[CANCER RESEARCH 59, 507–510, February 1, 1999]

Advances in Brief

Carboxymethyl Benzylamide Dextran Blocks Angiogenesis of MDA-MB435 BreastCarcinoma Xenografted in Fat Pad and Its Lung Metastases in Nude Mice1

Rozita Bagheri-Yarmand,2 Yamina Kourbali, Ana Maria Rath, Roger Vassy, Antoine Martin, Jacqueline Jozefonvicz,Claudine Soria, He Lu, and Michel CrepinLaboratoire d’Oncologie et Imagerie des Tumeurs Solides, Faculte´ de Medecine de Bobigny, Universite Paris 13, 93017 Bobigny Cedex [R. B-Y., Y. K., A. M. R., R. V., M. C.];Service d’Anatomie Pathologie, 93017 Bobigny Cedex [A. M.]; Laboratoire de Recherche sur les Macromolecules, Centre National de la Recherche Scientifique, URA 502,Universite Paris-Nord, 93430 Villetaneuse [J. J.]; and Institut National de la Sante et de la Recherche Medicale U353, Institut d’Hematologie, Hopital Saint-Louis, 75010 Paris[C. S., H. L.] France

Abstract

We previously showed that carboxymethyl benzylamide dextran(CMDB7) prevents tumor growth and tumor angiogenesis by binding toangiogenic growth factors, thereby preventing them from reaching theirreceptors on tumor or stromal cells (Bagheri-Yarmandet al. Br. J. Can-cer, 78: 111–118, 1998; Bagheri-Yarmandet al. Cell Growth Differ., 9:497–504, 1998). In this study, CMDB7 inhibited neovessel formationwithin the fibroblast growth factor 2-enriched matrigel in mice, and itsanticancer effect was then tested in a metastatic breast cancer model.Human MDA-MB435 cells were injected into the mammary fat pad ofnude mice, and breast tumors developed within 1 week; all of the mice hadlung metastases at 12 weeks. CMDB7 treatment (50, 150, or 300 s.c. or300 i.v. mg/kg/week for 10 weeks) reduced the incidence of lung metastasesto 12%. Histological analysis showed markedly less tumor neovascularizationin the CMDB7-treated mice. Pulmonary metastasis incidence was stronglydependent on the intratumoral neoangiogenesis in primary tumors.

Introduction

Tumor-associated neovascularization is a prerequisite of rapid tu-mor growth and metastasis (1, 2). Increased vascularity may allow fornot only enhanced tumor growth but also a greater chance of hema-togenous tumor embolization. Thus, it is hypothesized that inhibitingtumor angiogenesis will halt tumor growth and decrease metastaticpotential. Weidneret al. (3) demonstrated a statistically significantcorrelation between the incidence of metastases and microvesselcounts in invasive breast carcinoma. Thus, antiangiogenic agentstargeting the tumor vasculature are expected to block neovasculariza-tion and thereby prevent metastasis (4).

We have reported previously (5) that CMDB73 displays anin vitrogrowth-inhibitory activity in breast tumoral cells. A positive correla-tion was found between the inhibition of cell proliferation and theoverall content of benzylamide (5). Growth inhibition was associatedwith a decrease in the proportion of S-phase cells and an accumulationof G1 phase cells (5). CMDB7 specifically inhibited the mitogeniceffect and receptor binding of angiogenic growth factors, such asFGF-2, FGF-4, platelet-derived growth factor BB, and transforminggrowth factorb1, by forming complexes with them (6–8) and pre-vented endothelial cell proliferation and migration (7).In vivo studiesdemonstrated that growth of MCF-7ras (8) and FGF-4 transfected

HBL100 cell (7) tumors in nude mice was blocked by CMDB7treatment, and histological analysis showed much less neovascular-ization in these tumors.

We have now investigated the effect of CMDB7 on FGF-2-inducedangiogenesis in a matrigel angiogenic assay. Estrogen receptor-negative MDA-MB435 breast carcinoma cells were xenografted intonude mice to determine whether CMDB7 might have therapeuticutility in the prevention or treatment of metastatic breast cancer. Afterinjection into m.f.p., these cells gave rise to metastases in lung organand thus provided an experimental model for the metastasis of ahighly aggressive human breast carcinoma (9).

