delattre,

8Patrick H. Maxwell,

4

Mary J.C. Hendrix,2and Arjan W. Griffioen

1

1Angiogenesis Laboratory, Research Institute for Growth and Development, Departments of Pathology and Internal Medicine, MaastrichtUniversity and University Hospital Maastricht; 2Department of Physiology, Cardiovascular Research Institute Maastricht, MaastrichtUniversity, Maastricht, the Netherlands; 3Children’s Memorial Research Center, Robert H. Lurie Comprehensive Cancer Center,Feinberg School of Medicine, Northwestern University, Chicago, Illinois; 4Department of Renal Medicine, Imperial College London,London, United Kingdom; 5Department of Pathology, University Hospital Leuven, Leuven, Belgium; 6Stichting Laboratoria voorPathologische Anatomie en Medische Microbiologie, Eindhoven, the Netherlands; 7Department of Pathology, Leiden UniversityMedical Center, Leiden, the Netherlands; and 8Laboratoire de Pathologie Moleculaire des Cancers, Paris, France

Abstract

A striking feature of Ewing sarcoma is the presence of bloodlakes lined by tumor cells. The significance of these structures,if any, is unknown. Here, we report that the extent of bloodlakes correlates with poor clinical outcomes, whereas varia-bles of angiogenesis do not. We also show that Ewing sarcomacells form vessel-like tubes in vitro and express genesassociated with vasculogenic mimicry. In tumor models, weshow that there is blood flow through the blood lakes,suggesting that these structures in Ewing sarcoma contributeto the circulation. Furthermore, we present evidence thatreduced oxygen tension may be instrumental in tubeformation by plastic tumor cells. The abundant presence ofthese vasculogenic structures, in contrast to other tumortypes, makes Ewing sarcoma the ideal model system to studythese phenomena. The results suggest that optimal tumortreatment may require targeting of these structures incombination with prevention of angiogenesis. (Cancer Res2005; 65(24): 11520-8)

Introduction

Ewing sarcoma/primitive neuroectodermal tumor has beendefined as a round cell sarcoma showing different degrees ofneuroectodermal differentiation that present mostly in the bone orsoft tissues (1). Ewing sarcoma is a relatively rare highly aggressiveneoplasm with clinically evident metastatic disease at presentationinf25% of all patients. A striking feature of Ewing sarcoma, whichoften occurs at adolescence (2–4), is the presence of lakes of RBC.This was first recognized by James Ewing, which led him to regardthe tumor as an endothelioma (5). Furthermore, Ewing sarcomais characterized by a translocation that involves the Fli-1 gene,usually expressed specifically by endothelial cells. Prompted bythese striking features, we considered that these tumors couldprovide unusual insight into the relationship between a growingsolid tumor and the circulation.

When a growing tumor exceeds the size of f1 mm3, diffusionfails to provide the essential nutrients for continuous growth.Angiogenesis, a well-established paradigm in tumors, is subse-quently stimulated driven by tumor-derived cytokines, such asvascular endothelial growth factors (VEGF) and fibroblast growthfactors. Microvessels lined by endothelium grow into the tumor,providing a blood supply and allowing the tumor to growbeyond the limits imposed by substrate diffusion. It is observedin many tumor types that the extent of the capillary network, asassessed by the density of microvessels, is associated with poorprognosis (6, 7). There is considerable interest in the therapeuticpotential of targeting the growth of new vessels (antiangio-genesis) and the patency of those that have been formed(vascular targeting).Patients with Ewing sarcoma have previously been reported to

have high levels of circulating VEGF (8). In view of this and therelatively poor prognosis of these tumors, we anticipated that themicrovessel density would be high, supported by dynamic contrastenhanced magnetic resonance imaging studies (9). Surprisingly,we actually observed a relatively low microvessel density. Thisobservation, together with the fact that the tumors contain bloodlakes, led us to investigate the microcirculation of these tumors.We showed plasticity of tumor cells, using a combination ofapproaches, which we interpret as vasculogenic mimicry.Vasculogenic mimicry was initially recognized in aggressive

melanomas in 1999 (10), as a process in which tumor cells gaincharacteristics normally restricted to endothelial cells. Throughthis means, tumor cells could contribute to conducting blood invascular-like structures, a process that would be independent ofregular angiogenesis and endothelial cell proliferation. After theseinitial observations in melanoma, evidence for vasculogenicmimicry has been reported in other tumors (11–14). However,the mechanisms driving the vasculogenic mimicry process, and thecontribution of the tumor cell channels to the circulation, havebeen uncertain.

