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Tumor Biology |
Department of Pathology, State University of New York at Stony Brook, Stony Brook, New York 11794
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Brooks et al. (11) generated several mAbs6 against HEp-3 cell surface proteins using an approach termed subtractive immunization. Their protocol allowed them to produce mAbs with no preconceived notion as to the identity or function of the targeted antigen. Two of the antibodies, DM12-4 and 1A5, inhibited spontaneous HEp-3 metastasis in the chicken embryo metastasis assay by 86 and 90%, respectively. Neither antibody affected primary tumor growth on the chorioallantoic membrane or tumor cell growth in vitro, indicating that the mAbs specifically blocked metastatic behavior. The identification of the antigens recognized by the mAbs was not reported or was unknown.
In the present study, another monoclonal antibody generated by subtractive immunization, mAb 50-6, was used to clone and characterize a cell surface, metastasis-associated antigen expressed on HEp-3 cells. This antibody inhibits both spontaneous and experimental HEp-3 metastasis. Eukaryotic expression cloning of the antigen identifies it as PETA-3/CD151, a member of the tetraspanin family of proteins. We show that PETA-3/CD151 appears to be required at an early step in the formation of metastatic foci. Furthermore, this protein mediates tumor cell migration but does not appear to affect cell adhesion to various purified matrix proteins. The work described herein identifies PETA-3/CD151 as the first member of the tetraspanin family to be linked as a positive effector of metastasis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hybridomas producing mAb 50-6 and mAb 1A5 were generated by subtractive immunization (11) . Cultures of each hybridoma were maintained in one part DMEM, one part Hybridoma SFM (Life Technologies, Inc.), supplemented with 2.5% alpha calf serum (HyClone), sodium pyruvate, penicillin/streptomycin, and nonessential amino acids (Life Technologies, Inc.). Cultures were grown in spinner flasks in a humidified atmosphere of 5% CO2 at 37°C.
mAb Purification.
Conditioned media from the hybridoma cultures were centrifuged at 5000 x g for 20 min and then pumped over a column of GammaBind Plus Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ). The columns were washed with 10 column volumes of PBS, and the mAbs were eluted with 0.1 M glycine (pH 3.0). Purified mAbs were dialyzed against PBS, filter sterilized, then aliquoted and stored at -20°C.
Effect of mAb 50-6 on HEp-3 Growth in Vitro.
HEp-3 cells were plated into six-well culture plates (2.0 x 105 cells/well) in the presence of 50 µg/ml of mAb 50-6 or normal mouse IgG. At 24, 48, and 72 h, the cells in two wells from each culture condition were trypsinized and counted.
Inhibition of HEp-3 Metastasis in the Chicken Embryo Assay.
Antibody inhibition of HEp-3 spontaneous metastasis in the chicken embryo was conducted as described previously (11)
. Briefly, tumor cells were inoculated through a window 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 returned to the incubator. Twenty-four h later, a second window was carefully cut in the eggshell directly over a prominent blood vessel. The underlying, nonliving shell membrane was made transparent with a drop of paraffin oil, and 200 µg of purified mAb or normal mouse IgG (Sigma Chemical Co., St. Louis, MO) in 0.1 ml PBS were inoculated into the blood vessel with a 30-gauge needle. The window was sealed, and after an additional 6 days of incubation, the eggs were opened; the primary tumors were excised, trimmed of CAM tissue, and weighed as a measure of tumorigenicity. The lungs of the embryos were removed, finely minced, and passaged onto the CAMs of a second set of 10-day-old embryos. These embryos were incubated for an additional 7 days to allow any HEp-3 cells in the lungs to multiply. The "lung tumors" arising from the transferred lungs were then excised and finely minced, and the presence of HEp-3 was determined biochemically by quantitating human urokinase-type plasminogen activator activity present in detergent extracts of the lung tumors (8, 9, 10, 11)
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Antibody inhibition of HEp-3 experimental metastasis was determined by coinoculating 0.1 ml of PBS containing tumor cells (2.0 x 104) and 200 µg of purified mAb or normal mouse IgG (Sigma) directly into a prominent blood vessel (prepared as described above). For the time course study of inhibition of HEp-3 experimental metastasis, the antibodies were inoculated at different times before or after inoculation of the tumor cells, as indicated. The inoculated embryos were incubated for an additional 6 days, after which the lungs were excised, finely minced, and transferred to prepared CAMs of a second set of embryos. The assay was then completed as described above.
Eukaryotic Expression Cloning.
