[CANCER RESEARCH 58. 4468-4474. October I. 1998]

Selective Localization of Matrix Metalloproteinase 9, ß,Integrins, and Human

Lymphocyte Antigen Class I Molecules on Membrane Vesicles Shed by8701-BC Breast Carcinoma Cells1

Vincenza Dolo, Angela Ginestra, Donata Cassare, Stefania Violini, Giuseppe Lucania, Maria Rosaria Torrisi,Hideaki Nagase, Silvana Canevari, Antonio Pavan, and Maria Letizia Vittorelli2

Dipartimento di Medicina Sperimentale, Universitti ili L'Aquila, 67100 L'Aquila, Italy /V. D., S. V., A. P.j; Dipartimento di Biologia Cellulare e dello Sviluppo, Università diPalermo, WI28 Palermo, ltal\ ¡A.C., D. C., M. L V.¡;Dipartimene di Medicina Sperimentale e Patologia, Università "La Sapienza," 00161 Rome, Italy ¡G.L.. M. K. T.];

Department of Biochemistry and Molecular Biology, University of Kansas, Medical Center, Kansas City, Kansas 66160 [H. N.j; Divisione di Oncologia Sperimentale E, IstitutoNazionale Tumori. 20133 Milan. ltal\ /S. C.¡:and Centro di Oncohiologia Sperimentale. 90128 Palermo. ltal\ ¡M.L. V.}

ABSTRACT

The shedding of membrane vesicles from the cell surface is a vitalprocess considered to be involved in cell-cell and cell-matrix interactions

and in tumor progression. By immunoelectron microscopic analysis ofsurface replicas of 8701-BC human breast carcinoma cells, we observed

that membrane vesicles shed from plasma membranes contained denselyclustered gelatinase B [matrix metalloproteinase 9 (MMP-9)], ß,inte-

grins, and human lymphocyte antigen class I molecules. By contrast,«-folate receptor was uniformly distributed on the smooth cell membrane

and shedding areas. Both cell surface clustering of selected molecules andmembrane vesicle release were evident only when cells were cultured inthe presence of serum. Vesicle shedding occurred preferentially at theedge or along narrow protrusions of the cell. Specific accumulation ofproMMP-9 and active forms of MMP-9 in shed vesicles was also demon

strated by gelatin zymography. In addition. Western blotting analysisshowed the presence of a large amount of proMMP-9/tissue inhibitor of

metalloproleinase 1 complex. The release of selected areas of plasmamembranes enriched with MMP-9 and ß,integrins indicates that mem

brane vesicle shedding from tumor cells plays an important role in thedirectional protcolysis of the extracellular matrix during cellular migration. The presence of human lymphocyte antigen class I antigens suggestsa mechanism for tumor cells to escape from immune surveillance.

INTRODUCTION

The shedding of membrane vesicles into the extracellular space isa widespread phenomenon described in normal cells and more frequently in (umoral cells (1,2). It is an energy-requiring phenomenonfor which the synthesis of RNAs and proteins is essential (3). Char-

acteri/.ation of the shed materials indicates that there is no evidence ofsoluble cytosolic proteins or subcellular organeile markers within thevesicle (4). The shed extracellular vesicles are suggested to promotethe invasive activity of tumor cells. Early electron microscopic studiesof invasive murine skin and mammary carcinomas in vivo showed thedestruction of connective tissues by membrane vesicles in the immediate vicinity of the invading epithelial cells (5, 6). Poste and Nicolson(7) reported that fusion of the membrane vesicles shed from a highlymetastatic variant of murine melanoma cells with a poorly metastaticclone increased the invasive potential of the latter. Several proteinaseshave been detected in shed vesicles. Mouse melanoma cells in culturerelease a metalloproteinase activity (MT 59,000) as a component of

Received 4/30/98: accepted 7/30/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked (itlvrrlisenii'iil in accordance with

18 U.S.C. Section 1734 solely to indicate this fact.1Supported by grants from the Italian Association for Cancer Research and the Italian

Ministry of University and Scientific and Technological Research (to M. L. V.. A. P.. andM. R. T.). by NIH Grant AR39IK9 (to H. N.). and by a travel fellowship of the Gruppo diCoopera/ione in Immunologia (to V. D.). D. C. was supported by a scholarship from theItalian Association for Cancer Research.