Materials and Methods

Patient tissue materials. Tumor tissues from 33 patients from theUniversity of Leuven and the Leiden University Medical Center were

studied, who presented with Ewing sarcoma between 1987 and 2004. Most

patients were included in the European Intergroup Cooperative Ewing’sSarcoma Study and EuroEwing studies (15). Mean age was 23.8 years at

diagnosis (range: 3-59 years). Patient data were available from 31 patients;

11 died before the moment of data analysis. Mean follow-up was 50 months

Requests for reprints: Arjan W. Griffioen, Angiogenesis Laboratory, ResearchInstitute for Growth and Development, Departments of Pathology and InternalMedicine, Maastricht University and University Hospital Maastricht, Maastricht, theNetherlands. Phone: 31-43-3874630; Fax: 31-43-3876613; E-mail: [email protected].

I2005 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-2468

Cancer Res 2005; 65: (24). December 15, 2005 11520 www.aacrjournals.org

Research Article

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and mean survival was 34 months. Twenty patients of the 23 analyzed hadan 11;22 translocation. All patient materials were handled in a coded

fashion according to the protocols as detailed by the Dutch association of

Medical Scientific Associations.

Immunohistochemistry. Paraffin sections were dried for 48 hours at37jC before staining. Histochemical staining included H&E staining and

periodic acid Schiff ’s (PAS) reagent staining. The amount of blood lakes

and PAS-positive loops was semiquantitatively assessed by scoring 0-4

(0, absent; 1, <5%; 2, 5-20%; 3, 20-50%; 4, >50% of tissue area). Forimmunohistochemistry, primary antibodies used were CD31 (1:50

dilution, DAKOCytomation, Glostrup, Denmark), CD34 (1:50, Novocastra,

Valkenswaard, the Netherlands), Ki-67 (1:100, Labvision, Fremont, CA),

VEGF (1:100 dilution, Santa Cruz, Tebu Bio, Heerhugowaard, theNetherlands), endoglin/CD105 (1:50 dilution, Monosan, Uden, the Nether-

lands), VE-cadherin (1:50, Cayman, Ann Arbor, MI), and tissue factor

pathway inhibitor (TFPI, 1:40, American Diagnostica, Inc., Stamford, CT).After washing, the sections were incubated with antimouse immuno-

globulin (1:200, DAKOCytomation) or antirabbit immunoglobulin biotin-

labeled secondary antibody (1:200, DAKOCytomation) followed by avidin/

biotin-horseradish peroxidase (DAKOCytomation) and 3,3V-diaminobenzi-dine as substrate. For dual staining, sections were first labeled for Ki-67

using horseradish peroxidase and then CD31 and CD34 using alkaline

phosphatase reaction (DAKOCytomation). Microvessel density, proliferat-

ing tumor cells, and proliferating endothelial cells were assessed bycounting in quadruplicate randomly chosen fields at 200� magnification

(0.25 mm2) by three independent observers. Microvessel density was

assessed in Ewing sarcoma tissues and also in 117 colorectal carcinomas,211 renal cell carcinomas, 121 breast carcinomas, and 78 melanomas.

Cell culture and in vitro three-dimensional tube formation.

Melanoma cell lines MUM-2B, MUM-2C, C8161, and C81-61 were used.

Ewing sarcoma cell lines EW-7, A673, RD-ES, and SIM/EW27 were previously

characterized by Dr. O. Delattre, RD-ES was obtained from American Type

Culture Collection (Manassas, VA). Cells were maintained in RPMI 1640,

supplemented with 10% FCS and 2 mmol/L glutamine, except for C81-61

(HAM F-10) and A673 (DMEM; Life Technologies, Paisley, United Kingdom).

Cells grew on standard culture dishes except for the SIM-EW27, which were

grown on collagen-coated culture dishes. Human umbilical vein–derived

endothelial cells were cultured on gelatin-coated culture dishes in RPMI

1640, supplemented with 20% human serum, 100 units/mL penicillin, and

0.1 mg/mL streptomycin. For the three-dimensional culture, cells were

plated at 100,000 cells per well on 24-well dishes coated with rat tail collagen,

as described previously (10), and cultured for 10 days.Vasculogenic tube formation was tested using a commercial Matrigel

assay kit (BD Biocoat, Woerden, the Netherlands). Cells were plated at

20,000 cells per well and grown for 16 hours before calcein labeling

and fluorescence microscopy. The effect of VEGF at 1, 10, and 50 ng/mL(Peprotech, Rocky Hill, NJ) or VEGF blocking antibody HuMV833

(1:50 dilution, Protein Design Labs, Inc., Fremont, CA) was assessed on

Matrigel for 16 hours followed by counting the number of branch points per

microscopic field.RNA isolation and reverse transcription-PCR analysis. Total RNA

was isolated from tissue sections using RNeasy (Qiagen, Venlo, the

Netherlands), followed by RNase-free DNase treatment (Qiagen, Hilden,Germany). RNA concentration was measured by nanodrop (Nanodrop,