Materials and Methods

Dextran Derivative Preparation. A water-soluble dextran derivative(CMDB7) was prepared from dextran T40, as described previously (10), by astatistical substitution of dextran in two steps: carboxymethylation, followedby the coupling of benzylamide. This derivative was equilibrated and purifiedby ultrafiltration and then lyophilized. The chemical composition was deter-mined by acidimetric titration and elementary analysis of nitrogen (dextran,0%; carboxymethyl, 70%; benzylamide, 30%; mass apparent molecularweight 5 80,000 g/mol).

Cell Line and Cell Cultures. MDA-MB435 is an estrogen receptor-negative cell line isolated from the pleural effusion of a patient with breastcarcinoma (11). The cells were grown in DMEM (Life Technologies, Gaith-ersburg, MD) supplemented with 10% FCS, 2 mM L-glutamine, 50 IU/mlpenicillin, and 50 mg/ml streptomycin (Life Technologies) at 37°C in ahumidified atmosphere containing 5% CO2. Cells were routinely passed oncea week at a 1:10 split ratio.

Murine Angiogenesis Assay.Angiogenesis was assayed as the growth ofblood vessels from s.c. tissue into a solid gel of reconstituted basementmembranes containing the test sample (12). Matrigel (11.46 mg/ml; BectonDickinson Laware, Bedford, MA) in liquid form at 4°C was mixed with FGF-2(1 mg) with or without different concentrations of CMDB7 and injected intothe abdominal s.c. tissue of 5 mice/group. At body temperature, matrigelrapidly solidified, thereby trapping the factor, assuring its slow release, andprolonging exposure of surrounding tissues. Mice were killed 2 weeks later,and the matrigel plugs were exposed for photography. NIH image analysis (see“Image Analysis”) was applied to quantitate the vascularization in each tissuesection, the extent of which is expressed as the percentage area of labeledendothelial cells6 SE on each slide.

Animal Model for Metastases. Female athymic nude mice (nu/nu), 3weeks old, were obtained from Janvier Laboratory (Le-Genest-St-Isle, France).Animals were kept in a temperature-controlled room on 12 h/12 h light/darkschedule with food and waterad libitum. MDA-MB435 cells were grown inDMEM supplemented with 10% FCS in T150 plates and harvested at 80%confluence. The m.f.p. has been shown to be a more favorable graft site for thegrowth of mouse mammary tumors—because of its good blood supply ascompared with the subcutis—and also for the dissemination of metastases fromthe m.f.p tumors (9). Mice were anesthetized with Metofane, and a 5-mmincision was made in the skin over the lateral thorax to expose the m.f.p. Theinoculum (106 cells/0.1 ml) was injected into the tissue taking care to avoid thes.c. space. All of the mice developed tumors after about 1 week. CMDB7

Received 8/26/98; accepted 12/17/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Fondation Martine-Midy (Paris, France).2 To whom requests for reprints should be addressed, at Universite Paris 13, Labora-

toire d’Oncologie et Imagerie des Tumeurs Solides, Faculte´ de Medecine, 74, rue MarcelCachin, 93017 Bobigny Cedex, France. Phone: 33-1-48-38-77-20; Fax: 33-1-48-38-77-29.E-mail: bagheri@smbh.univ-paris13.fr.

3 The abbreviations used are: CMDB7, carboxymethyl benzylamide dextran; FGF-2,fibroblast growth factor 2; FGF-4, fibroblast growth factor 4; m.f.p., mammary fat pad;TBS, tris-buffered saline; GSL-1, Griffonia (Bandeiraea) simplicifolia lectin.

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treatment was initiated 2 weeks after cell inoculation, when tumors were wellestablished (approximately 0.34 cm3). Mice (n5 42) were arbitrarily assignedto receive 0.1 ml of PBS s.c. (controls,n 5 10) or CMDB7 injected s.c. closeto the tumor at 50 (n5 8), 150 (n5 8), or 300 mg/kg/week (n5 8) or i.v. (i.v.)at 300 mg/kg/week (n5 8) for 10 weeks. Tumors were measured along twomajor axes with calipers. Tumor volume was calculated as follows:

