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The Contribution of Bone Marrow–Derived Cells to the Tumor

Vasculature in Neuroblastoma Is Matrix

Metalloproteinase-9 Dependent

Sonata Jodele,1Christophe F. Chantrain,

5Laurence Blavier,

1Carolyn Lutzko,

2

Gay M. Crooks,2Hiroyuki Shimada,

3Lisa M. Coussens,

4and Yves A. DeClerck

1

Divisions of 1Hematology-Oncology and 2Research Immunology and Bone Marrow Transplantation, Departments of Pediatricsand Biochemistry and Molecular Biology and 3Pathology, Keck School of Medicine, University of Southern California and The SabanResearch Institute of Childrens Hospital Los Angeles, Los Angeles, California; 4Department of Pathology and Cancer Research Institute,University of California San Francisco, San Francisco, California; and 5Department of Pediatrics,Catholic University of Louvain Medical School, Brussels, Belgium

Abstract

The contribution of the tumor stroma to cancer progressionhas been increasingly recognized. We had previously shownthat in human neuroblastoma tumors orthotopicallyimplanted in immunodeficient mice, stromal-derived matrixmetalloproteinase-9 (MMP-9) contributes to the formationof a mature vasculature by promoting pericyte recruitmentalong endothelial cells. Here we show that MMP-9 ispredominantly expressed by bone marrow–derived CD45-positive leukocytes. Using a series of bone marrow trans-plantation experiments in MMP-9++/++ and MMP-9��/��/� micexenotransplanted with human neuroblastoma tumors, weshow that bone marrow–derived MMP-9 is critical for therecruitment of leukocytes from bone marrow into the tumorstroma and for the integration of bone marrow–derivedendothelial cells into the tumor vasculature. Expression ofMMP-9 by bone marrow–derived cells in the tumor stromais also critical for the formation of a mature vasculatureand coverage of endothelial cells with pericytes. Further-more, in primary human neuroblastoma tumor specimensof unfavorable histology, we observed a higher level oftumor infiltration with MMP-9 expressing phagocytic cellsand a higher degree of coverage of endothelial cells bypericytes when compared with tumor specimens with afavorable histology. Taken together, the data show that inneuroblastoma, MMP-9 plays a critical role in the recruit-ment of bone marrow–derived cells to the tumor microen-vironment where they positively contribute to angiogenesisand tumor progression. (Cancer Res 2005; 65(8): 3200-8)

Introduction

It is now well recognized that what happens outside the cancercell, in what is designated as the tumor stroma or microenviron-ment, plays an important role in tumor progression (1, 2).Endothelial cells, pericytes, fibroblasts, and inflammatory cellssuch as macrophages, neutrophils, and mast cells are not innocentbystanders at tumor sites and have a profound effect on the fate oftumor cells (3, 4). Many nonmalignant cells in the tumor stromaderive from adjacent tissues as is typically the case during

angiogenesis when the sprouting of endothelial cells from existingblood vessels is stimulated by a variety of angiogenic factors likevascular endothelial cell growth factor (VEGF) or basic fibroblastgrowth factor (5). However, distally located bone marrow–derivedcells also substantially contribute to the tumor microenvironmentby being a source of inflammatory cells and endothelial precursorcells (EPC) actively recruited in the tumor stroma by malignantcells (6–8).Matrix metalloproteinases (MMP) are a family of Zn2+

proteases involved in multiple steps of tumor progression wherethey process not only extracellular matrix proteins but alsogrowth factors, cytokines, and growth factor receptors (9–11).Among these proteases, MMP-9 (gelatinase B) plays a morespecific role in angiogenesis (12). Expression of this enzyme byinflammatory cells triggers an angiogenic switch critical duringcarcinogenesis where it releases VEGF (13, 14). In xenotrans-planted neuroblastoma tumors, MMP-9 contributes to tumorangiogenesis by promoting the recruitment of pericytes alongvascular endothelial cells (15). MMP-9 also plays a role in themobilization of hematopoietic stem cells into the circulation.In mice treated with the myelosuppresive agent 5-fluorouracil(5-FU), MMP-9 promotes the release of soluble c-kit ligand andpermits the transfer of hematopoietic and endothelial stem cellsfrom a quiescent to a proliferative compartment in the bonemarrow (16). This observation raises the possibility that MMP-9could also contribute to tumor progression by increasing thepresence of bone marrow–derived precursor cells available forrecruitment in the tumor stroma.Childhood neuroblastoma is the second most common

extracranial tumor in children (17). Based on their histopathology,neuroblastoma tumors are classified as having a favorable orunfavorable histology. Tumors with a favorable histology typicallycontain an abundant Schwannian stroma have a low mitosis-karyorrhexis index, are not MYCN amplified, are seen in youngerchildren, and have a favorable clinical outcome. In contrast,tumors with an unfavorable histology are typically characterizedby a paucity of stroma, contain few or no Schwann cells, have ahigh mitosis-karyorrhexis index, are MYCN amplified, and areassociated with a poor clinical outcome (18, 19).We have previously reported the presence of higher levels of

stromal-derived MMP-9 in primary human neuroblastoma tumorspecimens from patients with advanced stage (III and IV) diseasewhen compared with specimens from patients with localizeddisease (stage I or II; ref. 20). More recently, we have shown that inMMP-9–deficient mice orthotopically implanted with SK-N-BE (2)human neuroblastoma tumors there is a defect in the maturation

Requests for reprints: Yves A. DeClerck, Division of Hematology-Oncology,Childrens Hospital Los Angeles, 4650 Sunset Boulevard, Los Angeles, CA 90027. Phone:323-669-5648; Fax: 323-664-9455; E-mail: [email protected].

I2005 American Association for Cancer Research.