Wilmington, DE). cDNA was synthesized with 400 units of M-MLV RNase

H-reverse transcriptase (Promega, Leiden, the Netherlands) in 20 AL of 1�first strand buffer (Promega), and 1 mmol/L deoxynucleotide triphos-phates in the presence of 10 units RNase inhibitor RNasin (Promega), 0.5

Ag random primers (Promega), and 1 Ag total RNA. RNA from cell lines

was isolated using TRIzol (Invitrogen, Breda, the Netherlands). Semiquan-

titative PCR amplifications were done with primer sequences listed inTable 1 with glyceraldehyde-3-phosphate dehydrogenase primers (BD

Clontech, Mountain View, CA) as a control. PCR fragments were ligated

into pCR2.1-TOPO (Invitrogen) and two independent clones weresequenced from each primer set and shown to be identical to the

expected DNA sequence. Quantitative real-time reverse transcription-PCR

(RT-PCR) was done for cyclophylin A, h-actin, VEGF-A, VEGF-C, VEGF-D,

angiopoeitin-1, basic fibroblast growth factor, and placenta growth factoras described (16). All real-time PCR primers were synthesized by Sigma-

Genosys (Cambridgeshire, United Kingdom) except for VE-cadherin (a

FAM-TAMRA primer probe, Integrated DNA Technologies, Inc., Coralville,

IA). PCR was done using an iCycler MyIQ (Bio-Rad, Veenendael, theNetherlands) in 25 AL volume containing 0.8 AL cDNA, 1�SYBR Green

PCR master mix (Eurogentec, Liege, Belgium) added to 20 nmol/L

fluorescein (Bio-Rad) and 400 nmol/L of each primer. Data was analyzed

with the Sequence Detection System software (Applied Biosystems, FosterCity, CA). Experiments were done twice in duplicate.

Xenograft assays and intravital microscopy. All animal experiments

were approved by the local animal ethics committee. EW7 cells (106) were

injected s.c. into the flank of Swiss/nude mice and tumors measured withcalipers. On day 28, mice were anesthetized with ketamine/xylazine (17)

and intravital microscopy was done as described before (18). In two mice,

FITC dextran (MW 500 kDa, 500 Ag) was injected i.v. in the tail vein. In thesemice, imaging was done before and after injection. Two other mice were

given an i.p. injection with pimonidazole 60 minutes before sacrifice (2 mg

in 200 AL; Natural Pharmacia International, Research Triangle Park, NC).

Sections of xenografts were labeled with anti–von Willebrand factor(1:200, DAKOCytomation) and anti-CD31 antibody (1:250, PharMingen, BD

Company, Woerden, the Netherlands). Pimonidazole was detected using

FITC-labeled antipimonidazole antibody (1:50 dilution; Hypoxyprobe-1

Mab1; Chemicon International, Inc., Temecula, CA) and horseradishperoxidase–conjugated anti-FITC antibody (1:200 times dilution, clone

5D6.2; Chemicon International).

In some experiments, 200 Ag antiendothelial antibody (MECA20, a kindgift from Dr. A. Duijvestijn, Department of Immunology, University

Maastricht, Maastricht, the Netherlands; ref. 19) was administered i.v.

followed after 15 minutes by perfusion with Chinese ink (Pelikan, Hanover,

Germany). Sections from those xenografts were labeled with peroxidase-labeled goat anti-rat immunoglobulin and counterstained with hematoxylin.

Statistical analyses. Real-time RT-PCR data were statistically analyzed

using Mann-Whitney U tests (using SPSS-10 software). P values <0.05 were

considered statistically significant. Statistical significance of experimentalvariables to clinical outcome was assessed by Mann-Whitney U test to

compare between groups and log rank test and a Cox regression test were

done for survival analysis.

Results

The amount of blood lakes in Ewing sarcoma correlates toclinical outcome. In a unique series of Ewing sarcoma tissue biopsysamples, selected from patients before radiation or chemotherapywas started, we observed that microvessel density in these tumors,assessed by labeling of endothelial cells (microvessel density), wasrelatively low compared with other tumors (Fig. 1A). This led us tohypothesize that the blood lakes we observed in 92% of these tumorsmight play an important role in the tumor circulation (Fig. 1B).Supporting this notion, the blood lakes were not associated withevidence of coagulation and/or local necrosis. The cells lining theblood lakes did not label with the endothelial markers, CD31 andCD34. However, they expressed CD99, a marker of Ewing sarcomacells and light microscopic examination (Fig. 1B) and electron-microscopic examination (not shown) clearly showed that the lake-lining cells were tumor cells and not endothelial cells. The presenceof tumor cell–lined blood lakes has been described as vasculogenicmimicry. Vasculogenic mimicry was originally recognized inmelanoma as PAS-positive loops that contained RBC (10). Also, thepresence of PAS-positive loops was examined in Ewing sarcoma,which revealed that such loops were present in 68% of the patients(Fig. 1C). Notably, all tumors with PAS-positive networks alsocontained blood lakes.To address the significance of the blood lakes, we examined