V 54

3p R1

2 R2

whereR1 is radius 1,R2 is radius 2, andR1 , R2.Tissue Preparation and Immunohistochemical Analysis.Immediately

after surgical resection, primary tumor specimens were weighed and cut intosmall pieces; fragments were fixed with 4% formalin processed to paraffin inthe usual way, and 4-mm sections were stained with H&E. Endothelial cellswere specifically stained with GSL-1 (Vector Laboratories, Burlingame, CA).The GSL-1 lectin binds specifically to galactosyl residues and thus labels thevascular endothelium in mice (13). Sections were deparaffinized and rehy-drated. Endogenous peroxidase was inactivated with 3% H2O2 and washed inTBS (pH 7.6) followed by preincubation in FCS for 30 min at room temper-ature. The sections were then incubated for 45 min with biotinylated GSL-1(0.01 mg/ml), washed with TBS, and treated for 30 min with avidin-peroxidase(Vector Laboratories), and washed again with TBS. The peroxidase wasvisualized by incubation for 10 min in 0.1M acetate buffer (pH 5.2) containing3% H2O2 and 3% 3-amino-9-ethylcarbazole. Finally, the slides were washed indistilled water and tap water, counterstained with hematoxylin, dehydrated,and coverslipped with Permount.

Image Analysis. For each GSL-1-labeled section of control and CMDB7-treated tumors, five fields containing exclusively viable tumoral cells, asindicated by the hematoxylin stain, were selected randomly for analysis. Imageanalysis was performed on a Power Macintosh computer 8500/120 using thepublic domain NIH program.4 The endothelial cell area in each section wasdetermined, with an experimental error of,5%, by quantifying those areasemitting an intensity of GSL-1 labeling above a given threshold level. Thepercentage area of endothelial cells was then calculated as the ratio of the

labeled area:the total viewed area3 100; these values were then averaged foruntreated (control) and treated-CMDB7 tumors.

Statistical analysis. The results are presented as means6 SE. Multiplestatistical comparisons were performed using ANOVA and the Mann-WhitneyU tests in a multivariate linear model.

Results

CMDB7 Inhibits Angiogenesis Induced by FGF-2 in Matrigel.In the murine angiogenic assay, FGF-2 mixed with matrigel wasinjected s.c. into 20 mice. The mice were killed 2 weeks later for

4 Developed at NIH and available on the internet at http://rsb.info.nih.gov/nih-image/.

Fig. 1. The inhibition by CMDB7 of FGF-2-induced angiogenesis in mice. Mice received s.c. injections of 300ml of a matrigel mixture containing FGF-2 with or without CMDB7.The animals were killed and dissected 2 weeks later, and the matrigel plugs were exposed and photographed. Each group contained 5 animals, and a representative animal from eachgroup is shown. Matrigel containing: 1mg of FGF-2 (A); 1 mg of FGF-2 and 2 mg of CMDB7 (B); 1 mg of FGF-2 and 4 mg of CMDB7 (C); and 1mg of FGF-2 and 8 mg of CMDB7(D). E, the antiangiogenic effect of CMDB7 was quantitated by NIH image analysis, and data are expressed as the mean percentage areas6 SE (bars) of endothelial cells on day 14(n 5 5). p, significantly different from controls (P , 0.001).

Fig. 2. CMDB7 treatment does not affect tumor growth of MDA-MB435 carcinomacells. MDA-MB435 tumor cells (106 cells/site) were injected into the m.f.p. When tumorvolume reached 0.34 cm3 (n 5 42), CMDB7 was administrated s.c. at 50, 150, or 300mg/kg/week or i.v. at 300 mg/kg/week for 10 weeks. Tumor growth was measured for upto 10 weeks, and the results are presented as the mean volumes6 SE (bars) of tumorobtained from 10 control mice and from 8 mice in each treated group.

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morphometric analysis of matrigel plugs. Plugs containing FGF-2 (1mg) were bright red and often contained superficial blood vessels (Fig.1A). Growth of blood vessels from s.c. tissue and their penetration intothe solidified matrigel was totally dependent on FGF-2 (data notshown). When 2, 4, or 8 mg of CMDB7 were added to the matrigel,angiogenesis was greatly inhibited compared with the control (Fig. 1B-D). Image analysis quantification showed that neovascularizationwas 16 and 85% significantly lower when the matrix contained 2 and4 mg of CMDB7, respectively, and was almost completely inhibitedin the presence of 8 mg of CMDB7 (Fig. 1E).

