[CANCER RESEARCH 59, 3812–3820, August 1, 1999] …PETA-3 appears not to be involved in cell attachment because adhesion did not correlate with levels of PETA-3 expression and was

[CANCER RESEARCH 59, 3812–3820, August 1, 1999]

Eukaryotic Expression Cloning with an Antimetastatic Monoclonal AntibodyIdentifies a Tetraspanin (PETA-3/CD151) as an Effector of Human TumorCell Migration and Metastasis1

Jacqueline E. Testa,2,3 Peter C. Brooks,4 Jian-Min Lin, 5 and James P. Quigley3

Department of Pathology, State University of New York at Stony Brook, Stony Brook, New York 11794

ABSTRACT

A monoclonal antibody (mAb), 50-6, generated by subtractive immuniza-tion, was found to specifically inhibit in vivo metastasis of a human epider-moid carcinoma cell line, HEp-3. The cDNA of the cognate antigen of mAb50-6 was isolated by a modified eukaryotic expression cloning protocol froma HEp-3 library. Sequence analysis identified the antigen as PETA-3/CD151,a recently described member of the tetraspanin family of proteins. The clonedantigen was also recognized by a previously described antimetastatic anti-body, mAb 1A5. Inhibition of HEp-3 metastasis by the mAbs could not beattributed to any effect of the antibodies on tumor cell growth in vitro or invivo. Rather, the antibodies appeared to inhibit an early step in the formationof metastatic foci. In a chemotaxis assay, HEp-3 migration was blocked byboth antibodies. HeLa cells transfected with and overexpressing PETA-3/CD151 were more migratory than control transfectants expressing littleCD151. The increase in HeLa migration was inhibitable by both mAb 50-6and mAb 1A5. PETA-3 appears not to be involved in cell attachment becauseadhesion did not correlate with levels of PETA-3 expression and was unaf-fected by mAb 50-6 or mAb 1A5. The ability of PETA-3 to mediate cellmigration suggests a mechanism by which this protein may influence metas-tasis. These data identify PETA-3/CD151 as the first member of the tet-raspanin family to be linked as a positive effector of metastasis.

INTRODUCTION

Metastasis is a complex, multistep cascade of cellular events includingmigration of tumor cells through the surrounding stroma, entry into thecirculatory system, and finally arrest, extravasation, and growth at adistant secondary site (reviewed in Refs. 1–6). Given the complexity ofthe metastatic process, it is not surprising that a number of proteins havebeen associated with tumor cell dissemination including transcriptionfactors, signaling proteins, adhesion molecules, proteases, motility fac-tors, and others (1–6). Although nuclear, cytoplasmic, and secretedproteins have been associated with metastatic potential, tumor cell dis-semination is executed via the physical interactions of the cancer cellsurface with various host tissue elements. Not only is the cell membranethe interface at which cell-cell and cell-substrate contacts are made, it isthe portal through which external signals must pass. Activation of cellsurface receptors, by mutation or by ligand binding, initiates intracellularsignaling cascades that influence expression of genes that promote themalignant phenotype (2, 3, 6). Proteases expressed on or bound to the cellmembrane mediate degradation of tissue barriers. Integrins mediate tu-mor cell motility and transmit environmental cues by virtue of their

interactions with different matrix proteins. Our efforts to identify novelmetastasis-associated antigens have, therefore, focused on the tumor cellsurface. As our model system, we have used a highly metastatic humanepidermoid carcinoma cell line, HEp-3, which disseminates to host lungtissue. The distinct characteristics and behavior patterns of this tumor cellline have been described by Ossowski in a series of papers publishedbetween 1980 and 1998 (7–9). These cells are very aggressive and readilygive rise to metastasizing tumors in both the chicken embryo (10–12) andin the nude mouse model (13, 14). As such, these cells should possessdistinct surface antigens that are functionally involved in mediating tumorcell dissemination.

Brookset al.(11) generated several mAbs6 against HEp-3 cell surfaceproteins using an approach termed subtractive immunization. Their pro-tocol allowed them to produce mAbs with no preconceived notion as tothe identity or function of the targeted antigen. Two of the antibodies,DM12-4 and 1A5, inhibited spontaneous HEp-3 metastasis in the chickenembryo metastasis assay by 86 and 90%, respectively. Neither antibodyaffected primary tumor growth on the chorioallantoic membrane or tumorcell growth in vitro, indicating that the mAbs specifically blocked met-astatic behavior. The identification of the antigens recognized by themAbs was not reported or was unknown.

In the present study, another monoclonal antibody generated bysubtractive immunization, mAb 50-6, was used to clone and charac-terize a cell surface, metastasis-associated antigen expressed onHEp-3 cells. This antibody inhibits both spontaneous and experimen-tal HEp-3 metastasis. Eukaryotic expression cloning of the antigenidentifies it as PETA-3/CD151, a member of the tetraspanin family ofproteins. We show that PETA-3/CD151 appears to be required at anearly step in the formation of metastatic foci. Furthermore, this proteinmediates tumor cell migration but does not appear to affect celladhesion to various purified matrix proteins. The work describedherein identifies PETA-3/CD151 as the first member of the tet-raspanin family to be linked as a positive effector of metastasis.

MATERIALS AND METHODS

Cell Lines and Hybridomas. Human breast adenocarcinoma cells (MDA-MB-231), human cervical carcinoma (HeLa), human fibrosarcoma (HT1080),and monkey kidney cells (COS-7) were obtained from the American TypeCulture Collection (Rockville, MD). Metastatic human epidermoid carcinomacells (HEp-3) were obtained from solid tumors serially passaged on the CAMsof chicken embryos (10, 11). All cells were maintained as monolayer culturesin DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with10% FBS (HyClone, Logan, UT), sodium pyruvate, penicillin/streptomycin,and nonessential amino acids (Life Technologies, Inc.; growth medium).Cultures were grown in a humidified atmosphere of 5% CO2 at 37°C.

Hybridomas producing mAb 50-6 and mAb 1A5 were generated by sub-tractive immunization (11). Cultures of each hybridoma were maintained inone part DMEM, one part Hybridoma SFM (Life Technologies, Inc.), supple-mented with 2.5% alpha calf serum (HyClone), sodium pyruvate, penicillin/

Received 1/14/99; accepted 6/2/99.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 Grants RO1 CA60800 (to J. E. T.) and RO1 CA65660(to J. P. Q) from the National Cancer Institute at the NIH.

2 To whom requests for reprints should be addressed, at Department of VascularBiology, VB-1, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla,CA 92037. Phone: (619) 784-7188; Fax: (619) 784-7323; E-mail: [email protected].

3 Present address: Department of Vascular Biology, VB-1, The Scripps ResearchInstitute, 10550 North Torrey Pines Road, La Jolla, CA 92037.