whether clinical outcomes correlated with the number of blood

Tumor Cell Plasticity in Ewing Sarcoma

www.aacrjournals.org 11521 Cancer Res 2005; 65: (24). December 15, 2005



lakes. Interestingly, the number of blood lakes, quantified as thepercentage of tumor area containing lakes, was significantly higherin the patients that subsequently died (75% higher compared withthe group of patients still alive at the time of data analysis, P < 0.05;Fig. 1D).Ewing sarcoma tumors are angiogenic; lack of correlation

to clinical outcome. In tumors, we observed a high level ofproliferation (mean number of Ki-67-positive tumor cells 51.4%,SD F 30.4%). Highly proliferative tumors usually exhibit a highlevel of angiogenic signaling. On the other hand, the lowmicrovessel density that we observed in the tumors suggestedthat these tumors might have limited angiogenic potential. We,therefore, investigated angiogenesis using several approaches andfound evidence that Ewing sarcoma is highly angiogenic. First, wefound that 22% of CD31/CD34–positive vessels had one or moreKi-67-positive nuclei (Fig. 2A). Second, in all Ewing sarcomatissues, we found high mRNA expression levels of the angiogenicgrowth factors VEGF-A, basic fibroblast growth factor, placentagrowth factor, and angiopoietin-1 comparable with those found in

other angiogenic tumors, such as breast carcinoma and fibrosar-coma (Fig. 2B). Third, we observed high VEGF protein expressionin f100% of tumor cells (Fig. 2C), in line with high circulatingVEGF serum levels in Ewing sarcoma patients (8). Fourth, the celllines EW7 and RD.ES, which give rise to tumors with typicalEwing sarcoma morphology following injection into athymic mice(see also Fig. 4), express high levels of angiogenic growth factorsin vitro (Fig. 2B). Intriguingly, in the patient tissues, neithermicrovessel density nor the number of proliferating endothelialcells showed significant association with clinical outcome (Fig. 2Dand E).Ewing sarcoma cells form vasculogenic structures in vitro

and express vasculogenic mimicry–related genes. To investi-gate the capacity of different Ewing sarcoma cell lines to displayvasculogenic mimicry in vitro , we used three-dimensional collagenmatrix tube formation assays and done direct comparisons with arange of melanoma cell lines. The aggressive EW7 cell line (knownto have a high tumorigenicity in mice) efficiently formed a networkof tubular structures (Fig. 3A) comparable with the melanoma cell

Table 1. Primers used for semiquantitative and quantitative RT-PCR

Gene PCR primers (forward, reverse) Amplicon

size (bp)

Cycle

no.

Real-time PCR primers (forward, reverse)

EphA2 5V-GTTGAAGTTCACTACCGAGATCCATCCATCC-3V,5V-GAGCCGGATAGACACGCGG-3V

883 30 5V-AGACGCTGAAAGCCGGCTAC-3V,5V-CAGGGCCCCATTCTCCATG-3V

VE-cadherin 5V-CACCGGCGCCAAAAGAGAGA-3V,5V-CTGGTTTTCCTTCAGCTGGAAGTGGT-3V

1,032 30 5V-TCCCGGAGCAGAAGACGT-3V,5V-GAGAAAAGAAAGAGAGCATGGATTG-3V,probe 6-FAM/TGCATGACGGAG-CCGAGCCA/36-TAMRA

TIE-1 5V-CCCTGAGCTACCCAGTGCTAGAGTG-3V,5V-TGGTCACAGGTTAGACAGCAGAGTTTG-3V

1,000 30 5V-CCCCGCTGGTCTCGTTCTC-3V,5V-CACAATGGTCGACCAGTCC-3V

VEGF-C 5V-CAGGCAGCGAACAAGACCTGC-3V,5V-CAGATGAGCTCCAGTCCATTTCTGTAAAG-3V

721 30

Laminin5 c2 5V-TCAGCCAGAAGGTTTCAGATGCC-3V,5V-GGCAGCTTCACTGTTGCTCAAGCAG-3V

654 30 5V-GTTGATACCAGAGCCAAGAAC-3V,5V-GAAAGCTTCTGCTCCAGTAAG-3V

PAX 8 5V-CGATGCCTCACAACTCCATCAGAT-3V,5V-GCTCGCCTTTGGTGTGGCT-3V

694 30

Urokinase 5V-TGTGGCCAAAAGACTCTGAGGC-3V,5V-CTTGGTGTGACTGCGGATCCA-3V

771 27

Keratin 7 5V-CCAGCGGGTGCGCCA-3V,5V-CACTCCATCTCCAGCCAACCG-3V

973 30 (5% DMSO added)