CMDB7 Inhibition of Tumor Angiogenesis and Lung Metasta-sis of MDA-MB435 Human Breast Carcinomas.After injection of106 MDA-MB435 cells into the m.f.p.; all of the mice had developedtumors by the end of the first week and CMDB7 treatment wasinitiated 1 week later, when the tumors were well established.CMDB7 treatment (50, 150, or 300 mg/kg/week s.c. and 300 mg/kg/week i.v.) did not affect significantly primary tumor growth as com-pared with untreated control tumors (Fig. 2). At 12 weeks, all of themice were killed, and the numbers and locations of lung metastaseswere recorded. Although all (10 of 10) of the untreated mice bearing

primary breast carcinomas had microscopic lung metastases; lungmetastases developed in only 1 of the 8 mice in each CMDB7-treatedgroup (Fig. 3). No differences were found in the numbers of meta-static foci and their locations between lung metastasis in untreated andCMDB7-treated groups.

GSL-1 selectively labeled endothelial cells (Fig. 4,A and B) andthus enabled the relative density of endothelial cells (percentage ofarea occupied by endothelial cells) to be determined based on theimage analysis of the detection of the label. The mean percentage ofendothelial cell area in viable fields of control tumors (4.896 0.9; 50fields in 10 tumors)are compared with those in tumors treated withCMDB7 at 50, 150, or 300 mg/kg/week s.c. or 300 mg/kg/week byi.v., respectively: 1.716 0.76, 1.56 0.6, 1.286 0.51 (120 fields in24 tumors) and 0.536 0.21 (40 fields in 8 tumors; Fig. 4C). Theendothelial cell densities in all of the CMDB7 treatment groups weresignificantly lower (P , 0.001). Similar results were obtained withCD31 antibody but with nonspecific cell staining (data not shown). Ineach treated group, 1 tumor escaped CMDB7-induced inhibition andhad a high density of endothelial cells (Fig. 3). These results indicatethat the prevention of angiogenesis in primary tumors was responsiblefor this antimetastatic effect.

Dependence of the Incidence of Lung Metastases on PrimaryTumor Angiogenesis.The mean6 SE percentage of endothelial cellarea for 28 nonmetastatic tumors was significantly lower than that formicrometastasized 14 tumors: 0.86 0.5 and 4.826 2.7, respectively(P , 0.001). We showed that lung micrometastases developed whenthe percentage area of endothelial cells reached a threshold of 2%(Fig. 3). This finding confirmed the relationship between microvesseldensity in primary tumor and the development of metastases.

No Toxicity of CMDB7 in Nude Mice. The body weight of theinoculated mice (s.c. or i.v. injections) was not affected by CMDB7 atall of the doses tested after 10 weeks of treatment (data not shown).CMDB7 administrated i.v. or s.c. at 300 mg/kg produced no signs oftoxicity such as diarrhea, infection, weakness, and lethargy. All of the32 treated mice were alive at the end of 10 weeks.

Discussion

In this study, we assessed the antimetastatic activity of CMDB7against estrogen receptor-negative MDA-MB435 xenografted tumorsin nude mice. This model—based on tumor cell injection into m.f.p.followed by metastasis to the lung—mimics the clinical situation, inwhich tumors become estrogen receptor-negative and develop resist-ance to the antiestrogen tamoxifen after some duration of treatment.

Fig. 3. The inhibition of tumor angiogenesis and lung micrometastasis by CMDB7.Nude mice (n5 42) received MDA-MB435 xenografts in the m.f.p. and were treated withs.c. injections of CMDB7 at 50, 150, or 300 mg/kg/week or i.v. injections of 300mg/kg/week for 10 weeks. Tumor and lung metastases were assessed 12 weeks after cellinoculation, when all of the mice were killed and examined. The development of lungmicrometastasis is a function of primary tumor angiogenesis.F, mice with micrometas-tases;‚, mice without micrometastases.

Fig. 4. Immunohistochemical analysis of vascularization of xenografted human breast carcinoma in control and CMDB7-treated mice (3400). Tumor vascularization was analyzedimmunohistologically with the endothelial cell-specific marker GSL-1. Untreated tumors (A) and tumors treated with 300 mg/kg/week (i.v.) of CMDB7 (B). C, angiogenesis wasquantitated by image analysis of GSL-1-labeled endothelial cells, and the results are presented as the mean areas6 SE (bars)of endothelial cells in tumor section obtained from 10control mice and 8 mice in each treated group.p, significantly different from untreated controls (P , 0.001).

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This study confirmed that the injection of 106 viable MDA-MB435cells yielded a 100% tumor-take rate in the m.f.p. (9).