4 Present address: Department of Biochemistry and Molecular Biology, Norris CancerCenter, Topping Tower, Room 5409, 1441 Eastlake Avenue, University of SouthernCalifornia, Los Angeles, CA 90033.

5 Present address: Matrix Pharmaceuticals, Inc., 34700 Campus Drive, Fremont, CA94555.

6 The abbreviations used are: mAb, monoclonal antibody; CAM, chorioallantoicmembrane; FBS, fetal bovine serum; FN, fibronectin; LN, laminin; TM4SF, transmem-brane 4 superfamily; VN, vitronectin.

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streptomycin, and nonessential amino acids (Life Technologies, Inc.). Cultureswere grown in spinner flasks in a humidified atmosphere of 5% CO2 at 37°C.

mAb Purification. Conditioned media from the hybridoma cultures werecentrifuged at 50003 g for 20 min and then pumped over a column ofGammaBind Plus Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ).The columns were washed with 10 column volumes of PBS, and the mAbswere eluted with 0.1M glycine (pH 3.0). Purified mAbs were dialyzed againstPBS, filter sterilized, then aliquoted and stored at220°C.

Effect of mAb 50-6 on HEp-3 Growth in Vitro. HEp-3 cells were platedinto six-well culture plates (2.03 105 cells/well) in the presence of 50mg/mlof mAb 50-6 or normal mouse IgG. At 24, 48, and 72 h, the cells in two wellsfrom each culture condition were trypsinized and counted.

Inhibition of HEp-3 Metastasis in the Chicken Embryo Assay. Antibodyinhibition of HEp-3 spontaneous metastasis in the chicken embryo was conductedas described previously (11). Briefly, tumor cells were inoculated through awindow in the eggshell onto the surface of CAMs of 10-day-old chicken embryos(SPAFAS, Preston, CT). The window was sealed, and the embryos were returnedto the incubator. Twenty-four h later, a second window was carefully cut in theeggshell directly over a prominent blood vessel. The underlying, nonliving shell

membrane was made transparent with a drop of paraffin oil, and 200mg of purifiedmAb or normal mouse IgG (Sigma Chemical Co., St. Louis, MO) in 0.1 ml PBSwere inoculated into the blood vessel with a 30-gauge needle. The window wassealed, and after an additional 6 days of incubation, the eggs were opened; theprimary tumors were excised, trimmed of CAM tissue, and weighed as a measureof tumorigenicity. The lungs of the embryos were removed, finely minced, andpassaged onto the CAMs of a second set of 10-day-old embryos. These embryoswere incubated for an additional 7 days to allow any HEp-3 cells in the lungs tomultiply. The “lung tumors” arising from the transferred lungs were then excisedand finely minced, and the presence of HEp-3 was determined biochemically byquantitating human urokinase-type plasminogen activator activity present in de-tergent extracts of the lung tumors (8–11).

Antibody inhibition of HEp-3 experimental metastasis was determined bycoinoculating 0.1 ml of PBS containing tumor cells (2.03 104) and 200mg ofpurified mAb or normal mouse IgG (Sigma) directly into a prominent bloodvessel (prepared as described above). For the time course study of inhibition ofHEp-3 experimental metastasis, the antibodies were inoculated at differenttimes before or after inoculation of the tumor cells, as indicated. The inocu-lated embryos were incubated for an additional 6 days, after which the lungswere excised, finely minced, and transferred to prepared CAMs of a second setof embryos. The assay was then completed as described above.

Eukaryotic Expression Cloning. A custom-made, unidirectional cDNA li-brary was constructed in the eukaryotic expression vector pcDNA I (Invitrogen,San Diego, CA) using poly(A)1 RNA isolated from metastatic HEp-3 cells.Eukaryotic expression cloning in COS monkey kidney cells was conducted asdescribed previously (15) with some modifications. The first two rounds oftransfection and immunoselection were performed as described except that COScells were transfected using the calcium phosphate method (16). Plasmids recov-ered at the end of the second round were used to transfect COS cells growing ontissue culture plates. Twenty-four h later, the transfected cells were detached fromthe plates with nonenzymatic cell dissociation solution (Sigma) and plated ontopolycarbonate membranes (90 mm diameter, 0.4m pore size; Millipore, Bedford,MA). After an additional 24 h, the cells (attached to the membranes) were washedthree times with PBS, fixed with 0.25% glutaraldehyde in PBS for 5 min at roomtemperature, washed, quenched with 1.0M glycine (pH 8.0) for two h at roomtemperature, washed again, and then incubated with 10% normal goat serum inPBS (blocking solution) for 1 h atroom temperature. The membranes were thenincubated with mAb 50-6 (1mg/ml in blocking solution) overnight at 4°C withgentle agitation. As a control, one membrane was incubated with an isotype-matched control antibody (IgG1; Sigma; 1mg/ml in blocking solution). Theprimary antibody was removed by washing the membranes 33 10 min in PBS,and the membranes were then incubated with biotin-conjugated goat anti-mouseIgG (Southern Biotechnology Associates, Birmingham, AL; diluted 1:500 inblocking solution) for 1.5 h at room temperature with gentle agitation. Themembranes were washed and incubated with horseradish peroxidase-conjugatedstreptavidin (Southern Biotechnology Associates; diluted 1:500 in blocking solu-tion) for 45 min at room temperature with gentle agitation. After three washes inPBS, the membranes were developed with chloronaphthol to identify immunopo-sitive COS transfectants. No color reaction was seen on cells incubated withcontrol IgG1. With the aid of a dissecting microscope and a flame-drawn glassmicrocapillary pipette,;30 strongly immunopositive cells were detached from themembranes and transferred to a microcentrifuge tube. Episomal plasmid DNA wasrecovered from these cells as described previously (17) and used to transformbacteria (MC1061/p3; Invitrogen, San Diego, CA) by electroporation. The result-ing colonies were pooled, grown in liquid culture, and plasmid DNA was isolatedwith a Qiagen Plasmid kit (Qiagen, Chatsworth, CA). Plasmid DNA was frac-tionated by resolving 1mg on an agarose gel. The lane was cut into six segments,and DNA was isolated from each with a Gene Clean kit (Bio 101, La Jolla, CA)and used to electroporate MC1061/p3 (Invitrogen). Plasmids were isolated fromcultures of bacterial cells transformed with DNA from each of the six gel slices andthe fraction containing cDNA clones which directed the synthesis of a cell surfaceantigen recognized by mAb 50-6 was identified by transfecting and immuno-staining COS cells (growing on poly-L-lysine-coated coverslips) as describedabove. DNA from one positive gel fraction was used to transform bacteria, andmini-prep DNA from 10 individual colonies was used to transfect COS cells forimmunocytochemical analysis. Immunopositive cells were detected in 1 of the 10transfected cultures. The positive clone contained an insert of;1.5 kb, as deter-mined by restriction endonuclease digestion. The insert was sequenced with the T7Sequenase Quick-Denature plasmid sequencing kit (version 2.0; Amersham Phar-