CD13 5V-CTGCTCCAACGGAGTTCCAGAGTG-3V,5V-CTCAGGCAGCCTGGGTCATCA-3V

812 30

Vimentin 5V-CCTGAACCTGAGGGAAACTAATCTGGATT-3V,5V-CTAAATCTTGTAGGAGTGTC-GGTTGTTAAGAACTAGAG-3V

313 30 (5% DMSO added)

c-met 5V-CCAAGTCAGATGTGTGGTCCT-3V,5V-TCGTGTGTCCACCTCATCAT-3V

362 27 (annealingtemperature 62jC)

GAPDH 5V-TGAAGGTCGGAGTCAACGGATTTGGT-3V,5V-CATGTGGGCCATGAGGTCCACCAC-3V

983 25

TFPI-1 5V-TTTGTGSSGSTGGTCCGAAT-3V,5V-AGACACCATGAGGGACCG-3V

TFPI-2 5V-AGTGTGGACGACCAGTGTGAGG-3V,5V-TGCGCAGAAGCCCATACAAG-3V

Tie-2 5V-TTGAAGTGGAGAGAAGGTCTG-3V,5V-GTTGACTCTAGCTCGGACCAC-3V

Neuropilin 5V-ATGACGACCAGGCCAACT-3V,5V-AACTCTGATTGTATGGTGCTGT-3V

Endoglin 5V-GCCAGCATTGTCTCACTTCAT-3V,5V-TACCAGGGTCATGGCGTC-3V

Cancer Research




lines MUM-2B and C8161, which are aggressive and displayvasculogenic mimicry in vitro (data not shown; refs. 10, 20). Theless aggressive A673 and RD.ES Ewing sarcoma cell lines (knownto have a low tumorigenicity in mice) showed an intermediateactivity, forming a few tubular structures (Fig. 3B and C), whereasSIM/EW27 cells formed hardly any structures at all (Fig. 3D)comparable with the nonaggressive and vasculogenic mimicry–negative melanoma cell lines MUM-2C and C81-61 (not shown).Similar results were found on Matrigel (Fig. 3E-H).Using semiquantitative RT-PCR, we found that genes associated

with vasculogenic mimicry–related tumor cell plasticity inmelanoma (20) were expressed in the EW7 Ewing sarcoma cellline (Fig. 3I). We went on to perform quantitative real-time RT-PCR and found that integrin a3, VE-cadherin, TFPI-1 (Fig. 3J-L),EphA2, laminin5g2, Tie-1, neuropilin, and endoglin (data notshown) were all highly overexpressed in EW7 and melanomacell lines MUM-2B and C8161. Expression was lower or absent inEwing’s cell lines A673, RD.ES, and SIM-EW27 and the nonaggres-sive melanoma cell lines MUM-2C and C81-61, consistent withthe degree of vasculogenic mimicry in three-dimensional culture(Fig. 3J-L).To address whether these genes are expressed in vivo in Ewing

sarcoma, we used quantitative RT-PCR to assay TFPI-1, VE-cadherin, and EphA2 in frozen material from patients and found

that all three were expressed at a high level. Furthermore, usingimmunohistochemistry, we observed that TFPI-1/2, VE-cadherin,and EphA2 protein was present in tumor cells lining thevasculogenic mimicry structures (Fig. 2M).Blood lake structures in Ewing sarcoma are part of the

circulation. To study the functionality of blood lake structuresin vivo , EW7 tumors were grown s.c. in the flank of athymicmice. Tumors grew rapidly and we observed numerous lakes,especially in the outer rim of these tumors (Fig. 4A and B),a distribution that has been previously described in melanomas(21). The cells lining these lakes were negative for von Willebrandfactor and CD31. We used several approaches to investigatewhether blood was flowing through these lakes. First, micecarrying EW7 tumors were i.v. injected with MECA20, anantibody specifically recognizing mouse endothelial cells. After15 minutes, mice were sacrificed and perfused with an India inksuspension. We found ink both in MECA20 staining vessels aswell as in vascular structures negative for MECA20 and directlylined by tumor cells (Fig. 4C). Second, intravital microscopy wasdone. The vasculogenic structures were characterized by irregularprofiles and slow blood flow (Fig. 4D , a video image can beviewed on www.fdg.unimaas.nl/AngiogenesisLab). Such vasculo-genic structures were not present in LS174T colon carcinomatumors. In a third approach, the connection of vasculogenic

Figure 1. Vasculogenic mimicry in Ewing sarcoma. A, microvessel density quantification in several tumor types. *, P < 0.05, compared with the other tumor types.B, Ewing sarcoma patient tumor tissue stained with CD31/CD34 and counterstained with H&E. *, nonendothelial cell–lined structures containing erythrocytes orblood lakes. C, PAS staining of Ewing sarcoma. Bar, 100 Am (B and C ). Quantification of blood lakes (D , *P < 0.04) and PAS-positive loops (E) in relation with clinicaloutcome. dod, death of disease.