This study demonstrated a direct relationship between neovascu-larization within the primary tumors and the incidence of the percent-age distant micrometastases. The results showed that lung microme-tastases developed when the percentage area of endothelial cellsreached a threshold of 2%. The prevalence of metastases increased asthis area within the primary tumor increased. Our findings are con-sistent with previous studies (14–17) that demonstrated a direct cor-relation between blood vessel density in primary tumors and theirmetastasis dissemination. Tumor microvessels often had fragmentedbasal membranes, which suggested that these vessels were structurallyincomplete and leaky and, thus, that tumor cells could easily reach thecirculation (14, 18).

Untreated mice had highly vascularized tumors, and microscopiclung metastases were observed in all of them. In contrast, CMDB7-treated mice exhibited a very low incidence of lung metastases. Thislow metastatic rate could reflect to the inhibited intratumoral angio-genesis because, at 50 mg/kg/week, CMDB7 very significantlyblocked the growth of intratumoral vessels. CMDB7, when adminis-trated i.v. is also active and, moreover, is the most clinically applica-ble. CMDB7 as well as other polyanionic polysaccharides such asheparin could be active when given p.o. (19). Additional studies arenecessary to confirm this hypothesis.

It has been reported that MDA-MB435 cells produce angiogenicfactors, such as FGF-2 and vascular endothelial growth factor, that aredetectable in their culture surpernatants (20). Thus, CMDB7 couldexert its antiangiogenic action by disrupting the autocrine and para-crine effects of growth factors released by the tumor cells.

We also demonstrated that CMDB7 had strong antiangiogenic andantimetastatic effects without inhibiting the primary tumor growth ofxenografted MDA-MB435 tumors. This observation can be explainedby the fact that MDA-MB435 mammary tumors grow slowlyin vivo,and, thus, initial tumor growth may be independent of angiogenesiswhen the tumor size in# 3 mm3 (1). However, for rapidly growings.c. tumors, such as xenografted MCF-7ras (8) and FGF-4-trans-formed human breast cells (HH9; Ref. 7), CMDB7 inhibited tumorgrowth in parallel with less tumor angiogenesis.

The antiangiogenic activity of CMDB7 was further supported bythe results of experiments using the model of tumor-free angiogenesisFGF-2-enriched matrigel. Indeed, inclusion of CMDB7 in the matri-gel inhibited FGF-2-induced angiogenesis in a dose-dependent man-ner. These findings are in agreement with our earlier studies, whichshowed that CMDB7 inhibited thein vitro migration and proliferationof endothelial cells without acting on cell viability (7). Thus, CMDB7is cytostatic but not cytotoxic for endothelial cells. We reportedpreviously (6) that CMDB7 inhibited HBL100 cell proliferation byinterfering with the FGF-2 autocrine loop and that CMDB7 inhibitedthe paracrine mitogenic activity and receptor binding of FGF-2,FGF-4, transforming growth factorb1, and platelet-derived growthfactor BB (7, 8). Taken together, these new data confirmed thatCMDB7, by blocking the activities of angiogenic growth factors asdescribed in our previous reports, is a potent inhibitor of tumorangiogenesis and metastasisin vivo. We cannot exclude additionaltherapeutic effects of CMDB7 such as the inhibition of the number oftumor-associated macrophages infiltratingin vivo.

In conclusion, the data obtained with our model of human breastcancer highlighted: (a) the dependence of the incidence of distantmetastasis development on the intratumoral angiogenesis of the pri-mary tumors; and (b) the ability of CMDB7 to effectively inhibit

angiogenesis in these xenografted tumors and prevent their distantmetastasis to the lungs. Boehmet al. (21) demonstrated that a specificangiogenesis inhibitor (endostatin) did not induce drug resistance inthree different types of transplantable murine tumors. Thus, CMDB7and other antiangiogenic agents could be expected to be promising asnovel pharmaceutic agents in cancer therapy, especially when thepatients develop acquired drug resistance to cytotoxic drugs. CMDB7could prove beneficial in preventing the occurrence of secondarymetastases and in inducing tumor dormancy through its cytostaticaction on endothelial cells.

Acknowledgments

We thank Dr. Michel Kraemer for critical reading of the article.

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1999;59:507-510. Cancer Res   Rozita Bagheri-Yarmand, Yamina Kourbali, Ana Maria Rath, et al.   Lung Metastases in Nude MiceMDA-MB435 Breast Carcinoma Xenografted in Fat Pad and Its Carboxymethyl Benzylamide Dextran Blocks Angiogenesis of

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