Cancer Res 2005; 65: (8). April 15, 2005 3200 www.aacrjournals.org

Research Article

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of the tumor vasculature characterized by the presence of smallvessels and poor pericyte coverage (15). Here we have examined thestromal origin of MMP-9 expression in these tumors and haveobserved that MMP-9 is predominantly expressed by CD45-positivebone marrow–derived leukocytes. This observation led us toconduct a series of bone marrow transplantation experimentsin mice implanted with SK-N-BE (2) neuroblastoma tumors todetermine the contribution of bone marrow–derived MMP-9 totumor angiogenesis and vasculogenesis.

Materials and Methods

Cell culture. The human neuroblastoma cell line SK-N-BE (2) wasobtained from Dr. J. Biedler (Memorial Sloan-Kettering Cancer Center, New

York, NY). Cells were routinely grown in RPMI (Cellgro, Mediatech, Inc.,Herndon, VA) supplemented with 10% fetal bovine serum (Omega

Scientific, Inc., Tarzana, CA), penicillin-streptomycin (1%), and 2 mmol/L

L-glutamine (Irvine Scientific, Santa Ana, CA).

Bone marrow transplantation experiment. Eight-week-old mice inthe FVB/N background carrying homozygous null mutations in the Rag1

gene and the MMP-9 gene as previously reported (15) were used in all

experiments. The immunodeficient status of the mice was verified by

fluorescence-activated cell sorting (FACS) analysis (Becton Dickinson, SanJose, CA) of peripheral blood for the absence of T and B lymphocytes.

Animals were maintained in a germ-free barrier facility and procedures

were done according to the guidelines of the Institutional AnimalCare Utilization Committee at the Childrens Hospital Los Angeles

(Los Angeles, CA). Mice were arranged into four experimental groups for

the performance of syngeneic age-matched bone marrow transplantation

using MMP-9+/+ (RAG1ko/ko) and MMP-9�/� (RAG1ko/ko/MMP-9ko/ko)donor and recipient combinations (Table 1). Male mice were used as

bone marrow donors and were treated with 150 mg/kg of 5-FU

(American Pharmaceutical Partners, Inc., Los Angeles, CA) given i.p.

2 days before sacrifice and bone marrow harvest. Bone marrow cellswere harvested from both femurs and tibias by flushing the bone cavity

with basal bone marrow medium (BBMM): Iscove’s medium (Cambrex,

Walkersville, MD), supplemented with 30% fetal bovine serum (StemCell Technologies, Vancouver, British Columbia, Canada), 1% bovine

serum albumin (Sigma, St. Louis, MO), 100 mmol/L 2-mercaptoethanol

(Sigma), 2 mmol/L L-glutamine, 1% penicillin/streptomycin, and 1%

fungisone (Irvine Scientific). After washing with PBS, bone marrow cellswere resuspended at 1 to 2 � 106 nucleated cells/mL in BBMMsupplemented with 50 ng/mL recombinant human interleukin 6

(R&D Systems, Minneapolis, MN), 10 ng/mL murine interleukin 3, and

2.5 ng/mL murine stem cell factor (BioSource International, Camarillo,CA; BBMM-GF). The bone marrow cells were transduced with a

retroviral vector containing the cDNA of the enhanced green fluorescent

protein (EGFP). In brief, GPE packaging cells containing the MM-EGFP-

SAR retroviral vector (obtained from Dr. Donald Kohn, ChildrensHospital Los Angeles) were irradiated at 4,000 cGy once with

a Gammacell 1000 Cesium-137 cell irradiator (Atomic Energy of

Canada Ltd., Mississauga, Ontario, Canada) and plated at 1 � 106 cellsper 100-mm dish in DMEM (Cellgro, Mediatech) containing 10% newborn

calf serum (Cellgro). After 24 hours, 1 to 2 � 107 bone marrow cells wereadded to the packaging cells and incubated for 72 hours in BBMM-GF

containing 4 Ag/mL polybrene (Sigma). On the day of transplantation,nonadherent and adherent cells were collected, washed thrice with

PBS, and counted in an hemocytometer. Cells were injected at 3.5 � 106/mouse i.v. into female recipient mice that had been irradiated at 500 cGy

on two consecutive days using a MK-1-68A Cesium-137 Gamma animal

irradiator (J.L. Shepherd and Associates, San Fernando, CA). Recipient

mice were maintained in a germ-free environment and treated withoxytetracycline (Phoenix Pharmaceutical, Inc., St. Joseph, MO) added at

200 Ag/mL to the drinking water for the entire duration of theexperiment.

Xenotransplanted orthotopic neuroblastoma model and tissueharvest. After full bone marrow engraftment (day >30 after bone marrowtransplant) recipient mice were implanted with SK-N-BE (2) human

neuroblastoma tumors sown onto the left adrenal gland as previouslyreported (15, 21). Three weeks after tumor implantation, mice were

anesthetized with 2% Avertin, the chest was opened, and the vasculature

was washed free of blood by slow perfusion of 20 mL of 0.9% NaCl into the

left ventricle. Tumors were dissected from the kidneys and their volumedetermined with a caliper using the formula (H � L � l)/2.

Peripheral blood count. Twenty microliters of peripheral blood werecollected from the tail vein of each mouse and WBCs were manually

counted in a hemocytometer as per manufacturer’s instructions (UnopetteMicrocollection System, Becton Dickinson Vacutainer Systems, Franklin

Lakes, NJ).

FACS analysis of the peripheral blood nucleated WBC. Peripheralblood samples were incubated with RBC lysis buffer (156 mmol/L NH4Cl,

0.1 mmol/L EDTA, and 12 mmol/L NaHCO3) for 10 minutes. WBC were

washed and resuspended in PBS. The percentage of EGFP-labeled WBC

was quantified by FACS analysis using a FACSCalibur immunocytometerequipped with the Cellquest software (Becton Dickinson). For the

determination of WBC subclasses, cells were incubated with antibodies

against mouse CD4 and CD8 (T lymphocytes), CD19 (B lymphocytes),

Gr-1PE (granulocytes), CD11b (macrophages), and Dx5 (natural killercells; PharMingen, San Jose, CA) for 30 minutes before being examined

by FACS analysis.