A custom-made, unidirectional cDNA library was constructed in the eukaryotic expression vector pcDNA I (Invitrogen, San Diego, CA) using poly(A)+ RNA isolated from metastatic HEp-3 cells. Eukaryotic expression cloning in COS monkey kidney cells was conducted as described previously (15)
with some modifications. The first two rounds of transfection and immunoselection were performed as described except that COS cells were transfected using the calcium phosphate method (16)
. Plasmids recovered at the end of the second round were used to transfect COS cells growing on tissue culture plates. Twenty-four h later, the transfected cells were detached from the plates with nonenzymatic cell dissociation solution (Sigma) and plated onto polycarbonate membranes (90 mm diameter, 0.4 µ pore size; Millipore, Bedford, MA). After an additional 24 h, the cells (attached to the membranes) were washed three times with PBS, fixed with 0.25% glutaraldehyde in PBS for 5 min at room temperature, washed, quenched with 1.0 M glycine (pH 8.0) for two h at room temperature, washed again, and then incubated with 10% normal goat serum in PBS (blocking solution) for 1 h at room temperature. The membranes were then incubated with mAb 50-6 (1 µg/ml in blocking solution) overnight at 4°C with gentle agitation. As a control, one membrane was incubated with an isotype-matched control antibody (IgG1; Sigma; 1 µg/ml in blocking solution). The primary antibody was removed by washing the membranes 3 x 10 min in PBS, and the membranes were then incubated with biotin-conjugated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL; diluted 1:500 in blocking solution) for 1.5 h at room temperature with gentle agitation. The membranes were washed and incubated with horseradish peroxidase-conjugated streptavidin (Southern Biotechnology Associates; diluted 1:500 in blocking solution) for 45 min at room temperature with gentle agitation. After three washes in PBS, the membranes were developed with chloronaphthol to identify immunopositive COS transfectants. No color reaction was seen on cells incubated with control IgG1. With the aid of a dissecting microscope and a flame-drawn glass microcapillary pipette, 
30 strongly immunopositive cells were detached from the membranes and transferred to a microcentrifuge tube. Episomal plasmid DNA was recovered from these cells as described previously (17)
and used to transform bacteria (MC1061/p3; Invitrogen, San Diego, CA) by electroporation. The resulting colonies were pooled, grown in liquid culture, and plasmid DNA was isolated with a Qiagen Plasmid kit (Qiagen, Chatsworth, CA). Plasmid DNA was fractionated by resolving 1 µg 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 from cultures of bacterial cells transformed with DNA from each of the six gel slices and the fraction containing cDNA clones which directed the synthesis of a cell surface antigen recognized by mAb 50-6 was identified by transfecting and immunostaining COS cells (growing on poly-L-lysine-coated coverslips) as described above. DNA from one positive gel fraction was used to transform bacteria, and mini-prep DNA from 10 individual colonies was used to transfect COS cells for immunocytochemical analysis. Immunopositive cells were detected in 1 of the 10 transfected cultures. The positive clone contained an insert of 1.5 kb, as determined by restriction endonuclease digestion. The insert was sequenced with the T7 Sequenase Quick-Denature plasmid sequencing kit (version 2.0; Amersham Pharmacia Biotech, Arlington Heights, IL). The nucleotide sequence was compared with the National Center for Biotechnology Information database.
Transfection of HeLa Cells with the Cloned cDNA.
HeLa cells were cotransfected with the cloned PETA-3 cDNA and pSV2neo using the calcium phosphate method (16)
. Controls were cotransfected with vector (pcDNA I) containing no insert and pSV2neo. Forty-eight h later, Geneticin (G418; Life Technologies, Inc.) was added at a concentration of 400 µg/ml of medium, and the cultures were incubated for an additional 12 days. The resulting G418-resistant colonies were detached from the culture plates with nonenzymatic cell dissociation solution (Sigma), pooled, washed three times with serum-free DMEM, then blocked with 10% normal goat serum in PBS (blocking solution), on ice, for 30 min. The cells were then incubated with 1 µg/ml mAb 50-6 or an isotype-matched control (IgG1; Sigma) for 1 h on ice, washed three times with blocking solution, then incubated with a phycoerythrin-conjugated goat anti-mouse IgG (Southern Biotechnology Associates; diluted 1:500 in blocking solution) for 30 min on ice in the dark. The labeled cells were washed three times in PBS, and overexpressing PETA-3 transfectants were isolated by a fluorescence activated cell sorter. Control transfectants were also selected with a fluorescence-activated cell sorter. The cells collected were and then subcloned by limiting dilution. Approximately 50 subclones 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 2 x 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 of cells/well.