Fig. 1. mAb 50-6 recognizes aMr 29,000 cell surface antigen. Live, unfixed HEp-3cells were immunostained with normal mouse IgG (a, NM) or mAb 50-6 (b) and analyzedby flow cytometry.Horizontal axis,fluorescence intensity;vertical axis,numbers of cellsanalyzed. HEp-3 lysates were also analyzed by Western blotting (c). Proteins (10mg)were resolved by SDS-PAGE, transferred to nitrocellulose, then incubated with mAb 50-6or normal mouse IgG. The signal was visualized with a peroxidase-conjugated goatanti-mouse IgG by chemiluminescence.Left, molecular weight standards (in thousands).

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PETA-3/CD151 IS AN EFFECTOR OF MIGRATION AND METASTASIS



macia Biotech, Arlington Heights, IL). The nucleotide sequence was comparedwith the National Center for Biotechnology Information database.

Transfection of HeLa Cells with the Cloned cDNA. HeLa cells werecotransfected with the cloned PETA-3 cDNA and pSV2neo using the calciumphosphate method (16). Controls were cotransfected with vector (pcDNA I)containing no insert and pSV2neo. Forty-eight h later, Geneticin (G418; LifeTechnologies, Inc.) was added at a concentration of 400mg/ml of medium, and thecultures were incubated for an additional 12 days. The resulting G418-resistantcolonies were detached from the culture plates with nonenzymatic cell dissociationsolution (Sigma), pooled, washed three times with serum-free DMEM, thenblocked with 10% normal goat serum in PBS (blocking solution), on ice, for 30min. The cells were then incubated with 1mg/ml mAb 50-6 or an isotype-matchedcontrol (IgG1; Sigma) for 1 h on ice, washed three times with blocking solution,then incubated with a phycoerythrin-conjugated goat anti-mouse IgG (SouthernBiotechnology Associates; diluted 1:500 in blocking solution) for 30 min on ice inthe dark. The labeled cells were washed three times in PBS, and overexpressingPETA-3 transfectants were isolated by a fluorescence activated cell sorter. Controltransfectants were also selected with a fluorescence-activated cell sorter. The cellscollected were and then subcloned by limiting dilution. Approximately 50 sub-clones isolated from each of the two populations of cells were expanded in culture,then rescreened by whole-cell ELISA to verify the levels of PETA-3 expression.In vitro growth rates of the HeLa transfectants were determined by plating 23 104

cells into each of two wells in a 24-well plate. At 48-, 72-, and 96-h time points,the cells were trypsinized and counted. Results are reported as numbers ofcells/well.

Whole-Cell ELISA. The levels of cell-surface PETA-3 expression weremeasured by whole-cell ELISA. Subconfluent cultures HEp-3 or HeLa cells weredetached from the culture plates, washed three times in serum-free DMEM, thenresuspended in growth medium. Cells (2.03 104/0.1 ml) were added to each well

in a 96-well culture plate and cultured for 36 h. The cells were then washed threetimes with PBS, fixed with 0.25% glutaraldehyde in PBS for 5 min at roomtemperature, washed again, then quenched with 1.0M glycine (pH 8.0) for 2 h atroom temperature. The plates were washed three times with PBS and usedimmediately or stored in 0.1% sodium azide in PBS at 4°C. Stored plates werewashed three times with PBS prior to use to remove the sodium azide.

For the assay, wells were incubated with 0.2 ml of blocking solution overnightat 4°C. Purified mAb or isotype-matched control antibody (0.1 ml, 1mg/ml inblocking solution) was added to the appropriate wells and incubated for 2 h atroom temperature. The plates were washed three times with PBS, and thenhorseradish peroxidase-conjugated goat anti-mouse IgG (Southern BiotechnologyAssociates) was added to the wells (0.1 ml, 1mg/ml in blocking solution) andincubated for 2 h at room temperature. The plates were washed, and 0.1 ml of thesubstrate,o-phenylene diamine (0.34 mg/ml, 0.1M sodium citrate, pH 4.5, 0.012%H2O2) was added. After a 10-min incubation at 37°C, the plates were read at 405nm using a Titer Tek Multiscan plate reader. The nonspecific signal from theisotype-matched control was subtracted from the experimental wells. Cell surfacelevels of thea3b1 integrin on HEp-3, HeLa, MDA-MB-231, and HT1080 cellswere measured in the same manner, using mAb 1992 (Chemicon, Temecula, CA),which specifically recognizes the heterodimer.

Migration (Chemotaxis) Assay. HEp-3 cells were detached from cultureplates with Versene (Life Technologies, Inc.), washed twice with serum-freeDMEM (Life Technologies, Inc.), and resuspended in AIM-V medium (LifeTechnologies, Inc.). Cells (1.43 104) were added to the upper reservoir ofBioCoat control chambers (uncoated; 8mm pore size; Becton DickinsonLabware, Bedford, MA) in AIM-V medium, and 50mg/ml of mAb 50-6, mAb1A5, or normal mouse IgG (Sigma). The lower reservoirs contained DMEMsupplemented with 10% FBS (HyClone) and 50mg/ml of the appropriateantibody. After 6 or 12 h of incubation, the microporous inserts were fixedwith 10% neutral buffered formalin and stained with hematoxylin. Cells on theupper surfaces of the membranes were removed with cotton swabs, and themembranes were excised and mounted on microscope slides in Permount.Cells on the underside of one quadrant of each filter were counted. Experi-ments were conducted in triplicate. Chemotaxis assays with the HeLa trans-fectants were similarly conducted, except that 2.03 104 cells were added tothe upper reservoirs, and the experiments were terminated after 18 h.