2 To whom requests for reprints should be addressed, at Dipartimento di BiologiaCellulare e dello Sviluppo, Viale delle Scienze, 90128 Palermo, Italy. Phone: 39-91-425081; Fax: 39-91-420897: E-mail: mlvitKs'mbox.unipa.it.

membrane vesicles (8), and human coloréela!carcinoma cells shedvesicles containing collagenolytic activity (9). In our previous study(10), we demonstrated that vesicles shed by 8701-BC human breastcarcinoma cells are rich in gelatinase B (MMP-9)3 and its zymogen.

More recently, we have shown that vesicles shed by HT-1080, ahighly invasive human fibrosarcoma cell line, carry MMP-9. gelatinase A (MMP-2), and the receptor-bound uPA (11).

Membrane vesicles were also suggested to play a relevant role inhelping the tumor escape from immune responses (12). It was shownthat membrane vesicles shed by murine melanoma cells inhibited theexpression of a MHC class I molecule in macrophages (13); weobserved that vesicles shed by breast carcinoma cells inhibited theproliferation of phytohemagglutinin-stimulated peripheral blood lym

phocytes, and that their inhibitory effect was completely abrogated bythe addition of antitransforming growth factor ßantibodies (14).These observations suggest that shedding vesicles and their contentinto the extracellular milieu influences cellular behavior, possibly byaltering cell-cell and cell-matrix interactions.

Although shed vesicles are derived from the plasma membrane, theyhave different compositions of lipids (15) and proteins (16) than those ofthe plasma membrane. Using immunoprecipitation and dot-blot analyses,

we have shown that membrane vesicles from two breast carcinoma celllines, MCF-7 and 8701-BC. carry most antigens expressed on the cellsurface (ß,,a,, and as integrin chains, tumor-associated antigens. HLA

class I molecules, and so forth), but specific localization of these antigenson the vesicle was not demonstrated. (17).

In this work, we show that vesicle shedding is a release of selectedareas of plasma membrane where specific molecules involved in cellmatrix interactions and degradation (ß,integrins and MMP-9) or

responsible for antigen presentation (HLA class I molecule) are clustered.

MATERIALS AND METHODS

Reagents and Antibodies. Gelatin-Sepharose 4B was from PharmaciaFine Chemicals (Uppsala, Sweden). Human proMMP-9, proMMP-2, TIMP-1.and TIMP-2 were purified from the culture medium of HT-1080 cells and

uterine ftbroblasts (18, 19). and specific polyclonal antibodies to these proteinswere obtained as described previously ( 18-20). Monoclonal antibodies againstMMP-9. MMP-2. and TIMP-1 were obtained from Oncogene Science (Cambridge. MA). W6/32 antibodies (anti-HLA class I hybridoma) were obtained

from the American Type Culture Collection (Rockville, MD). Monoclonalantibodies [MOV 18 against human folate receptor (21) and MAR4 againsthuman ßtintegrin (22)] as well as the antibody from W6/32 were purifiedaccording to their isotype by affinity chromatography on protein A or G(Pharmacia Fine Chemicals).

Cell Culture. The 8701-BC cell line, which is derived from a ductal

infiltrating human breast carcinoma, was kindly provided by Prof. S. Minafra(University of Palermo, Palermo, Italy: Ref. 23). Unless otherwise specified.

' The abbreviations used are: MMP. matrix metalloproteinase: T1MP. tissue inhibitor

of metalloproteinasc: uPA. urokinase-type plasminogen activator: HLA. human lymphocyte antigen; ECM, extracellular matrix: TSA. tumor-specific antigen.

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Fig. 1. Electron microscopic analysis of surfacereplicas of breast cancer 8701-BC cells. Cells werecultured in serum-free medium (a and <•)or in 10%

PCS (b and d) for 2 h. Conspicuous vesicle sheddingwas observed when cells were cultured in PCS, butnot when cells were cultured in serum-free medium.