structures in EW7 tumors with the circulation was shown whenfluorescence was visualized in the vasculogenic structures afteri.v. injection of a FITC-conjugated dextran (Fig. 4E).Blood flow in vasculogenic structures is inefficient; role of

hypoxia. Somewhat unexpectedly, the abundant presence of bloodlakes and PAS-positive loops in the patient tissues coincided withhigh levels of VEGF (and other angiogenic growth factors) andongoing angiogenesis (high numbers of proliferating endothelialcells). We, therefore, considered whether VEGF is instrumental inthe induction of blood lakes and tubes by tumor cells. However,this seems unlikely because (a) VEGF did not augment tubeformation of EW7 or MUM-2B cells in the in vitro tube formationassay as examined at concentrations of 1 to 50 ng/mL (Fig. 5F); (b)VEGF did not increase expression of genes involved in tubeformation by tumor cells, neither in tube forming nor in non–tube-forming tumor cells (Fig. 5G); and (c) addition of a VEGF blockingantibody was also not able to block or inhibit in vitro tubeformation by the tumor cells; (Fig. 5H). Results are shown for TFPI-1 (Fig. 5F), similar results were found for VEGF receptor-1 andVEGF receptor-2, Tie-1, Neuropilin-1, laminin 5g2, and EphA2.Furthermore, when comparing the VEGF RNA expression levelsbetween tumors with higher amounts of blood lakes to tumors withlower numbers of lakes, this did not yield a difference in expression(data not shown).Although we did not find a relationship between tube

formation by tumor cells and VEGF, a factor that is regulatedby hypoxia, we investigated the expression of hypoxia-induciblefactor 1a (HIF1a). This is a transcription factor potently stabilizedunder hypoxic conditions, which acts as a master regulator ofgene expression in response to changes in oxygen tension (22–24).Interestingly, we found HIF1a protein around the blood lakes butnot around CD31+ vessels (Fig. 5B and C). Further evidence ofHIF1a activation was provided by the observation that theglucose transporter, GLUT1 , another HIF1a target gene, was alsohighly expressed in these regions (Fig. 5D). Because HIF1a can beactivated by other variables besides low oxygen, we injected

pimonidazole in Ewing sarcoma tumor-bearing mice to assesslocal oxygenation. This nitroimidazole forms adducts with cellularproteins only when oxygen tension is reduced. Like HIF1aprotein, pimonidazole adducts were observed around the bloodlakes, consistent with the notion that these regions are hypoxic(Fig. 5E , the tumors from mice not injected with pimonidazoledid not stain positive; data not shown). Taken together, thesefindings suggested that hypoxic activation of HIF1a might beinvolved in driving vasculogenic mimicry. To test this idea, theexpression of the above-mentioned genes that are involved invasculogenic mimicry was investigated in aggressive and nonag-gressive tumor cell lines cultured under hypoxic and standardconditions. We found that culturing for 16 hours under hypoxiasignificantly increased the expression of laminin5g2, EphA2, Tie-1,and TFPI-1 ( for all these molecules P < 0.01) in all cell lines tested(Fig. 5I-L).

Discussion

Despite the clinically aggressive behavior of Ewing sarcoma, arelatively low number of microvessels was observed in this tumortype compared with other malignancies. We showed using severaltechniques that this was not due to a low angiogenic potential.Instead, the high angiogenic potential was illustrated by the highnumbers of proliferating endothelial cells (although total numberof vessels was low) and the high expression level (both RNA andprotein) of angiogenic growth factors in the patient tissues aswell as in the cell lines. The low amount of regular blood vessels,together with the abundant presence of blood lakes and PAS-positive loops (vascular-like tube formation by tumor cells),suggested a contribution of the blood lakes to circulation thatmight be considered vasculogenic structures as described earlier(10, 12, 13). We, therefore, suggest that Ewing sarcoma tumorcells cooperate in the formation of a circulatory system such ashas been described as vasculogenic mimicry in aggressivemelanoma. Interestingly, whereas in most tumors vasculogenic

Figure 2. Ewing sarcoma is highly angiogenic.A, double staining of Ewing sarcoma tissue withCD31/34 (blue ) and Ki-67 (brown ), indicating highnumbers of proliferating endothelial cells (arrows ).Bar, 100 Am. B, mean relative expression(in 2�DC t, corrected for housekeeping gene b-actin )VEGF-A, basic fibroblast growth factor (bFGF ),placenta growth factor (PlGF ), and angiopoietin-1(ang-1 ) expression in Ewing sarcoma, fibrosarcoma,breast cancer tissue, and Ewing sarcoma tumorcell lines EW7 and RD.ES as measured byquantitative real-time RT-PCR. C, VEGF expressionin Ewing sarcoma (conjugate control in top rightcorner ; inset, scale bar represents 100 Am).Angiogenesis variables microvessel density(MVD ; D ) and proliferating endothelial cells (EC ; E)do not correlate with clinical outcome.