Whole-Cell ELISA.
The levels of cell-surface PETA-3 expression were measured by whole-cell ELISA. Subconfluent cultures HEp-3 or HeLa cells were detached from the culture plates, washed three times in serum-free DMEM, then resuspended in growth medium. Cells (2.0 x 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 three times with PBS, fixed with 0.25% glutaraldehyde in PBS for 5 min at room temperature, washed again, then quenched with 1.0 M glycine (pH 8.0) for 2 h at room temperature. The plates were washed three times with PBS and used immediately or stored in 0.1% sodium azide in PBS at 4°C. Stored plates were washed 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 overnight at 4°C. Purified mAb or isotype-matched control antibody (0.1 ml, 1 µg/ml in blocking solution) was added to the appropriate wells and incubated for 2 h at room temperature. The plates were washed three times with PBS, and then horseradish peroxidase-conjugated goat anti-mouse IgG (Southern Biotechnology Associates) was added to the wells (0.1 ml, 1 µg/ml in blocking solution) and incubated for 2 h at room temperature. The plates were washed, and 0.1 ml of the substrate, o-phenylene diamine (0.34 mg/ml, 0.1 M sodium citrate, pH 4.5, 0.012% H2O2) was added. After a 10-min incubation at 37°C, the plates were read at 405 nm using a Titer Tek Multiscan plate reader. The nonspecific signal from the isotype-matched control was subtracted from the experimental wells. Cell surface levels of the 
3ß1 integrin on HEp-3, HeLa, MDA-MB-231, and HT1080 cells were 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 culture plates with Versene (Life Technologies, Inc.), washed twice with serum-free DMEM (Life Technologies, Inc.), and resuspended in AIM-V medium (Life Technologies, Inc.). Cells (1.4 x 104) were added to the upper reservoir of BioCoat control chambers (uncoated; 8 µm pore size; Becton Dickinson Labware, Bedford, MA) in AIM-V medium, and 50 µg/ml of mAb 50-6, mAb 1A5, or normal mouse IgG (Sigma). The lower reservoirs contained DMEM supplemented with 10% FBS (HyClone) and 50 µg/ml of the appropriate antibody. After 6 or 12 h of incubation, the microporous inserts were fixed with 10% neutral buffered formalin and stained with hematoxylin. Cells on the upper surfaces of the membranes were removed with cotton swabs, and the membranes were excised and mounted on microscope slides in Permount. Cells on the underside of one quadrant of each filter were counted. Experiments were conducted in triplicate. Chemotaxis assays with the HeLa transfectants were similarly conducted, except that 2.0 x 104 cells were added to the 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, 0.1 M Tris (pH 8.0), 5 mM EDTA, 10 µM E64, 20 units/ml aprotinin, and 20 µg/ml soybean trypsin inhibitor (all from Sigma)] on ice for 10 min with vortexing at 5-min intervals. Insoluble material was removed by centrifugation at 12,000 x g for 5 min at 4°C. The protein concentration of the cleared lysates was measured with the bicinchoninic acid system (Pierce Chemical Co., Rockford, IL). Proteins were resolved on 10% SDS-PAGE gels and then transferred to nitrocellulose. The blots were blocked with a solution of 5% nonfat milk, 5% FBS, and 0.1% Tween 20 in PBS (Western blocking solution) for 1 h at room temperature. The blots were then incubated with either mAb 50-6, mAb 1A5, or normal mouse IgG (1 µg/ml in Western blocking solution) overnight at 4°C. The blots were washed three times for 5 min with 0.1% Tween 20 in PBS and then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (diluted 1:2500 in Western blocking solution) for 2 h at room temperature. The blots were then washed three times for 10 min with 0.1% Tween 20 in PBS, and the signals were visualized with the ECL system (Amersham Pharmacia Biotech, Arlington Heights, IL) according to the manufacturer’s directions.
Immunoprecipitation.