Angiography. Biotinylated Lycopersicon esculentum (tomato) lectin

(50 AL) and Texas red avidin D (100 AL; Vector Laboratories, Inc.,Burlingame, CA) were mixed for 20 minutes before i.v. injection in tumor

bearing mice (150 AL per mouse). Five minutes after injection, animals wereanesthetized with 2% Avertin, perfused with 0.9% NaCl as described above

and the tumors were harvested and examined unfixed under fluorescent

confocal microscopy using a Leica SP confocal DM IRBE-inverted

microscope equipped with a 488-nm argon ion laser and RSP500 beam

splitter as previously described (15).SDS-PAGE. Frozen tumor tissues were homogenized in lysis buffer

[10 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 10% Glycerol, 1% Triton

X-100]. Aliquots of tumor lysates containing 20 Ag of proteins were loadedon 0.1% SDS, 8% polyacrylamide gels containing 1% gelatin. After

electrophoresis, gels were incubated overnight at 37jC in substrate buffer[50 mmol/L Tris-HCl (pH 7.4) and 10 mmol/L CaCl2], stained with 0.25%

Coomassie brilliant blue, and destained in methanol/acetic acid/water

(50:10:40). Aliquots of serum-free conditioned medium from 12-O -

tetradecanoylphorbol-13-acetate–treated human HT1080 cells and recom-

binant murine MMP-9 (R&D Systems) were used as markers for human and

murine MMP-9, respectively.

Immunofluorescence. Primary antibodies used for immunofluores-cence were a goat polyclonal antibody against CD31/platelet-endothelialcell adhesion molecule 1 (PECAM-1) for endothelial cells (Santa Cruz

Biotechnology, Santa Cruz, CA), a mouse monoclonal anti-human smooth

muscle actin (SMA) antibody for pericytes (DakoCytomation, Carpinteria,

CA), a rabbit polyclonal anti-EGFP antibody (Novus Biologicals, Littleton,CO), goat anti-mouse and goat anti-human MMP-9 antibodies (R&D

Systems), a mouse anti-mouse CD45.1/Ly-5.1 monoclonal antibody for

leukocyte detection (SouthernBiotech, Birmingham, AL), a rabbitpolyclonal antibody against human lysozyme for phagocytic cells

Table 1. Donor and recipient combinations used in bonemarrow transplantation experiments

Group Donor Recipient

1 MMP-9+/+ MMP-9+/+

2 MMP-9+/+ MMP-9�/�

3 MMP-9�/� MMP-9+/+

4 MMP-9�/� MMP-9�/�

MMP-9, Bone Marrow Cells, and Tumor Vasculature

www.aacrjournals.org 3201 Cancer Res 2005; 65: (8). April 15, 2005



detection (EC 3.2.1.17 from DakoCytomation; ref. 22), and a rabbit anti-human S100B antibody for Schwann cells (DakoCytomation). Cy3 or

FITC-conjugated anti-IgG secondary antibodies (Jackson ImmunoRe-

search, West Grove, PA) were selected according to the primary antibody.

Five-micrometer paraffin-embedded sections of tumors or organs weredeparaffinized, microwaved in antigen unmasking solution (Vector

Laboratories), and incubated with the primary antibody overnight at

4jC. The secondary antibody was applied for 45 minutes at roomtemperature. Sections were mounted with 4V,6-diamidino-2-phenylindole–containing medium (Vector Laboratories) and viewed with a Leica

DMRA microscope (Wetzlar, Germany) using Plan Apo 20�/1.6 NA phasedifferential interference contrast microscopy or Plan Apo 100�/1.6 oilimmersion objective lenses. The microscope was equipped with a SutterLS175W ozone-free xenon arc lamp. Images were acquired with an

Applied Spectral Imaging Ltd. SkyVision-2/VDS camera from Easy

fluorescence in situ hybridization software (Migdae Ha’Emek, Israel).Quantitative analysis was done using MetaMorph 6.2 software. Vessel

maturation was quantified by calculating the ratio of pericyte area/

endothelial cell area.

Human neuroblastoma tumor samples. Paraffin-embedded sections ofarchival primary adrenal and paraspinal human neuroblastoma tumor

samples were obtained by Dr. H. Shimada and assigned into favorable

histology and unfavorable histology groups according to their histopathol-

ogy (19). Twelve human cases (7 favorable histology tumors and5 unfavorable histology tumors) were examined. All the favorable histology

cases had nonamplified MYCN gene and included three ganglioneuroblas-

toma (Schwannian stroma rich; diagnosed at the age of 1 year 10 months,3 years 2 months, and 14 years 6 months, respectively), and four tumors

showing an appearance of neuroblastoma (Schwannian stroma poor),

poorly differentiated subtype with a low mitosis-karyorrhexis index

(diagnosed at the age of 28 days, 2, 5, and 6 months, respectively). Twounfavorable histology tumors showed the same appearance of poorly

differentiated neuroblastoma with a low mitosis-karyorrhexis index

(diagnosed at the age of 4 years 8 months and 10 years 7 months) and

had nonamplified MYCN gene. The three other unfavorable histologytumors had amplified MYCN gene: two of them were neuroblastoma,

undifferentiated subtype with a high mitosis-karyorrhexis index (diagnosed

at the age of 1 year and 6 months and 4 years and 4 months), and one wasneuroblastoma, poorly differentiated subtype with an intermediate mitosis-

karyorrhexis index (1 year and 6 months of age at the time of diagnosis). All

these unfavorable histology tumors were classified as Schwannian stroma

poor. Specimens were randomly coded and blindly analyzed. The study wasapproved by the Committee of Clinical Investigations at Childrens Hospital

Los Angeles.