Bar, 0.1 urn.

cells were cultured in tissue culture flasks (Falcon: Becton Dickinson) in RPMI1640 (Sigma) with 10% PCS (Celbio Hyclone) after filtration through 0.22-fj,m-pore membranes to avoid vesicle contamination of serum sedimentingmaterial; cells at 22-26 passages were used. The cells were negative for

Mycoplasma contamination, as routinely tested by Hoechst 33258 (Sigma)staining.

Immunolabeling. Cells grown on 4-mm glass coverslips to semiconflu-

ence were washed three times in PBS (pH 7.4), fixed with \% glutaraldehydein the same buffer, and incubated with the first antibody for l h at 4°C.After

washing extensively, cells were labeled with colloidal gold conjugated withprotein A (Pharmacia Fine Chemicals) for 2 h at 4°C.Cells were washed three

times with PBS and subjected to surface replication.Surface Replication. Monolayers of 8701-BC cells on coverslips were

dehydrated in a series of ethanol washes and air-dried. Platinum-carbon replicas were obtained in a Balzers BAF 300 freeze-etching apparatus (Balzers

AG, Liechtenstein. Liechtenstein) by shadowing the cell surface with platinum-carbon evaporation. The replicas were cleaned overnight in household

bleach, washed extensively in distilled water, and examined using a transmission Electron Microscopy (EM 420 Philips Electron Instruments, Eindhoven,the Netherlands).

Isolation of Membrane Vesicles from the Cell-conditioned Medium.Vesicles were prepared from the 8701-BC cell-conditioned medium asdescribed previously (10). Briefly, after a 3-h culture with subconfluent

cells, in the presence of 10% PCS unless otherwise stated, the conditionedmedia were harvested and centrifuged at 500 X g for 10 min and 800 X gfor 15 min. The supernatant was then centrifuged at 100,000 X g for l h at4°C,and the vesicle pellets were resuspended in PBS. Protein concentra

tions were measured by the method of Bradford (Ref. 24; Bio-Rad), using

BSA (Sigma) as a standard.Preparation of Plasma Membranes. Cell plasma membranes were iso

lated using nitrogen cavitation as described previously (8). Briefly, cells werescraped and resuspended in 0.15 M NaCl, 20 ITIMHEPES. 5 mM CaCl2, 20mg/ml leupeptin, 2.5 mg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride and exposed to nitrogen cavitation. After differential centrifugation,membranes were pelleted at 100,000 X g. Protein concentration was measuredby the method of Bradford (24).

Gelatin-Sepharose Chromatography of FCS. A 10-ml aliquot of PCSwas incubated with 1 ml of gelatin-Sepharose equilibrated with 50 mM Tris-

HC1, 150 mM NaCl, 5 mM CaCU, 0.02% Tween 20, and 10 mM EDTAovernight at 4°Cto adsorb the gelatinases present in FCS. The supernatant was

recovered by centrifugation and then filtered through a 0.22-/j.m-pore membrane. Gelatin-Sepharose filtered serum is referred as GS/FCS.

Gelatin Zymography. Zymography was performed as described previously (10). Briefly, samples were subjected to electrophoresis in SDS- 7.5%

polyacrylamide gels impregnated with I mg/ml gelatin type B (Sigma); afterelectrophoresis, gels were washed in 2.5% Triton X-IOO. incubated overnightat 37°Cin the assay buffer for gelatinases [50 mM Tris-HCl (pH 7.5), 10 mM

CaCI2, and 150 mM NaCl] and stained in Coomassie Blue R 250 (Sigma).Gelatinase activities were visualized as clear zones in a blue background,indicating the proteolysis of the substrate.

Reverse Zymography for TIMPs. TIMPs present in plasma membranesand membrane vesicles were analyzed by reverse Zymography. Gels wereprocessed as described for regular gelatin Zymography except for the incorporation of 30% (v/v) serum-free culture medium of HT-1080 into the sepa

rating gel. Gels were incubated overnight in the assay buffer for gelatinases.Dark-staining bands were verified as inhibitors for MMP activity.