Western Blot Analysis. Cells were lysed in Triton X-100 lysis buffer[0.5% Triton X-100, 0.1M Tris (pH 8.0), 5 mM EDTA, 10mM E64, 20 units/mlaprotinin, and 20mg/ml soybean trypsin inhibitor (all from Sigma)] on ice for10 min with vortexing at 5-min intervals. Insoluble material was removed bycentrifugation at 12,0003 g for 5 min at 4°C. The protein concentration of thecleared lysates was measured with the bicinchoninic acid system (PierceChemical Co., Rockford, IL). Proteins were resolved on 10% SDS-PAGE gelsand then transferred to nitrocellulose. The blots were blocked with a solutionof 5% nonfat milk, 5% FBS, and 0.1% Tween 20 in PBS (Western blockingsolution) for 1 h at room temperature. The blots were then incubated witheither mAb 50-6, mAb 1A5, or normal mouse IgG (1mg/ml in Westernblocking solution) overnight at 4°C. The blots were washed three times for 5min with 0.1% Tween 20 in PBS and then incubated with horseradish perox-idase-conjugated goat anti-mouse IgG (diluted 1:2500 in Western blockingsolution) for 2 h atroom temperature. The blots were then washed three times

Fig. 2. Effect of mAb 50-6 on HEp-3 growthin vitro. HEp-3 cells were plated intosix-well culture plates (2.03 105 cells/well) in the presence of 50mg/ml of mAb 50-6 ornormal mouse IgG (NM). At 24, 48, and 72 h, the cells in two wells from each culturecondition were trypsinized and counted. Data are reported as numbers of cells/well.

Table 1 Inhibition of HEp-3 metastasis by mAb 50-6

Inhibition of spontaneous metastasis was determined by Brookset al. (11), inhibition of experimental metastasis was determined by i.v. inoculating 11-day-old chicken embryoswith 0.1 ml of PBS containing mAb 50-6 (200mg) and HEp-3 cells (23 104). The inoculated embryos were incubated for an additional 7 days, at which time the embryonic lungswere removed, finely minced, and transferred to the CAMs of a second set of embryos, which were incubated for an additional 7 days. The extent of lung metastasis in both assayswas measured by determining the amount of human urokinase-type plasminogen activator activity, which has been shown to be a quantitative reflection of the numbers of human cellsin the lung (8–10). Data are the combined results of two separate spontaneous assays and two separate experimental metastasis assays.

Antibody No. of embryosPrimary tumor weighta

(mean6 SEM)Lung metastasis (huPA in lung)

(mean6 SEM) % inhibition

Spontaneous metastasisNormal mouse IgG 11 274.1 (651.2) 1015.8 (6249.4)b 0mAb 50-6 13 253.9 (646.7) 261.2 (6117.2)b 74.30%

Experimental metastasisNormal mouse IgG 11 2169.3 (6601.8)c 0mAb 50-6 10 937.8 (6299.9)c 56.80%

a P 5 0.689.b P 5 0.022.c P 5 0.044.

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for 10 min with 0.1% Tween 20 in PBS, and the signals were visualized withthe ECL system (Amersham Pharmacia Biotech, Arlington Heights, IL) ac-cording to the manufacturer’s directions.

Immunoprecipitation. HEp-3 cells were in lysed in Brij lysis buffer [1% Brij98, 25 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 10mM E64, 20 units/mlaprotinin, and 20mg/ml soybean trypsin inhibitor (all from Sigma)] for 1 h withconstant rocking at 4°C (18). Insoluble material was removed by centrifugation at12,0003 g for 5 min at 4°C. The lysate was precleared with GammaBind PlusSepharose beads. Aliquots of the extract representing 107 cells were incubatedwith either 25mg of mAb 1A5, 10mg of mAb 1992 (Chemicon, Temecula, CA),which specifically recognizes thea3b1 integrin heterodimer, or 25mg of normalmouse IgG (Sigma). Fiftyml of packed GammaBind Plus Sepharose beads wereadded to each sample, and the mixtures were incubated at 4°C overnight withconstant rocking. The beads were then washed with the Brij lysis buffer, and theimmune complexes were eluted with Laemmli sample buffer at 95°C for 2 min.The eluted proteins were resolved on a 10% SDS-PAGE gel, transferred tonitrocellulose. Because biotinylation of both mAb 1A5 and mAb 50-6 destroystheir immunoreactivity, PETA-3 was detected by incubating the blots with unla-beled mAb 1A5, followed by horseradish peroxidase-conjugated goat anti-mouseIgG as described above.

Cell Attachment to Purified Matrix Proteins. For the cell attachmentassays, 96-well plates precoated with purified FN were purchased from BectonDickinson Labware (Bedford, MA). Purified VN (Becton Dickinson Labware,Bedford, MA) was used to coat 96-well plates as described previously (19).Cells were detached from culture plates with nonenzymatic cell dissociationsolution (Sigma) and washed three times in PBS containing 0.1% heat dena-tured (60°C for 30 min) BSA. The cells were then resuspended to a concen-

tration of 2 3 105 cells/ml in PBS/0.1% BSA containing 50mg/ml of mAb50-6, mAb 1A5, or normal mouse IgG (Sigma). After a 5-min incubation atroom temperature, 0.1 ml of the cell suspension was added to the appropriatewell and incubated for 15 and 30 min at 37°C. At the end of the incubationperiod, unattached cells were removed by gently washing the plates three timeswith PBS. Adherent cells were fixed with 0.25% glutaraldehyde for 1 h atroom temperature, followed by three washes in PBS. Cell adhesion wasquantitated adding 50ml of crystal violet (0.1% in H2O) to each well. After 10min at room temperature, the plates were washed in PBS, and the dyeincorporated by the attached cells was released by adding 0.1 ml of 10% aceticacid, then quantitated by spectrophotometry in a Titer Tek Multiscan platereader at 595 nm.

RESULTS

mAb 50-6 Recognizes aMr 29,000 Cell Surface Antigen.MAb50-6 was generated by subtractive immunization (11) using intactHEp-3 cells as the immunogen. Localization of the antigen to the cellsurface of HEp-3 cells was determined by whole-cell ELISA (notshown) and flow cytometry (Fig. 1). When compared with cellsincubated with normal mouse IgG (Fig. 1a), the fluorescence intensitysignal from live, nonpermeablized HEp-3 cells immunostained withmAb 50-6 (Fig. 1b) is significantly higher. The molecular weight ofthis cell surface protein was determined by Western blot analysis. Asseen in Fig. 1c, mAb 50-6 recognizes a single broad protein bandhaving an apparent molecular weight ofMr 29,000.

Fig. 3. Immunocytochemical localization of PETA-3on HEP-3 cells and COS transfectants. Cells were platedonto poly-lysine-coated glass coverslips, fixed 24 hlater, and then immunostained with mAb 50-6 or normalmouse IgG. Signals were detected with horseradish per-oxidase-conjugated goat anti-mouse IgG and chlo-ronaphthol. All images were photographed with trans-mitted light.a, HEp-3 cells incubated with mAb 50-6.b,HEp-3 cells incubated with normal mouse IgG.c, COScells transiently transfected with PETA-3 cDNA andincubated with mAb 50-6.d, COS cells transiently trans-fected with PETA-3 cDNA and incubated with normalmouse IgG.e, COS transiently transfected with vector(pcDNA I) alone and incubated with mAb 50-6.Bars,20 mm.