Statistical analysis. The two-sample t test was used in all experimentsfor comparison of the average values among four bone marrow transplantgroups.

Results

Expression of matrix metalloproteinase-9 in orthotopicallyxenotransplanted neuroblastoma tumors. To better define thecellular source of stromal expression of MMP-9 in xenotrans-planted tumors, we did dual immunofluorescence analysis onsections of SK-N-BE (2) neuroblastoma tumors orthotopicallyimplanted in RAG-1�/� mice. In these tumors, large nests oftumor cells were separated by connective tissue septae thatcould be easily identified on H&E-stained sections (Fig. 1A).MMP-9 expressing cells were identified along CD31/PECAMexpressing endothelial cells (Fig. 1B) and SMA (Fig. 1C)expressing pericytes but neither endothelial cells nor pericytesshowed MMP-9 expression. In contrast, staining for MMP-9 andCD45, an epitope present on the surface of leukocytes, includinglymphocytes, polymorphonuclears, and monocytes, indicated thatthese cells were the predominant source of MMP-9 (Fig. 1D).

(Note that because mice were RAG-1�/�, lymphocytes areabsent.) The expression of MMP-9 by CD45-positive leukocytesthus suggested that transplantation of tumor bearing MMP-9�/�

recipient mice with bone marrow cells from MMP-9 expressingdonor mice may correct the defect in the tumor vasculature.Transplantation of MMP-9++/++ bone marrow cells corrects the

vascular defect in tumors of MMP-9�/��/� mice. To test thishypothesis, we did a series of bone marrow transplantationexperiments in MMP-9+/+ and MMP-9�/� mice that wereorthotopically implanted with SK-N-BE (2) neuroblastoma tumorsafter successful bone marrow engraftment. We used MMP-9+/+ andMMP-9�/� donor and recipient combinations to generate fourexperimental groups (Table 1). To monitor engraftment and thefate of these cells post-transplantation, harvested donor bonemarrow cells were transduced in vitro with an EGFP expressingretroviral vector before being injected into sublethally irradiated(500 cGy daily � 2) mice. After successful engraftment (4 weeks),recipient mice were orthotopically implanted with SK-N-BE (2)neuroblastoma tumors. Three weeks after tumor implantation,in vivo fluorescence angiography was done and tumors wereharvested. Analysis of the tumor vasculature by fluorescenceangiography indicated the presence of a well-developed network ofvessels in tumors derived from group 1 and 2 mice (transplantedwith bone marrow cells from MMP-9+/+ donor mice) whencompared with tumors derived from group 3 and 4 mice(transplanted with MMP-9�/� donor bone marrow cells), whichconsistently exhibited a poorly developed vasculature with smallblood vessels (Fig. 2A).Next, we examined the level of pericyte coverage of endothelial

cells in these tumors by double immunofluorescence histologyusing CD31/PECAM as a marker for endothelial cells and SMAas a marker for pericytes (Fig. 2B). This analysis revealed ananticipated significant lack of pericyte coverage in tumors derived

Figure 1. MMP-9 in xenotransplanted orthotopic neuroblastoma tumors isexpressed by CD45-positive inflammatory cells. MMP-9 expression inneuroblastoma tumors (n = 5) was examined by immunofluorescence asdescribed in Materials and Methods. A, H&E stain of a xenotransplantedneuroblastoma tumor. Tumor area (T ) and stromal tissue (S). B, frozen tumorsection immunostained for MMP-9 (green ) and PECAM (red ). C, paraffin-embedded tumor section immunostained for MMP-9 (red ) and SMA (green ).D, frozen tumor section immunostained for MMP-9 (red ) and CD45 (green ). Bar ,50 Am in all pictures. Open arrowheads, cells coexpressing MMP-9 and CD45.

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from MMP-9�/� recipient mice transplanted with bone marrowcells from MMP-9�/� donor mice (group 4) which had a pericytearea of 1,120 F 407 Am2 when compared with tumors derivedfrom MMP-9+/+ recipient mice transplanted with bone marrowcells from MMP-9+/+ donor mice (group 1) which had a pericytearea of 4,808 F 1,027 Am2 (P < 0.001). These data are consistentwith our previously published observations (15). Tumors derivedfrom group 4 mice also had significantly lower values ofendothelial cell area (618 F 233 Am2) and exhibited a lowerpericyte/endothelial cell ratio (1.98 F 0.7) compared with tumorsderived from group 1 (3.95 F 1.08; P < 0.001). Restoration ofpericyte coverage was achieved by transplanting MMP-9�/�

recipient mice with bone marrow cells from MMP-9+/+ donormice (group 2) as tumors from this group exhibited pericyte areaand endothelial cell area values (5,060 F 596 and 1,325 F 154 Am2)and a pericyte/endothelial cells ratio (4.5 F 0.77) similar to valuesobserved in tumors from group 1 (P = 0.4; Fig. 2C). In thesetumors, vessels were well developed with multiple layers ofpericytes surrounding endothelial cells as observed in tumorsderived from group 1 mice. In contrast, in tumors derived fromMMP-9+/+ recipient mice transplanted with bone marrow cellsfrom MMP-9�/� donor mice (group 3), significant lower values forpericyte and endothelial cell areas were observed (1,433 F 377 and673 F 252 Am2 respectively; P < 0.001) and the pericyte/