Western Blotting. Vesicle proteins (20 fig) were electrophoresed in aSDS-PAGE (7.5% acrylamide) under nonreducing conditions and transferred

onto a nitrocellulose membrane (Hybond; Amersham). The membrane wasfirst incubated with 5% horse serum and 0.1% Tween 20 in PBS for 2 h andthen incubated with the indicated antibody ( 1:200) for l h and with horseradishperoxidase-conjugated anti-IgG antibody (1:7500; Sigma) for l h at roomtemperature. Immunoreactive bands were visualized by the enhanced chemi-

luminescence Western blotting kit (Amersham) using Hyperfilms.

RESULTS

Demonstration of Vesicle Shedding from 8701-BC Cells bySurface Replica. The surface replica technique provides images oflarge areas of the cell surface. Using this technique, we were able toacquire images showing the membrane shedding activity of 8701-BC

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a v

Fig. 2. Immunoclectron microscopic analysis of surface replicas of breast cancer 8701-BC cells. Cells

cultured in GS/FCS were fixed with \7t glularaldehydeand incubated with the following antibodies: a. none: b.anli-HLA class 1 molecule: <•,anti-a-folale receptor;and i/. ,11111/; ¡ntcgrin.Colloidal gold-conjugated pro

tein A was added to all samples. Bar. 0.1 firn.

cells (Fig. 1). As we reported previously, the shedding of membranevesicles from 8701-BC cells was greatly promoted by the addition of

PCS (10, 25). Shedding activity was negligible when cells werecultured in serum-free media (Fig. 1, a and c). Maximal sheddingactivity was observed 2-3 h after the addition of PCS to the medium

(Fig. 1, b and d). The release of membrane vesicles occurred primarilyat the edge or along narrow protrusions of the cell surface (Fig. id).

Uneven Distribution of HLA Class I Molecules and of ß,Integrins on the Vesicle and the Cell Membrane. We reported thatmembrane vesicles shed by 8701-BC cells were rich in integrins, HLA

class I antigens, and most of the plasma membrane antigens, but ourprevious methods did not demonstrate the distribution of these antigens between plasma membranes and vesicle membranes (17). Toanalyze the movement and distribution of membrane antigens duringvesicle shedding, we used the immunolabeled surface replica technique, which enabled us to evaluate the distribution of cell membranecomponents and the vesicle shedding event simultaneously. As shownin Fig. 2, 2 h after the addition of fresh serum-containing medium to

the cell, antibodies against HLA class I molecules and ß,integrinswere found to be more densely packed on vesicles compared to thebulk of the cell membrane. In Fig. 2b, a vesicle densely labeled by

anti-HLA antibodies appears in the center of the field above the plane

of the cell surface. The clustering of the ß,integrin subunit onvesicles shed from cell protrusions is shown in Fig. 2d. On thecontrary, folate receptor was evenly distributed on the cell surface andthe vesicles (Fig. 2c). No packing phenomena of the tested antigens onspecific cell surface areas were observed when cells were cultured inserum-free medium (data not shown).

Clustering of MMP-9 and TIMP-1 on Vesicle Membranes. Asreported previously (10), vesicles shed in the serum-containing medium by 8701-BC and MCF-7 breast carcinoma cells carried a largeamount of MMP-9 that was easily detected by gelatin zymography.We therefore analyzed the location of MMP-9 using surface replicasof the cell surface decorated with anti-MMP-9 antibody/colloidalgold-conjugated protein A. To establish whether MMP-9 bound to

vesicle membranes was produced by the tumor cell or derived fromserum, cells were cultured in either normal PCS or the gelatinase-deprived serum (GF/FGS). MMP-9 was readily detected using this

technique by either polyclonal or monoclonal antibodies, and it wasfound to be clustered on the vesicle membranes regardless of whetherthe cells were cultured in complete PCS (data not shown) or inGS/FCS (Fig. 3, a and b). This indicates that MMP-9 localized on the