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mAb 50-6 Inhibits Spontaneous and Experimental Metastasisof HEp-3 Cells. To determine whether theMr 29,000 cell surfaceprotein was involved in HEp-3 dissemination, the effect of mAb 50-6 onspontaneous metastasis was measured using anin vivomodel, the chickenembryo metastasis assay (11, 20). In the spontaneous metastasis assays,purified antibody (mAb 50-6 or normal mouse IgG) was inoculated i.v.24 h after metastatic HEp-3 cells were implanted onto prepared CAMs,and the extent of tumor cell dissemination to the lungs was measured (see“Materials and Methods”). As shown in Table 1, spontaneous metastasiswas reduced by 74% in embryos that received mAb 50-6. The effect ofmAb 50-6 on experimental metastasis also was tested by coinoculatingthe tumor cells with the antibody directly into the circulatory system. Inthese experiments, dissemination to the embryonic lungs was inhibited by57% when compared with controls. The antimetastatic properties of mAb50-6 cannot be attributed to a cytostatic or cytotoxic effect, because thisantibody had no effect on proliferation of HEp-3 cellsin vitro (Fig. 2), nordid it result in a decrease in the size of the primary tumor on the CAM(Table 1). These results indicate that mAb 50-6 specifically blocks astep(s) in the metastatic cascade.

Eukaryotic Expression Cloning Identifies theMr 29,000 Metas-tasis-associated Antigen as PETA-3/CD151.The antibody inhibitionstudies demonstrated a functional role for theMr 29,000 protein inmediating metastasis. To identify the protein, mAb 50-6 was used in aeukaryotic expression cloning strategy to isolate the cDNA from a full-

length HEp-3 cDNA library. COS cells were subjected to several roundsof transfection and immunoselection with mAb 50-6. In the final round ofthe cloning protocol, immunopositive cells showing a strong cell surfacesignal were selected for isolation of cDNA clones because these cellswould more likely harbor full-length inserts (see “Materials and Meth-ods”). Indeed, a clone that directed the synthesis of a cell surface antigenrecognized by mAb 50-6 was isolated and sequenced. The open readingframe of this clone encodes a core protein having a predicted molecularmass of 27.8 kDa. There are four distinct hydrophobic domains, and asingleN-glycosylation site in a large hydrophilic region that separates thethird and fourth hydrophobic domains. Comparison of the sequence ofthis clone with the National Center for Biotechnology Information data-base identified the metastasis-associated antigen as PETA-3/CD151, arecently described Member of the tetraspanin family of proteins, which isalso known as the TM4SF. The coding sequence was identical to thatreported by Fitteret al. (21). The cell surface distribution of PETA-3 onHEp-3 cells and COS cells transfected with the cloned cDNA wasanalyzed by immunocytochemistry. On HEp-3 cells incubated with mAb50-6, there was a strong staining pattern over the entire surface of the cellmembrane, including filopods extending from the cell body (Fig. 3a). Thesame cell surface staining pattern was seen on COS cells transientlytransfected with PETA-3 cDNA (Fig. 3c). No signal was observed onHEp-3 or PETA-3-transfected COS cells incubated with normal mouseIgG (Fig. 3,b andd) or on control COS transfectants incubated with mAb50-6 (Fig. 3e). Western blot analysis of the cell lysates from PETA-3-transfected COS cells also demonstrated that the encoded protein comi-grates with theMr 29,000 antigen expressed on HEp-3 cells (Fig. 4A).

Brookset al. (11) had described an antimetastatic mAb, mAb 1A5,which also recognizes aMr 29,000 HEp-3 cell surface antigen. Todetermine whether this mAb also recognizes PETA, mAb 1A5 wasused to probe Western blots of COS cells transiently transfected withthe PETA-3/CD151 cDNA (Fig. 4B). No signal was observed inlysates from control transfectants (Lane 2); however, mAb 1A5 rec-ognized PETA-3/CD151 expressed by the COS cells (Lane 3). AminorMr 25,000 band, immunostained with mAbs 50-6 and 1A5, wasalso evident in lysates of transfected COS cells (Fig. 4) and in HEp-3lysates (seen upon prolonged exposure of the Western blot; data notshown). This is likely the nonglycosylated form of the protein, be-cause cultivation of HEp-3 cells with tunicamycin or treatment ofHEp-3 cell lysates withN-glycanase generates aMr 25,000 band thatis immunoreactive with mAb 1A5 (data not shown). This is smallerthan the predicted mass of 27.8 kDa, and may be due to the hydro-phobic nature of the protein and/or the presence of disulfide bondsresulting from 15 cysteine residues. These results demonstrate thatboth mAb 50-6 and mAb 1A5 recognize PETA-3 and suggest that theepitope(s) is resident in the protein core.

PETA-3/CD151 Mediates an Early Event in the Formation ofMetastatic Foci. To examine the possible mechanisms by whichPETA-3/CD151 effects metastasis, a time course study of inhibitionof experimental metastasis was carried out. As shown in Table 2,

Fig. 4. PETA-3/CD151 is recognized by mAb 50-6 and mAb 1A5. COS-7 cells weretransiently transfected with the cloned PETA-3/CD151 cDNA or vector (pcDNA I) alone.Lysates of HEp-3 cells and the transfected COS cells were resolved by SDS-PAGE andanalyzed by Western blotting.a, Western blot of HEp-3 cell lysate (10mg; Lane 1) andlysates from control (COS/pcDNA I; 20mg; Lane 2) and PETA-3-transfectants (COS/PETA-3; 20mg; Lane 3) probed with mAb 50-6.b, Western blot of HEp-3 cell lysate (0.5mg; Lane 1) and lysates from control (COS/pcDNA I;Lane 2; 2.5mg) and PETA-3transfectants (COS/PETA-3;Lane 3; 2.5mg) probed with mAb 1A5. In both panels, thesignals were visualized with a peroxidase-conjugated goat anti-mouse IgG by chemilu-minescence.Arrow, PETA-3, Mr 29,000. AMr 25,000 signal is also seen in COS celllysates probed with both mAb 50-6 and mAb 1A5.Right,molecular weight markers.

Table 2 Time course of inhibition of HEp-3 experimental metastasis

Purified mAb 1A5 (180–200mg) was inoculated i.v. into 11-day-old chicken embryos either 2 h before or 2, 6, 10, or 20 h after inoculation of 23 104 HEp-3 cells. As a control,normal mouse IgG was inoculated 2 h before the HEp-3 cells. The inoculated embryos were incubated for an additional 6 days, at which time the embryonic lungs were removed, finelyminced, and transferred to the CAMs of a second set of embryos, which were incubated for an additional 7 days. The extent of lung metastasis was measured by determining the amountof human urokinase-type plasminogen activator activity, which has been shown to be a quantitative reflection of the numbers of human cells in the lung (8–10).