endothelial cell ratio was lower (2.37 F 0.72; P = 0.03) whencompared with group 1 or 2 tumors. These differences in pericytecoverage among the four experimental groups were not observedin the vasculature of normal organs like muscle, liver, lungs, andkidney (data not shown), which points to a more specific role forMMP-9 in pathologic rather than normal angiogenesis. The lack ofpericyte recruitment in tumors derived from group 3 mice whencompared with tumors derived from group 2 mice suggests thatbone marrow–derived cells are the major source of MMP-9 in thetumor stroma and that cells from neighboring tissue do notsubstantially contribute to MMP-9 expression. To confirm thispossibility, we did an analysis of MMP-9 expression in tumorlysates by gelatin zymography (Fig. 3A). All tumors in group1 and 2 mice showed, as anticipated, the presence of a Mr 95,000gelatinolytic band that comigrated with recombinant murineproMMP-9. This band was not detected in tumors derived fromgroup 4 mice. Whereas the majority of tumors derived from group3 mice were negative for MMP-9, some tumors contained lowlevels of murine proMMP-9, an observation that suggested thatbone marrow cells may not be the only source of MMP-9, becausethese mice were transplanted with MMP-9�/� bone marrow cells.However, an immunohistologic analysis of the bone marrow ofthese mice revealed the presence of MMP-9-positive cells that didnot express EGFP, indicating the presence of a mixed chimerism

Figure 2. Transplantation of MMP-9+/+ bonemarrow cells corrects the vascular defect oftumors implanted in MMP-9�/� mice. A, tumorvascular architecture examined by in vivoangiography as described in Materials andMethods. Representative pictures are areconstruction of the maximum intensity in 50Z-series sections at 2-Am intervals obtained byconfocal microscopy. Three fresh tumors in eachgroup were examined. Bar , 200 Am.B, representative pictures of paraffin-embeddedtumor sections evaluated by doubleimmunofluorescence for pericytes (SMA, green )and endothelial cells (EC , PECAM, red ). Bar ,50 Am. Insets, high magnification (100/�1.6) of avessel cross-section. C, amount of pericytesand endothelial cells in the tumor sections wasquantified by measuring the amount of green andred fluorescent area as indicated in Materials andMethods in 30 histological fields for each tumorsection. The level of pericyte coverage wasexpressed as pericyte/endothelial cell ratio andwas calculated from the data above. Average areaper field in each tumor generated from threeindependent BMT experiments. Number of tumorsexamined in each group: group 1, n = 5; group 2,n = 7; group 3, n = 5; and group 4, n = 6.





with the persistence of some MMP-9-positive recipient cells post-BMT (Fig. 3B). In the tumor tissue, these cells were also detectedas MMP-9-positive, EGFP-negative cells that coexpressed CD45(Fig. 3C). The presence of MMP-9 in some tumors derived fromMMP-9+/+ recipient mice transplanted with bone marrow cellsfrom MMP-9�/� donor mice is therefore attributed to thepresence of a bone marrow mixed chimerism where somerecipient bone marrow cells survived despite sublethal irradiation.Altogether, the data support our hypothesis that bone marrow–derived cells are the primary source of MMP-9 in xenotrans-planted tumors and show that expression of MMP-9 by these cellsis critical for the establishment of a mature vasculature andpericyte recruitment.Matrix metalloproteinase-9 contributes to the recruitment

of bone marrow–derived cells to the tumor microenviron-ment. It has recently been suggested that MMP-9 contributesto the mobilization of bone marrow stem cells into thecirculation (16). We therefore asked the question whether

MMP-9 could affect the recruitment of bone marrow–derivedcells from the blood circulation into the tumor microenviron-ment. To address this question, we examined the presence ofEGFP expressing cells in the stroma of tumors obtained ineach experimental group (Fig. 4). This analysis indicated thepresence of numerous EGFP-positive bone marrow–derived cells inthe stroma of tumors derived from group 1 (19.4F 4.9 per field) andgroup 2 (19.4 F 5 per field) recipient mice transplanted with bonemarrow cells from MMP-9+/+ donor mice (Fig. 4A and B). Incontrast, significantly fewer EGFP-positive bone marrow–derivedcells were present in tumors derived from group 3 (2.7 F 0.6 perfield) and group 4 (0.97 F 0.7 per field) recipient mice transplantedwith bone marrow cells from MMP-9�/� donor mice (P < 0.01). Todetermine whether these differences were due to differences inengraftment and release of bone marrow–derived cells into thecirculation, we analyzed the number of EGFP-positive WBC in theperipheral blood 4 weeks after transplantation when the mice wereimplanted with SK-N-BE (2) tumors (Fig. 4C). The number of WBCwas significantly higher in group 1 mice compared with the otherthree groups (8.1 mm3, P < 0.01) but was not different betweengroup 2 and 3 (3.35 and 2.9 mm3, P = 0.1). There was no differencein the relative number of granulocytes, macrophages, and naturalkiller cells between wild-type and MMP-9–deficient mice. In allexperimental groups, the percentage of peripheral blood WBCexpressing EGFP was similar, ranging between 75% and 80%(reflecting similar levels of transduction efficiency). The differencein the number of EGFP-positive WBC between groups 1 and 2might suggest that stromal-derived MMP-9 contributes to themobilization of WBC from the bone marrow space to the bloodcirculation. However, the degree of infiltration of these cells intothe tumor tissue was not different. The difference in the amount ofEGFP-positive cells present in the tumor stroma between group 2and 3 mice in the absence of a similar difference in the peripheralblood (P = 0.2) suggests that MMP-9 expression by WBCcontributes to their recruitment from the peripheral blood intothe tumor stroma.Hematopoietic stem cells are a source of EPC that contribute to

the tumor vasculature (6). To determine whether bone marrow–derived endothelial cells contribute to the tumor vasculature in ourmodel and whether MMP-9 was involved in this process, weexamined tumors in the four experimental groups for the presenceof EGFP-positive cells also expressing CD31/PECAM (Fig. 5A).By double immunofluorescence analysis, we detected CD31/PECAM and EGFP coexpressing cells in the vasculature of tumorsderived from group 1 and 2 mice where they represented 13.3 F 2%and 14F 3.9% of the total CD31/PECAM positivity, respectively. Incontrast, no EGFP-positive cells coexpressing CD31/PECAM couldbe detected in group 3 and 4 tumors (Fig. 5C). To determinewhether bone marrow–derived cells could also differentiate intopericytes, tumor sections were double stained for EGFP and SMA.This analysis (Fig. 5B) failed to identify EGFP-positive cells thatwere also SMA positive in tumors of the four experimental groups.Thus, in addition to promoting pericyte recruitment, MMP-9 alsoplays a role in the recruitment of bone marrow–derived endothelialcells in the tumor microenvironment where these cells contributeto vasculogenesis.Primary neuroblastoma tumors with an unfavorable

histology have higher infiltration with matrix metalloprotei-nase-9–expressing inflammatory cells and a more maturevasculature. From our experiments in xenotransplanted tumors,we would anticipate that in advanced-stage human neuroblastoma