Cancer Research




Figure 3. Ewing sarcoma cells form tubular structures in vitro and express vasculogenic mimicry–specific genes. Ewing sarcoma cell lines EW7 (A), A673 (B ),RD.ES (C ), and SIM-EW27 (D ) in three-dimensional culture on collagen type I matrix. EW7 cells (E ), A673 Ewing sarcoma cells (F ), MUM-2B aggressivemelanoma cells (G ), and MUM-2C nonaggressive melanoma cells (H ) on a Matrigel three-dimensional matrix stained with calcein. I, gene expression measured bysemiquantitative RT-PCR in the Ewing sarcoma cell lines compared with the vasculogenic mimicry–positive melanoma cell line MUM-2B and the vasculogenicmimicry–negative cell line MUM-2C. The housekeeping gene GAPDH was used to control for equal loading. Quantitative real-time RT-PCR confirmation of VE-cadherin(J ), integrin a3 (K ), and TFPI-1 (L) gene expression of VE-cadherin in melanoma and Ewing sarcoma cell lines. The C t values are corrected for the expressionof housekeeping genes. Statistical significance determined by Mann-Whitney test. M, staining of Ewing sarcoma tissue sections with TFPI, VE-cadherin, and EphA2antibodies. Bar, 100 Am [A, E (for A-H ), and M ].





mimicry is present in 10% to 40% of cases, Ewing sarcoma is thefirst tumor in which vasculogenic mimicry is so abundantlypresent (blood lakes in 92% of cases). In Ewing sarcoma, theamount of blood lakes and presence of PAS-positive loops, incontrast to the classic angiogenesis variables, correlated withsurvival, confirming that vasculogenic mimicry is an indicator ofpoor prognosis (10, 25).The vascular-like tube formation by Ewing sarcoma tumor cells

was confirmed in a three-dimensional culture system using humancell lines, such as EW7, grown on a collagen matrix. AggressiveEW7 Ewing sarcoma cells rapidly formed vascular-like tubes inthis system. In addition, EW7 cells injected into athymic mice gaverise to tumors with Ewing sarcoma morphology and blood lakesin vivo .Furthermore, based on the similarities with the gene expression

profiles in vasculogenic melanoma cells (13, 26), it was suggestedthat tube formation in Ewing sarcoma was due to a similarplasticity and dedifferentiation of tumor cells, which gained anendothelial phenotype. We showed that the genes involved in thisprocess, such as TFPIs, EphA-2 , and VE-cadherin (10, 27), are alsohighly expressed in the patient tissues and aggressive vascular-liketube-forming EW7 cells. In contrast, less aggressive Ewingsarcoma cells did not or less efficiently form these tubes anddo not overexpress these genes. It is interesting in this contextthat one of the diagnostic indicators for Ewing sarcoma is atranslocation of chromosomes 11 and 22, which involves the Fli-1gene on chromosome 11 and the EWS gene on chromosome 22(28, 29). In diagnostic pathology, Fli-1 antibodies are used asmarkers for Ewing sarcoma and for endothelial cells or vasculartumors. The protein that is formed by the Fli-1 gene is usuallyspecifically expressed by endothelial cells. This translocationmight play a role in the tumor cell plasticity seen in Ewingsarcoma.Critical proof for the contribution of nonendothelial cell–lined

structures in circulation was the demonstration of blood flow inthese structures. Using intravital microscopy and immunohisto-chemistry after injection of antiendothelium antibody and India

ink, we were able to show blood flow in the nonendothelial cell–lined vascular structures. This blood flow was observed to be veryslow, which urged us to study oxygenation in the tumor tissue.When assessing the Ewing sarcoma tissue samples for hypoxia, wefound that the tumor cells surrounding the blood lakes did expressHIF1a, a transcription factor known to play a role in the expressionof VEGF, as well as GLUT1, indicative of inefficient oxygen deliveryby these structures. Possible explanations for this would includethe very slow blood flow through these channels, and/or thepossibility that they act primarily as a circulatory system drainingblood from the tumor.It is likely that the hypoxia that is induced in these tumors has