Antibody Time of antibody inoculation No. of embryosLung metastasis (huPA in lung)

mean6 SEM % inhibition

Normal mouse IgG 22 h 47 15226 54 0mAb 1A5 22 h 36 2956 60 81mAb 1A5 16 h 21 4016 36 74mAb 1A5 110 h 8 14716 36 3mAb 1A5 120 h 20 14086 67 7

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HEp-3 dissemination to the embryonic lungs was markedly inhibitedwhen mAb 1A5 was administered as early as 2 h before or as late as6 h after inoculation of the tumor cells. In contrast, there was littleeffect on HEp-3 colonization of the lungs when mAb 1A5 wasadministered 10 or 20 h after the tumor cells were inoculated. Theinability of mAb 1A5 to inhibit lung colonization at the latter timepoints (when tumor cells would likely have extravasated already)indicates that the antibody does not block metastasis by inhibiting cellgrowth at the secondary sites. This is consistent with our observationsthat the anti-PETA-3 mAbs have no effect on HEp-3 growthin vivoor in vitro (Table 1; Fig. 2). These experiments suggest that PETA-3/CD151 mediates HEp-3 dissemination by affecting an earlier event,such as cell adhesion to the vessel wall, extravasation, and/or tumorcell migration to selective sites of secondary growth.

PETA-3/CD151 Is Involved in Cell Migration in Vitro. Thetetraspanins have been described as molecular facilitators that influ-ence cell adhesion and migration, presumably through their interac-tions with integrins (reviewed in Refs. 22–24), and it is possible thatPETA-3/CD151 affects migration of HEp-3 cells. Therefore, the an-timetastatic mAbs were tested for their ability to inhibit HEp-3 mi-gration in a chemotaxis assay. Fig. 5 demonstrates that both mAb 50-6and mAb 1A5 inhibited HEp-3 migration by approximately 45 and44%, respectively, when compared with controls (P 5 0.034).

To further demonstrate the role of PETA-3/CD151 in tumor cellmigration, HeLa cells were transfected with the cloned cDNA or withvector alone. Two clones from each group were selected for testing ina chemotaxis assay on the basis of their PETA-3 expression levels(Fig. 6a), their similar rates of growthin vitro (Fig. 6b) and similarityin morphological appearance (not shown). The levels of cell surfacePETA-3 on the two overexpressing clones, PB6HI and PC3HI, wereabout 2-fold higher than the HEp-3 cells, whereas the control trans-fectants, NB11LO and NB17LO, expressed approximately one-quarterto one-half the amount detected on HEp-3 cells (Fig. 6a). The growthrates of the four clones were identical (Fig. 6b). Migration by theoverexpressing clones PB6HI and PC3HI was significantly higher thanthe underexpressing control transfectants NB11LO and NB17LO (Fig.6c; P 5 0.001). When mAb 50-6 or mAb 1A5 (50mg/ml) was addedto the chambers, migration of clone PB6HI was significantly reduced

Fig. 5. Inhibition of HEp-3 migration (chemotaxis) by mAb 50-6 and mAb 1A5. HEp-3cells were detached from culture plate with Versene (Life Technologies, Inc.), washed twicewith serum-free DMEM (Life Technologies, Inc.), and resuspended in AIM-V medium (LifeTechnologies, Inc.). Cells (1.43 104) were added to the upper reservoir of the migrationchambers in AIM-V medium and 50mg/ml of mAb 50-6, mAb 1A5, or normal mouse IgG.The lower reservoir contained DMEM supplemented with 10% FBS and 50mg/ml of theappropriate antibody. After 6 h of incubation, the microporous inserts were fixed with 10%neutral buffered formalin and stained with hematoxylin, and cells on the underside of onequadrant of each filter were counted. Experiments were conducted in triplicate;bars,SEM.

Fig. 6. Effects of PETA-3/CD151 expression on HeLa cell migration. HeLa cells werecotransfected with the cloned PETA-3/CD151 cDNA and pSV2neo. Controls were co-transfected with pcDNA I and pSV2neo. Two PETA-3 overexpressing clones (PB6HI andPC3HI) and two control clones (NB11LO and NB17LO) were selected.a, the amount of cellsurface PETA-3 expressed by the HeLa transfectants and HEp-3 cells was determined bywhole-cell ELISA on fixed, nonpermeabilized cells and is expressed as the absorbance(OD) of the chromogen at 405 nm (b). In vitro growth rates of the HeLa transfectants weredetermined by plating 23 104 cells into each of two wells in a 24-well plate. At the timesindicated, the cells were trypsinized and counted. Results are reported as number of cellsper well.c, inhibition of HeLa migration (chemotaxis) by mAb 50-6 and mAb 1A5. HeLacells were detached from culture plate with Versene (Life Technologies, Inc.), washedtwice with serum-free DMEM (Life Technologies, Inc.), and resuspended in AIM-Vmedium (Life Technologies, Inc.). Cells (2.03 104) were added to the upper reservoir ofthe migration chambers in AIM-V medium and 50mg/ml of mAb 50-6, mAb 1A5, ornormal mouse (NM) IgG. The lower reservoirs contained DMEM supplemented with 10%FBS (HyClone) and 50mg/ml of the appropriate antibody. After 18 h of incubation, themicroporous inserts were fixed and stained, and the cells on the underside of one quadrantof each filter were counted. Experiments were conducted in triplicate;bars,SEM.

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(P 5 0.034) by 51.6 and 52.8%, respectively, whereas migration ofclone PC3HI was significantly reduced (P5 0.007) by 36.7 and50.6%, respectively, lowering the motility of these cells to the level ofcontrol NB11LO cells. Neither mAb 50-6 nor mAb 1A5 inhibitedmigration of the weakly expressing clones NB11LO (P 5 0.729) orNB17LO (P 5 0.337), indicating that migration by these cells ismediated by a PETA-3-independent mechanism.