Figure 3. MMP-9 expression in xenotransplanted tumors and bone marrowcells. A, presence of MMP-9 in tumor lysates in the four experimental groupsdetected by SDS-PAGE zymography. Two representative samples for eachgroup. Number of samples in each group: group 1, n = 5; group 2, n = 9;group 3, n = 5; and group 4, n = 7. Each lane contained 20 Ag of proteins.Recombinant murine MMP-9 and supernatant of 12-O -tetradecanoylphorbol-13-acetate–treated HT1080 cells were used as controls for murine and humanMMP-9, respectively. B, representative pictures of paraffin-embedded mousebone marrow samples immunostained for EGFP (green ) and murine MMP-9(red) in group 2 and 3 mice. Insets, presence of cells coexpressing EGFP andMMP-9 in group 2 (yellow in left) but not in group 3. Bars , 50 Am.C, representative pictures of paraffin-embedded orthotopic tumor samplesimmunostained for EGFP (green ) and murine MMP-9 (red ). Note the presenceof cells coexpressing EGFP and MMP-9 in group 2 but not in group 3. Inset,presence of cells coexpressing MMP-9 and CD45 in a tumor of group 3.Bars , 50 Am.

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tumors, there would be a higher level of infiltration with MMP-9–expressing cells and a higher level of pericyte coverage ofendothelial cells. To test this hypothesis, we examined a seriesof primary human neuroblastoma tumors for the presence ofpericytes and MMP-9–expressing cells. We analyzed five tumorswith unfavorable histology and seven tumors with a favorablehistology. Analysis of the pericyte/endothelial cell ratio in thetumor samples indicated the presence of more mature vesselsin unfavorable histology tumors with an average pericyte/endothelial cell ratio of 3.0 F 0.7 (Fig. 6A and C), whereasfavorable histology tumors exhibited immature vessels poorlycovered with pericytes as indicated by a significantly lowerpericyte/endothelial cell ratio (1.8 F 0.45; P = 0.01). Unfavorablehistology tumors had a lower microvessel density (19 F 7.8vessels per field) than favorable histology tumors (48 F 11vessels per field; P < 0.001). However, the average tumor vesselsize in unfavorable histology tumors was 2.3� larger (550 F 296pixels per field) than in favorable histology tumors (239 F 90pixels per field; P = 0.07; Fig. 6C). The endothelial cell areabetween these two types of tumors was however no different(906.6 versus 1,177 Am2; P = 0.4) thus indicating that for thesame area of endothelial cells, unfavorable histology tumorscontained larger and more mature vessels in smaller numbersthan favorable histology tumors which were rich in small butimmature vessels. When examined for the presence of Schwanncells with an anti-S100B antibody unfavorable histology tumorsdid not stain positive, whereas favorable histology tumors, and inparticular ganglioneuroblastoma, stained strongly positive (Fig.6B ). When examined for MMP-9 expression, unfavorablehistology tumors also contained a substantially higher numberof MMP-9 expressing cells than favorable histology tumors(24 F 8.9 versus 2.7 F 0.9 cells per field, P < 0.01; Fig. 7A and C),and the majority of these MMP-9-positive cells containedlysozyme, indicating they were phagocytic cells, (Fig. 7B). Insummary, we have shown that bone marrow–derived inflamma-tory cells are the primary source of MMP-9 expression inneuroblastoma tumors where MMP-9 has a dual role: itpromotes pericyte coverage of endothelial cells and it is

important for the recruitment of inflammatory cells to thetumor microenvironment. Both roles are associated with themore aggressive behavior and poor clinical outcome thatcharacterize unfavorable histology tumors.

Discussion

The contribution of the tumor stroma as a source of MMP-9expression in animal models of tumorigenesis and in humancancer has been well recognized. In murine models of squamousepithelial carcinoma and pancreatic cancers, MMP-9 is expressedby inflammatory cells like mast cells, neutrophils, and macro-phages where it plays a critical role in initiating an angiogenicswitch (13, 14). In ovarian cancer cells xenotransplanted in nudemice, MMP-9 is expressed by tumor-associated macrophageswhere it promotes angiogenesis and growth (23). MMP-9expression is also specifically induced in lung endothelial cellsand macrophages by distant primary tumors via VEGF receptor(VEGFR-1) and promotes the establishment of lung metastasis(24). In human cancers, like breast, colon, bladder, skin, andprostate cancers, MMP-9 is predominantly expressed by stromalfibroblasts and its expression correlates with clinical outcome(25–29). We previously reported that in both primary humanneuroblastoma tumors and in orthotopically xenotransplantedtumors, MMP-9 is expressed by the stroma and that MMP-9contributes to the tumor vasculature by promoting the coverageof endothelial cells by pericytes (15, 20). In this article, weprovide three lines of evidence indicating that in neuroblastomabone marrow–derived leukocytes are the primary source ofMMP-9 expression. In our xenotransplanted orthotopic tumormodel, we show that CD45-positive leukocytes (granulocytes andmonocytes) express MMP-9 whereas CD31/PECAM-positiveendothelial cells or SMA-positive pericytes do not express theenzyme. Second, our transplantation experiments clearly indicatethat transplantation of MMP-9-positive bone marrow cells inMMP-9 null mice results in tumor colonization by MMP-9-positive leukocytes and can restore the vascular architecture.Third, in unfavorable histology human neuroblastoma tumor