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Fig. 3. Immunolocalization of MMP-9 andTIMP-1 on surface replicas of breast cancer8701-BC cells. Cells cultured in GS/FCS werefixed with 1% glutaraldehyde and incubated withthe following antibodies: a and b, anti-MMP-9; c.anti-MMP-2; and d, anti-TIMP-1. Colloidal gold-conjugated protein A was added to all samples. Bar,0.1 ¡aa.

surface of vesicles derived from the tumor cells. MMP-9 was not

detected on the smooth area of plasma membranes.The presence and localization of MMP-2, TIMP-1, and TIMP-2

were analyzed with the same technique. No labeling was observedwith 8701-BC plasma membranes and vesicle membranes when anti-TIMP-2 polyclonal antibody and anti-MMP-2 monoclonal antibody(Fig. 3c) or polyclonal antibodies were used. The absence of MMP-2in 8701-BC vesicles agrees with the results of Western blotting (seeFig. 5). Anti-TIMP-1 antibodies gave a moderate positive reaction,

and they were found to be clustered on vesicle membranes (Fig. 3d).Clustering of MMP-9 and TIMP-1 on specific areas of the cell

surface was not observed when cells were cultured in serum-free

medium (data not shown).Gelatinases and TIMPs in Isolated Vesicles and Plasma Mem

branes. To quantify the amount of gelatinolytic activities associatedwith the vesicles and the plasma membranes, the 8701-BC cells weregrown in gelatinase-free GS-FCS, and vesicles released in the medium

and the plasma membranes of the cultured cells were prepared. Anequal amount of proteins from each preparation was than subjected togelatin zymography. As shown in Fig. 4a, only faint gelatinolytic

activities were detected in the plasma membranes, but the purifiedvesicles exhibited a large amount of gelatinolytic activities around Mr140,000, M, 97,000, Mt 67,000, and M, 64,000. These activities areprimarily due to proMMP-9 and the active form of MMP-9 (see

below). Similar results were obtained with cells cultured in the medium containing complete FCS.

Free TIMP-1 was also detected by reverse zymography. A higheramount of TIMP-1 was found in vesicles than in the plasma mem

brane preparation (Fig. 4b).Presence of the proMMP-9/TIMP-l Complex, proMMP-9, and

the Active Form of MMP-9 in Membrane Vesicles. To bettercharacterize the gelatinolytic enzymes present in the membrane vesicles, we performed Western blotting analyses with antibodies againstMMP-9 and MMP-2 and their inhibitors, TIMP-1 and TIMP-2. Polyclonal antibody against MMP-9 showed a prominent band at about A/r

140,000 and a less pronounced band at M, 67,000 (Fig. 5a. Lime 1).A band at Mr 97,000 was recognized only when a higher proteinconcentration was used (data not shown). On the contrary, bothpolyclonal and monoclonal antibodies against MMP-2 failed to recognize any band in 8701-BC vesicles (Fig. 5b, Lane I). These anti-

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a

205 -

116 -

97 -

66 -

Fig. 4. Zymographic analyses of plasma membrane- and vesicle-associated gelatinasesand TIMPs. Plasma membranes and vesicles released from 8701-BC cells were preparedas described in "Materials and Methods." a. gelatin zymography. Lane I. vesicle proteins;

Lane 2. plasma membrane proteins. Samples (30 Mg °f proteins/lane I were separatedusing a 7.5% polyacrylamide gel with SDS. The positions of molecular mass markers areindicated, b. reverse zymography for TIMPs. Lane 1. vesicle proteins: Lane 2, plasmamembrane proteins. Samples (30 /ig of proteins/lane) were separated using a 14%polyaerylamide gel with SDS. The positions of molecular mass markers are indicated.Htitttim [Hint'l. densitometric analysis.

a

180-

116 -

97 -

66 -

II180-

116-

97 -

66 -

45 -

Fig. 5. Immunological identification of MMP-9 in vesicles shed by 8701-BC cells, a.Western blotting analyses of vesicles shed by 8701-BC cells (Lane /) and HT-1080 cells(iMtie 2) with antibodies against proMMP-9. b. Western blotting analyses of vesicles shedby 8701-BC cells (Lane I) and HT-1080 cells (Lane 2) with antibodies againstproMMP-2. Samples (20 fig of proteins/lane) were separated by a 7.5% polyacrylamidegel with SDS. The positions of molecular mass markers are indicated.