PETA-3/CD151 has been shown to associate with thea3b1 integrinin several cell types (25, 26). Using a monoclonal antibody, mAb1992, which specifically recognizes thea3b1 heterodimer, we havedetected, by whole-cell ELISA, thea3b1 integrin on HEp-3 cells (Fig.7) at levels equivalent to known positive controls, MDA-MB-231, ahuman breast carcinoma cell line, and HT1080, a human fibrosarcomacell line (18). A physical interaction betweena3b1 and PETA-3 inHEp-3 cells was demonstrated by the ability of mAb 1992 to coim-munoprecipitate these two molecules from HEp-3 cell lysates. West-ern blot analysis of immunoprecipitates obtained with mAb 1992shows aMr 29,000 protein that immunostains with mAb 1A5 (Fig. 8,Lane 3). This protein comigrates with the authentic antigen present inthe HEp-3 lysates (Fig. 8,Lane 1) and in immunoprecipitates obtainedwith mAb 1A5 (Fig. 8,Lane 2). No PETA-3 signal was observed incontrol normal mouse IgG “immunoprecipitates” probed with mAb1A5 (Fig. 8, Lane 4) or normal mouse IgG (Fig. 8,Lane 5). The

background bands observed in the immunoprecipitated samples aredue to the reactivity of the secondary antibody to mouse IgG proteinsand IgG fragments as seen in the control lanes.

When the PETA-3-transfected HeLa clones were analyzed bywhole-cell ELISA, there was no detectablea3b1 signal. Weak signalswere seen with the control HeLa transfectant (Fig. 7). This suggeststhat PETA-3 may interact with a different integrin in the transfectedHeLa cells to effect the observed increase in cell migration (Fig. 6c).

PETA-3/CD151 Appears Not to Be Associated with Cell Adhe-sion. Because cell adhesion is a prerequisite to cell migration, it ispossible that the inhibitory effects of mAbs 50-6 and 1A5 in thechemotaxis assays are related to antibody inhibition of cell attach-ment. Therefore, to quantitate the role of PETA-3 in cell attachment,HEp-3 cells and the HeLa transfectants were tested for adhesion tovarious purified matrix proteins in the presence of mAb 50-6, mAb1A5, or normal mouse IgG. As shown in Fig. 9, at the 30-min timepoint, HEp-3 attachment to both FN (Fig. 9a) and VN (Fig. 9b) wasunaffected by the presence of either of the antimetastatic mAbs.Likewise, attachment of the overexpressing and weakly expressing

Fig. 7. Whole-cell ELISA of expression of thea3b1 integrin heterodimer by equalnumbers (23 104) of MDA-MB-231 human breast carcinoma cells, HT1080 humanfibrosarcoma cells, HEp-3 cells, and the PETA-3 overexpressing HeLa clones (PB6HI andPC3HI) and control HeLa clones (NB11LO and NB17LO). mAb 1992 was used tospecifically identify the heterodimer. Data represent the amount of chromogen released inthe assay and are expressed as the absorbance (OD) at 405 nm.

Fig. 8. PETA-3 expressed by HEp-3 cells associates with thea3b1 integrin heterodimer.HEp-3 cell lysates were incubated with mAb 1A5 (Lane 2), mAb 1992 (specific for thea3b1heterodimer;Lane 3), or normal mouse IgG (NM; Lane 4). Immune complexes were collectedon GammaBind Plus Sepharose, resolved by SDS-PAGE, and transferred to nitrocellulose.The blot was incubated with unlabeled mAb 1A5 as a primary antibody, followed byhorseradish peroxidase-conjugated goat anti-mouse IgG. Signals were visualized by chemi-luminescence. As a control, a NM “immunoprecipitate” was probed with NM IgG andperoxidase-conjugated goat anti-mouse IgG (Lane 5). A sample of the original HEp-3 lysateis shown inLane 1. p, PETA-3 signal.Left, molecular weight markers (in thousands).

Fig. 9. Effects of mAb 50-6, mAb 1A5, and PETA-3 expression on cell adhesion to variousmatrix proteins. HEp-3 cells, PETA-3-overexpressing HeLa clones (PB6HI and PC3HI) andcontrol HeLa clones (NB11LO and NB17LO) were added to the wells of 96-well plates coatedwith either fibronectin (a) or vitronectin (b) in the presence of 50mg/ml mAb 50-6, mAb 1A5,or normal mouse IgG and incubated for 30 min. Nonadherent cells were gently washed away,and the amount of cell adhesion was determined by crystal violet staining. Data are expressedas the absorbance (OD) at 595 nm of the incorporated crystal violet dye and are representativeof two separate experiments conducted in triplicate;bars,SEM.

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HeLa transfectants to FN or VN was not blocked by mAb 50-6 ormAb 1A5. There was also no correlation between levels of PETA-3expression and cell attachment. Similar results were obtained whenthe adhesion assays were terminated after 15 min (not shown) andwhen cell attachment was tested on plates coated with laminin, type Icollagen, and type IV collagen (not shown). These data suggest thatPETA-3 is not functionally involved in cell attachment.

DISCUSSION

The metastatic process has been correlated with expression of awide variety of cellular proteins, including adhesion molecules,growth factors, motility factors, proteases, transcription factors, andsignaling molecules. However, a correlative association does notnecessarily imply that a protein is functionally relevant to tumor cellmetastasis. In the present study, mAb 50-6 was used to immunolog-ically target a cell surface antigen that is mechanistically involved intumor cell dissemination. This mAb was generated by subtractiveimmunization against a highly metastatic human epidermoid carci-noma cell line, HEp-3, without bias as to the nature or identity of theantigen. The ability of mAb 50-6 to block metastasis specifically(Table 1) strongly suggests that its cognate antigen is functionallyinvolved in the metastatic process. Using mAb 50-6 in a modifiedeukaryotic expression cloning protocol, the full-length cDNA encod-ing theMr 29,000 metastasis-associated HEp-3 antigen was isolated,sequenced, and identified as PETA-3/CD 151 (21), also known asSFA-1 (27). We also demonstrated that PETA-3 is recognized by apreviously described metastasis-inhibiting antibody, mAb 1A5 (Fig.4), also generated by subtractive immunization (11), but the cognateantigen of which was unknown. The unbiased selection of two inde-pendently generated anti-PETA-3 mAbs that block metastasis indi-cates that the subtractive immunization approach may be a powerfultool to identify functionally important antigens.

PETA-3 is a TM4SF protein. Also known as tetraspanins, membersof this protein family are characterized by having four hydrophobic,transmembrane domains, two short cytoplasmic tails, and one smalland one large extracellular loop (reviewed in Refs. 22–24). PETA-3 isexpressed by a variety of cell types including the basal cells of theepidermis, epithelial cells, skeletal, smooth and cardiac muscle,Schwann cells, platelets, and endothelial cells (28). Ya´ñez-Mó et al.(25) have shown that PETA-3 expressed by cultured endothelial cellsis localized at cell-cell junctions. However, our immunocytochemicalanalysis of fixed, nonpermeabilized HEp-3 cells show that PETA-3 isdistributed over the entire cell surface (Fig. 3a). Furthermore, thisexpression pattern was not restricted to HEp-3 cells because immu-nostaining over the entire cell surface was also observed on COS-7transfectants expressing PETA-3 (Fig. 3b). Whether there is a func-tional relevance associated with the different patterns of PETA-3distribution remains to be determined.