Figure 4. MMP-9 contributes to the recruitmentof bone marrow–derived cells to the tumormicroenvironment. A, representative picturesof paraffin-embedded tumor sectionsimmunostained for EGFP (green ) and PECAM(red). Bar, 50 Am. B, quantification ofEGFP-positive bone marrow–derived cells intumor sections. Average number of EGFPpositive cells per field from 10 microscopicfields examined each in two tissue sections.Number of tumors examined: group 1, n = 4;group 2, n = 8; group 3, n = 5; and group 4,n = 5. C, number of WBC in the peripheral bloodin each experimental group on day 28 after bonemarrow transplantation. Average number of WBC(closed column ) and EGFP-positive cells (opencolumn ). Number of blood samples examined:group 1, n = 10; group 2, n = 12; group 3,n = 10; and group 4, n = 12.





samples, MMP-9 is expressed by phagocytic cells (activatedmacrophages). Whereas we cannot entirely rule out somecontribution of other non-bone marrow–derived cells as asource of MMP-9, our data support the concept that bonemarrow–derived cells are the predominant contributor of MMP-9expression. The fact that we observed some MMP-9 expressionin the tumor tissues of neuroblastoma tumors implanted inMMP-9+/+ recipient mice transplanted with MMP-9�/� bonemarrow cells (group 3) is not inconsistent with our hypothesis

because we documented the presence of a bone marrow–mixedchimerism in these mice.Our data also show that the recruitment of bone marrow–

derived cells into the tumor is dependent on MMP-9 expressionby these cells because the number of MMP-9-positive cells thatcolonized neuroblastoma tumors implanted in mice was afunction of the genotype of the bone marrow donor and not ofthe genotype of the recipient. Expression of MMP-9 by bonemarrow cells releases the soluble c-kit ligand permitting thetransfer of endothelial cells and hematopoietic stem cells from aquiescent to a proliferative niche in the bone marrow and to thecirculation, resulting in a more rapid recovery post myeloablativetherapy (30). Consistent with these data, we observed a largernumber of EGFP-positive bone marrow–derived cells in the tumorstroma of MMP-9�/� mice transplanted with MMP-9+/+ bonemarrow cells (group 2) than in MMP-9+/+ mice transplanted with

Figure 5. Presence of bone marrow–derived endothelial cells inxenotransplanted tumors. A, representative pictures of paraffin-embeddedxenotransplanted tumor sections immunostained for EGFP (green ) and PECAM(red). White arrowheads, presence of EGFP and PECAM coexpressing cells inthe endothelium layer of blood vessels. B, representative pictures ofparaffin-embedded tumor samples immunostained for EGFP (red) and SMA(green ). Bars , 50 Am in all pictures. C, quantification analysis of the amount ofbone marrow–derived endothelial cells incorporated into the tumor vasculature.Average percentage of PECAM and EGFP colocalization area from the totalPECAM-positive area per microscopic field (vasculogenesis). Ten microscopicfields were examined in two tissue sections for each tumor. Number of tumorsexamined: group 1, n = 4; group 2, n = 8; group 3, n = 5; and group 4, n = 5.

Figure 6. Primary neuroblastoma tumors with an unfavorable histology havebetter endothelial cell (EC ) coverage by pericytes. A and B, representativepictures of sections of primary human neuroblastoma tumors immunostained forpericytes (green ) and endothelial cells (red) in (A) and for Schwann cells (red )and pericytes (green ) in (B ). Bar , 50 Am. C, quantitative analysis of pericyte/endothelial cell ratio, microvessel density (MVD), microvessel size (MVS ),and endothelial cell area per field in the tumors. Average values from thirtymicroscopic fields examined in each tumor. Number of tumors examined:unfavorable histology (UH ), n = 5 and favorable histology (FH ), n = 7.

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MMP-9�/� bone marrow cells (group 3). The analysis of theperipheral blood of the mice at the time the tumors wereimplanted showed no difference in the number and percentage ofEGFP-positive cells between these two groups, indicating thatMMP-9 contributed to the recruitment of bone marrow–derivedinflammatory cells into the tumor. The presence of CD31/PECAMand EGFP coexpressing cells in the tumor microvasculature ofmice transplanted with MMP-9+/+ bone marrow (groups 1 and 2)was also consistent with MMP-9 playing a role in the recruitmentof bone marrow–derived endothelial cells into the tumor stroma.Because we did not perform an analysis of circulating EPC, wecannot conclude at this point whether MMP-9 is also involved inthe release of EPC from the bone marrow into the circulation inour model. However, the data show that the presence of bonemarrow–derived endothelial cells in the tumor vasculaturedepends on MMP-9 expression.MMP-9 has been shown to contribute to tumor angiogenesis in

a complex way. Most of the mechanisms have pointed toendothelial cells where MMP-9 promote their ability to formtubes on Matrigel (31), trigger an angiogenic switch by increasingthe solubilization of VEGF (13), or as discussed above, bypromoting the mobilization and recruitment of EPC. UsingMMP-9�/� mice, we have recently shown that pericytes wereanother target for MMP-9 in angiogenesis as we had shown thatneuroblastoma tumors xenotransplanted in MMP-9–deficientmice had an immature vasculature that lacked pericyte coverage(15). Our data provide further support to our observations bydemonstrating that reestablishing MMP-9 expression by bonemarrow transplantation restores a normal vascular phenotype inthe tumors of MMP-9�/� recipient mice. Our data are alsoconsistent with the observation of other investigators who haveshown that forced expression of the inhibitor of MMP, tissueinhibitor of metalloproteinase-3, in murine neuroblastoma andmelanoma tumors resulted in an unanticipated increase in CD31/