bodies, however, identified proMMP-2 in vesicles from HT-1080

cells (Fig. 5h, Lune 2).Although the gelatinase activity detected around M, 67,000 in

8701-BC membrane vesicles (Fig. 4) seems to correspond toproMMP-2 from its electrophoretic migration, it reacted with anti-MMP-9 antibody (Fig. 5«,Lane I) and was not recognized by anti-MMP-2 antibodies (Fig. 5b, Lime I). Furthermore, as reported previ

ously (10), the electrophoretic mobility of the Air 67,000 gelatinasedid not shift after treatment with 4-aminophenylmercuric acetate. Wetherefore conclude that it is an active form of MMP-9 ( 18, 26). The M,

97.000 band observed on gelatin zymography was identified asproMMP-9. As reported previously (10), the electrophoretic mobilities of this band before and after 4-aminophenylmercuric acetatetreatment corresponded to those of purified proMMP-9, and the bandwas recognized by anti-MMP-9 antibodies in Western blotting per

formed at a high concentration of proteins. The Mr 140,000 banddetected on the vesicle by zymography (Fig. 4) and by Westernblotting with anti-MMP-9 antibody (Fig. 5a) is considered to be acomplex of proMMP-9/TIMP-l. Whereas antibodies against TIMP-2

did not show any reactivity (data not shown), Western blotting withanti-TIMP-1 antibodies demonstrated the strongest staining around Mr140,000 (Fig. 60). The association of TIMP-1 with proMMP-9 in a

complex running at an apparent molecular mass of 140 kDa was alsoconfirmed by reverse zymography (Fig. 6b, Lane 1). When the sameamount of vesicle proteins was separated by SDS-PAGE and stained

with Coomassie Blue, no protein staining was observed in the areawhere inhibitory activity was detected by reverse zymography (Fig.6b, Lane 2).

DISCUSSION

In this report, we have shown that the nonrandomized distributionof HLA class I molecules, ß,integrin subunit, proMMP-9, andTIMP-1 occurs on the plasma membrane of breast carcinoma8701-BC cells when vesicle shedding takes place. Both vesicle shed

ding and clustering of selected molecules are greatly induced by theaddition of FCS. Molecular mechanisms and specific serum components that induce this effect have been partially investigated (25) butat present are incompletely understood. As in the case of virusbudding during the propagation of viral infection (27), vesicle shedding is more evident along the microvilli and along free margins of thecell surface. Such events probably have specific biological roles in thecell surrounded by ECM.

a

1 2

180-

116-

97 -

66 -

29 -

I

I

180-

*

116 -

97 -

66 -

Fig. 6. Detection of MMP-9/TIMP-I complexes in vesicles shed by 8701-BC cells, a.Western blotting analyses of TIMP-I. Lane I. purified T1MP-I: Lane 2, vesicles, b.reverse zymography for TIMPs (Ltine 1} and Coomassie Blue staining (Lime 2) ofmembrane vesicles. Samples (20 /xg of proteins/lane) were separated by a 7.5% polyacrylamide gel with SDS. The positions of molecular mass markers are indicated. Arrows,the M, 140.000 band corresponding to the proMMP-9/TIMP-l complex.

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The degradation of the ECM requires the concerted actions of anumber of extracellular proteinases. The increased expression andactivity of serine and metalloproteinases have been shown to becorrelated with the metastatic behavior of tumor cells. In particular,MMP-2 and MMP-9 are thought to be involved in the invasive

phenotype of tumor cells (28, 29). Our finding that vesicle membranesare the only areas of cell membranes where MMP-9 is localized

suggests that vesicle formation and shedding is associated with theprogression of tumor cell metastasis.