led to the high expression of VEGF (and maybe other angiogenicfactors) and resultant endothelial cell proliferation as observed inthe patient tissues. An important possibility is that vascular-liketube formation by tumor cells might be induced by VEGF orother angiogenic factors. In fact, tube formation has previouslybeen reported to be enhanced by VEGF in vitro (30). In contrast,in our study, we were not able to show that VEGF enhances orinduces vascular-like tube formation by tumor cells. Although thiscould reflect the difference in tumor models and assay systems,we were also not able to show differences in expression of thegenes involved in tube formation in response to VEGF (Fig. 5).Therefore, we favor the view that in Ewing sarcoma, blood lakeand PAS-loop formation is not induced or supported by VEGF.However, we did find an increased expression of genes involved invasculogenic mimicry when cells were cultured under low oxygentension. Thus, we anticipate that hypoxia via induction of HIF1ais able to enhance vasculogenic mimicry. Interestingly, bothHIF1a and vasculogenic mimicry associated genes (i.e., EphA2and laminin-5c2 ) signal via the PI3/K pathway (31, 32).Furthermore, blockade of this signal transduction pathway blocksvasculogenic mimicry and the expression of the genes involvedin this process (33). In addition, Tie-1 can be up-regulated byHIF1a (34).The results of this study strongly suggest that plasticity of

Ewing sarcoma tumors is associated with the contribution oftumor cells to contribute to circulation. This presumablyexplains, at least in part, why the microvessel density is unusuallylow for such an aggressive tumor. This scenario may have effecton the treatment of tumors with angiogenesis inhibitors thatact directly on endothelial cells. As we have shown before,dedifferentiating tumor cells do not acquire sensitivity to angio-genesis inhibitors (35), suggesting that an antiangiogenesisprotocol may lead to only a partial regression of the tumor.Because vascular-like tube formation is much less frequent and/or less well recognized in other tumor types, it is possible thatthere is an important relationship between these structure andresponse to therapy, which has been overlooked in trials withangiogenesis inhibitors. Up to now, no data are available on suchtrials in Ewing sarcoma patients. We suggest that it would beparticularly informative to seek a relationship between thepresence of vasculogenic structures and the response toantiangiogenesis therapy. Furthermore, an interesting possibilityis that antiangiogenesis therapy may result in a selective growthadvantage for cells exhibiting vasculogenic mimicry, allowingdrug-induced resistance to occur.It seems likely that angiogenesis therapy may be more effective

when combined with other forms of cancer therapies to eradicatevasculogenic tube formation, explaining the good results ofcombination between antiangiogenesis therapy and conventional

Figure 4. Vasculogenic mimicry structures in Ewing sarcoma are part of thecirculation. EW7 Ewing sarcoma xenograft tumor tissue stained with H&E(A, paraffin-embedded tissue) and CD31 (B, frozen tissue). C, EW7 tumor ofa mouse sacrificed 10 minutes after injection of MECA-20 and India ink.Arrows, blood lakes (A-C ). Bar, 100 Am (B). D, an intravital microscopicimage of Ewing sarcoma (a video image can be viewed on www.fdg.unimaas.nl/AngiogenesisLab). E, intravital microscopic image of an Ewing sarcoma tumorn a mouse injected i.v. with FITC-dextran. Bar, 100 Am (B , valid for D and E ).

Cancer Research




cancer therapies in (pre)clinical studies (36–38). The Ewing sarcomamodel with its abundant vasculogenic structures may be an idealmodel to develop and test therapies designed to counteractvasculogenic tube formation by attacking tumor cells that takepart in the formation of vascular lakes (e.g., through CD99-directedtherapy; ref. 39).

Acknowledgments

Received 7/18/2005; revised 9/16/2005; accepted 9/27/2005.Grant support: Institute of Growth and Development at the University and the

University Hospital of Maastricht.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Figure 5. Blood lakes and hypoxia. A, Ewing sarcoma H&E-stained tumor section. B to D, CD31, HIF1a, and GLUT1 stainings, respectively. Arrows, regular bloodvessels. E, pimonidazole adduct formation around blood lakes in a Ewing sarcoma mouse tumor. F, VEGF regulation of tube formation by EW7 cells cultured onMatrigel. G, regulation of vasculogenic mimicry–associated gene (TFPI-1 ) expression in EW7 by incubation for 48 hours in 10 ng/mL VEGF, MUM-2B, and MUM-2Ccells. H, incubation of EW7 cells on a three-dimensional matrix with blocking VEGF antibody as a positive control human umbilical vein–derived endothelial cells(HUVEC ) were used. Regulation of expression of vasculogenic mimicry–associated genes laminin 5c2 (I ), Tie-1 (J ), TFPI-1 (K), and EphA2 (L ) by hypoxia in EW7,A673, and C8161 cells. *, P < 0.05. Bar, 50 Am.



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2005;65:11520-11528. Cancer Res Daisy W.J. van der Schaft, Femke Hillen, Patrick Pauwels, et al. Circulatory System Stimulated by HypoxiaTumor Cell Plasticity in Ewing Sarcoma, an Alternative

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