Several TM4SF proteins have been implicated as regulators of cellproliferation. Cell proliferation can be either stimulated (29–31) orslowed (32) in the presence of anti-TM4SF antibodies. Transfection withseveral tetraspanin family members also retards growth (32–35). Ouranti-PETA-3 mAbs 50-6 and 1A5 were shown not to affectin vitrogrowth of HEp-3 cells (Fig. 2; Ref. 11) norin vivogrowth in the primarytumor (Table 1; Ref. 11) or at the secondary site (Table 2). In addition, thein vitro growth rates of HeLa transfectants overexpressing PETA-3 are nodifferent than control clones (Fig. 6b). These results indicate that PETA-3does not affect tumor cell proliferation but functions specifically in one ormore steps in the metastatic process itself.

Metastatic success by tumor cells has been shown to be dependenton initial arrest in the secondary organ (36), as well as events thatoccur after extravasation, such as migration through the stroma to sites

of preferred secondary tumor growth (37–39). Our studies on the timecourse of inhibition of HEp-3 experimental metastasis (Table 2)suggest that PETA-3 is involved in an early step in the formation ofmetastatic foci, such as arrest, extravasation, and/or migration into theconnective tissue stroma of the secondary organ. Although PETA-3 isknown to be expressed on endothelial cells (25, 28), neither mAb 50-6nor mAb 1A5 react with the endothelium of the chicken embryo, thehost in the present metastatic model.7 Thus, the antimetastatic effectof mAb 50-6 and mAb 1A5 is the result of antibody binding to theHEp-3 cells themselves and not to host endothelial cells.

Tumor invasion of tissue elements is one hallmark of the malignantphenotype and is dependent on the ability of tumor cells to transientlyadhere to various matrix proteins and to migrate into the surroundingstroma. TM4SF proteins are known to associate with other tetraspanins,integrins, and potential signaling molecules and are believed to facilitatethe formation and stabilization of these macromolecular complexes andthus influence a number of cellular functions including migration andadhesion (Refs. 25, 33, and 40–44; reviewed in Refs. 22–24). In thepresent report, several experiments demonstrate a positive role forPETA-3 in mediating cell migration: (a) we were able to inhibit HEp-3chemotaxis with mAb 50-6 and mAb 1A5 (Fig. 5); (b) we showed thatHeLa cells transfected with and overexpressing PETA-3 were moremigratory than control transfectants (Fig. 6c); and (c) the increase inmotility by PETA-3-transfected HeLa clones was inhibitable by bothmAb 50-6 and mAb 1A5 (Fig. 6c). The results of our antibody inhibitionstudies are consistent with recent observations that random migration ofendothelial cells (25) and polymorphonuclear chemotaxis (26) are sensi-tive to inhibition by anti-PETA-3 mAbs. The mechanism by whichPETA-3 influences migration of these cells is apparently related to theassociation of this tetraspanin with thea3b1 integrin (25, 26) and poten-tial signaling molecules (26). In the present study, we have demonstrated,by coimmunoprecipitation, a physical association between PETA-3 anda3b1 in HEp-3 cells (Fig. 8). However, there is little or no detectablea3b1 expressed on our HeLa transfectants (Fig. 7). TM4SF proteins caninteract with several different integrin molecules, primarily those in theb1 class (22–24), and it is possible that PETA-3 expressed by the HeLacells associates with another integrin to effect the observed increase inmotility.

The effects of PETA-3 on migration of HEp-3 and HeLa cells appearnot to be related to changes in cell adhesion. We found no correlationbetween levels of cell surface PETA-3 expression and adhesion to wellscoated with theb1 substrates FN (Fig. 9a), LN, collagen type I, orcollagen type IV (not shown) or theb5 substrate VN (Fig. 9b). Inaddition, there was no significant difference in adhesion when cells wereplated onto these same substrates in the presence of mAb 50-6 or mAb1A5 (Fig. 9 and data not shown). Ya´ñez-Mó et al. (25) found thatendothelial cell adhesion to FN, LN, and collagen type I increasedslightly but significantly in the presence of their anti-PETA-3 mAbs. Thereason for the differences between the results of this latter study and ourobservations remains to be determined.

Several members of the tetraspanin family of proteins have beenassociated with the metastatic phenotype, but these associations havebeen, for the most part, negative. KAI-1/CD82 expression suppressedexperimental metastasis of rat prostate tumor cells (45), decreased mo-tility and invasion of colon carcinoma cells (44), and decreased invasionand metastasis of mouse melanoma cells (46). Likewise, experimentalmetastasis of mouse melanoma was reduced in cells expressing motility-related protein (MRP)-1/CD9 (33). In addition, (over)expression of CD9slowed growth and blocked migration of CHO cells, and human lungadenocarcinoma and myeloma cells (33). CD63 expression also resulted

7 Quigley, unpublished results.

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in decreasedin vivo growth of human melanoma cells (34) and NIH3T3cells (35) and blocked experimental metastasis of human melanoma cells(34). Claaset al. (32) recently cloned the rat homologue of CO-029, atetraspanin that appears to affect metastasis by altering the homing patternof tumor cells. Transfection of BSp73AS cells, a weakly metastatic ratpancreatic adenocarcinoma cell line, with the homologue shifted themetastatic burden from the lymph nodes to the lungs and resulted in anincreased survival rate of animals inoculated with the transfectants. Amonoclonal antibody to the CO-029 homologue partially reduced aconsumptive coagulopathy associated with expression of this protein;however, the effect of the antibody on metastatic dissemination was notreported (32). In contrast to the reports cited above, we have, in thepresent study, exploited the techniques of subtractive immunization andeukaryotic expression cloning to detect, clone, and identify PETA-3/CD151 as a metastasis-associated antigen that appears to contributepositively to the metastatic phenotype. PETA-3 does not affect tumor cellproliferation but rather appears to be specifically involved in an early stepin the formation of secondary metastatic lesions. The ability of PETA-3to mediate tumor cell migration provides a possible mechanism for therole of this protein in effecting metastatic dissemination. Our studiesidentify PETA-3 as the first member of the tetraspanin family of proteinsto be linked as a positive effector of metastasis.

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1999;59:3812-3820. Cancer Res Jacqueline E. Testa, Peter C. Brooks, Jian-Min Lin, et al. as an Effector of Human TumorCell Migration and MetastasisMonoclonal Antibody Identifies a Tetraspanin (PETA-3/CD151) Eukaryotic Expression Cloning with an Antimetastatic

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[CANCER RESEARCH 59, 3812–3820, August 1, 1999] …PETA-3 appears not to be involved in cell attachment because adhesion did not correlate with levels of PETA-3 expression and was