The catabolism of ECM is regulated not only by the amount ofspecific proteolytic enzymes but also by their location. The binding ofuPA to uPA receptor accelerates plasminogen activation on the cellsurface. This localizes the enzyme to focal contact sites (30-32). An

increasing body of experimental evidence has been accumulated thatdemonstrates that MMP-2 is localized and modulated on specificregions of the cell surface (33-36). Brooks et al. (37) reported thatMMP-2 binds to the integrin avß3.ProMMP-2 bound to the cellsurface is activated by membrane-bound MT1-MMP (38-40). Forms ofMMP-9 with different molecular weights have been found to be associ

ated with the cell surface (41), Olson et al. (42) have recently shown thatproMMP-9 binds with high affinity to the cell surface of human breast

epithelial cells, and the mechanism of this association seems to bemediated by the presence of the a2 (IV) chain of collagen IV.

Vesicles are also enriched with the j3, integrin subunit. Becauseintegrins are the major class of receptors for ECM macromolecules,packing of integrins on the membrane of shed vesicles indicates thatthese vesicles can readily bind to ECM components, thus allowingproteolytic enzymes to act focally on their substrates. Integrins arealso signaling molecules that regulate cell proliferation, differentiation, and survival as well as cell adhesion (43-45). The shedding of

cell surface integrins in response to extracellular signals may thereforemodify cellular behavior. Additional investigations are needed toestablish which integrin dimers are clustered on membrane vesiclesand identify their possible interaction with MMP-9.

It has been suggested that membrane vesicles play a role in thetumoral escape from immune surveillance. Vesicle shedding isthought to be a mechanism for tumor cells to remove antigens thatcould stimulate an immune response against tumors. Indeed, sometumor-associated antigens are present on vesicle membranes (12, 46,

17). However, most TSAs identified thus far are cytoplasmic ornuclear proteins that can be recognized only by HLA class I-restricted

CTLs (47). The present study, which demonstrates that the majorrestriction element for T-cell cytotoxicity is clustered in the vesicles,

further supports this hypothesis. Moreover, vesicles shed from breasttumor cells inhibit lymphocyte proliferation (14). Therefore, in vivo,subsets of lymphocytes may adhere to membrane vesicles releasedfrom tumor cells by recognizing TSAs. Such an interaction may resultin the inhibition of proliferation of anti-TSA lymphocytes.

It is notable that the molecule that was not specifically sorted invesicles but was randomly distributed on the cell surface, i.e., a-folatereceptor, is anchored on the cell surface by a glycosylphosphatidyli-

nositol tail. This type of molecule is generally localized in membranemicrodomains rich in sfingolipids and cholesterol (48). Additionalstudies analyzing other glycosylphosphatidylinositol-linked proteins

are necessary to evaluate the relevance of this observation.We recently reported that membrane vesicles from HT-1080

fibrosarcoma cells contain MMP-9 and MMP-2 in both the active

and pro forms, and the receptor bound uPA (11). In this study, wewere unable to detect vesicle-bound MMP-2 and uPA (data notshown) on 8701-BC breast carcinoma vesicles. These findings,together with the previous observation that 8701-BC and MCF-7vesicles, but not HT-1080 vesicles, inhibited lymphocyte prolifer

ation (14), indicate that the properties of shed vesicles vary ac

cording to the cell origin. However, we also observed that theamounts and the lytic content of shed vesicles correlate with thediffering in vitro invasiveness of cells (49).

In conclusion, our studies suggest that the shedding of membranevesicles is an active phenomenon with specific pathophysiologicalimplications such as directional proteolysis during cell migration andtumor invasion and the cellular escape from immune surveillance.Further understanding of the molecular mechanisms by which migrating cells localize metalloproteinases and other selected molecules onthe cell surface and subsequently shed them as membrane vesiclesmay suggest strategies to control tumoral progression and invasion.

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1998;58:4468-4474. Cancer Res   Vincenza Dolo, Angela Ginestra, Donata Cassarà, et al.   Membrane Vesicles Shed by 8701-BC Breast Carcinoma Cells

onIntegrins, and Human Lymphocyte Antigen Class I Molecules 1βSelective Localization of Matrix Metalloproteinase 9,

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