PECAM expressing endothelial cells but a decrease in pericyterecruitment (32). The mechanism by which MMP-9 is involvedin the recruitment of pericytes along microvascular endothelialcells is not known. Pericytes are recruited by endothelial cellsvia two major mechanisms. Upon stimulation by VEGF, endo-thelial cells express platelet-derived growth factor (PDGF) B,which stimulates the migration and proliferation of PDGFreceptor h expressing pericytes (33). Pericytes also expressepithelial growth factor (EGF) receptor ErbB1 and ErbB2 andendothelial cell express heparin-bound EGF upon exposure toangiopoietin-1 (34). It is conceivable that MMP-9 promotespericyte recruitment along endothelial cells by increasing thesolubility of PDGFB or heparin-bound EGF, a possibility currentlyexplored in our laboratory.Pericytes are present in most tumor blood vessels but are

often abnormal (35). Interestingly, they have been recentlyidentified as a novel and important target for antiangiogenictherapies. Bergers et al. have shown in a mouse model ofpancreatic islet cancer that targeting only VEGFR in endothelialcell with the receptor tyrosine kinase inhibitor SU5416 iseffective against early-stage tumors but not against large andwell-vascularized tumors. In contrast, a PDGF receptor kinaseinhibitor SU6668 targeting pericytes is effective in end-stagetumors and a combination of VEGFR and PDGFR inhibitors hasthe maximum antiangiogenic effect (36). A similar observationhas been made in a rat brain tumor model (37). Targeting bothendothelial cells and pericytes or blocking pericyte recruitmentto the tumor vessels may therefore be a powerful antiangiogenicstrategy by inducing endothelial cell apoptosis and tumor vesselregression. This approach could be particularly effective intumors that exhibit mature vessels rich in pericytes. It isconceivable that inhibiting pericytes in the tumor vasculaturemight negatively contribute to tumor progression by increasingvascular permeability and facilitating the diffusion of cytotoxicdrugs.It is interesting to note that our analysis of human neuro-

blastoma tumors points to a deficit in angiogenesis in favorablehistology tumors that are also rich in Schwannian stroma. Otherinvestigators have shown that Schwann cells actively contributeto inhibition of angiogenesis by secreting antiangiogenic factorslike tissue inhibitor of metalloproteinase inhibitor-2, pigmentepithelium-derived factor, and SPARC (38).The same group of investigators recently reported that

treatment of mice with a folded synthetic peptide correspondingto the epidermal growth factor-like module of SPARC decreasesthe size and the density of microvessels that formed in a plug ofMatrigel implanted s.c. (39). Consistent with their data, we alsoobserved smaller blood vessels in favorable histology tumorswhich contained Schwann cells when compared with unfavorablehistology tumors which did not contain Schwann cells and hadthe same endothelial cell area but contained larger and moremature vessels at a lower density. The observation that a moreaggressive type of tumor (unfavorable histology) has a lowermicrovessel density than a less aggressive type illustrates howthe measurement of microvessel density along cannot beconsidered as a single and reliable marker for angiogenesis. Aspointed out by several laboratories, microvessel density alone isnot a measure of the angiogenic dependence of a tumor and ahigh vascular density does not necessarily imply a rapid tumorgrowth (40). The degree of maturation of blood vessels asexamined by their degree of coverage by pericytes may thus

Figure 7. Primary neuroblastoma tumors with an unfavorable histology havehigher infiltration with MMP-9 expressing inflammatory cells. Representativepictures of primary human neuroblastoma tumors immunostained for humanMMP-9 (red ) and SMA (green ) in (A ) and for MMP-9 (red) and Lysozyme(green ) in (B). Bar , 50 Am. Inset, high magnification of coexpression of MMP-9and lysozyme. C, average number of MMP-9 expressing cells per field from20 microscopic fields examined in each tumor.





provide an additional indicator of tumor aggressiveness andsuggests a potential benefit from antiangiogenic therapies thattarget pericytes.The significance of our observation in the context of clinical

treatment of neuroblastoma should be discussed. Many patientswith high-risk neuroblastoma are treated with intensivemyeloablative chemotherapy followed by bone marrow trans-plantation (41). Intensive myeloablative chemotherapy is typi-cally followed by an increased mobilization of bone marrowcells and EPC into the peripheral blood circulation (42). Asshown by our data, these cells contribute to tumor progressionby promoting the formation of a more mature vasculature. Bydemonstrating that MMP-9 is critical for the recruitment ofthese cells into the tumor stroma, our data suggest thatinhibition of MMP-9 activity after bone marrow transplantationmay be of therapeutic benefit by preventing the infiltration of

residual tumors by bone marrow–derived cells. Inhibition ofMMP-9 may also interfere with the recruitment of pericytesalong newly formed microvessels.In summary, our data provide a new insight into the

contribution of bone marrow–derived cells to tumor angiogenesisin which MMP-9 plays a key role.

Acknowledgments

Received 10/20/2004; revised 12/23/2004; accepted 2/3/2005.Grant support: NIH grants PO1 CA81403 (Y.A. DeClerck and H. Shimada), P01

CA72006, and R01 CA94168 (L.M. Coussens) and T.J. Martell Foundation.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.

We thank Drs. G. McNamara (Julian Dixon Cell Imaging Core facility of the SabanResearch Institute) and P. Malik for helpful advice, Shundi Ge for technical assistance,and J. Rosenberg for her excellent assistance in typing the article.

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2005;65:3200-3208. Cancer Res Sonata Jodele, Christophe F. Chantrain, Laurence Blavier, et al. DependentVasculature in Neuroblastoma Is Matrix Metalloproteinase-9

Derived Cells to the Tumor−The Contribution of Bone Marrow

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