HEp-3 cells were in lysed in Brij lysis buffer [1% Brij 98, 25 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 10 µM E64, 20 units/ml aprotinin, and 20 µg/ml soybean trypsin inhibitor (all from Sigma)] for 1 h with constant rocking at 4°C (18)
. Insoluble material was removed by centrifugation at 12,000 x g for 5 min at 4°C. The lysate was precleared with GammaBind Plus Sepharose beads. Aliquots of the extract representing 107 cells were incubated with either 25 µg of mAb 1A5, 10 µg of mAb 1992 (Chemicon, Temecula, CA), which specifically recognizes the 3ß1 integrin heterodimer, or 25 µg of normal mouse IgG (Sigma). Fifty µl of packed GammaBind Plus Sepharose beads were added to each sample, and the mixtures were incubated at 4°C overnight with constant rocking. The beads were then washed with the Brij lysis buffer, and the immune 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 to nitrocellulose. Because biotinylation of both mAb 1A5 and mAb 50-6 destroys their immunoreactivity, PETA-3 was detected by incubating the blots with unlabeled mAb 1A5, followed by horseradish peroxidase-conjugated goat anti-mouse IgG as described above.
Cell Attachment to Purified Matrix Proteins.
For the cell attachment assays, 96-well plates precoated with purified FN were purchased from Becton Dickinson 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 dissociation solution (Sigma) and washed three times in PBS containing 0.1% heat denatured (60°C for 30 min) BSA. The cells were then resuspended to a concentration of 2 x 105 cells/ml in PBS/0.1% BSA containing 50 µg/ml of mAb 50-6, mAb 1A5, or normal mouse IgG (Sigma). After a 5-min incubation at room temperature, 0.1 ml of the cell suspension was added to the appropriate well and incubated for 15 and 30 min at 37°C. At the end of the incubation period, unattached cells were removed by gently washing the plates three times with PBS. Adherent cells were fixed with 0.25% glutaraldehyde for 1 h at room temperature, followed by three washes in PBS. Cell adhesion was quantitated adding 50 µl of crystal violet (0.1% in H2O) to each well. After 10 min at room temperature, the plates were washed in PBS, and the dye incorporated by the attached cells was released by adding 0.1 ml of 10% acetic acid, then quantitated by spectrophotometry in a Titer Tek Multiscan plate reader at 595 nm.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. When compared with cells incubated with normal mouse IgG (Fig. 1a)
, the fluorescence intensity signal from live, nonpermeablized HEp-3 cells immunostained with mAb 50-6 (Fig. 1b)
is significantly higher. The molecular weight of this cell surface protein was determined by Western blot analysis. As seen in Fig. 1c
, mAb 50-6 recognizes a single broad protein band having an apparent molecular weight of Mr 29,000.
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, spontaneous metastasis was reduced by 74% in embryos that received mAb 50-6. The effect of mAb 50-6 on experimental metastasis also was tested by coinoculating the tumor cells with the antibody directly into the circulatory system. In these experiments, dissemination to the embryonic lungs was inhibited by 57% when compared with controls. The antimetastatic properties of mAb 50-6 cannot be attributed to a cytostatic or cytotoxic effect, because this antibody had no effect on proliferation of HEp-3 cells in vitro (Fig. 2)
, nor did 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 a step(s) in the metastatic cascade.
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. The same cell surface staining pattern was seen on COS cells transiently transfected with PETA-3 cDNA (Fig. 3c)
. No signal was observed on HEp-3 or PETA-3-transfected COS cells incubated with normal mouse IgG (Fig. 3, b and d)
or on control COS transfectants incubated with mAb 50-6 (Fig. 3e)
. Western blot analysis of the cell lysates from PETA-3-transfected COS cells also demonstrated that the encoded protein comigrates with the Mr 29,000 antigen expressed on HEp-3 cells (Fig. 4A)
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. No signal was observed in lysates from control transfectants (Lane 2); however, mAb 1A5 recognized PETA-3/CD151 expressed by the COS cells (Lane 3). A minor Mr 25,000 band, immunostained with mAbs 50-6 and 1A5, was also evident in lysates of transfected COS cells (Fig. 4)
and in HEp-3 lysates (seen upon prolonged exposure of the Western blot; data not shown). This is likely the nonglycosylated form of the protein, because cultivation of HEp-3 cells with tunicamycin or treatment of HEp-3 cell lysates with N-glycanase generates a Mr 25,000 band that is immunoreactive with mAb 1A5 (data not shown). This is smaller than the predicted mass of 27.8 kDa, and may be due to the hydrophobic nature of the protein and/or the presence of disulfide bonds resulting from 15 cysteine residues. These results demonstrate that both mAb 50-6 and mAb 1A5 recognize PETA-3 and suggest that the epitope(s) is resident in the protein core.
PETA-3/CD151 Mediates an Early Event in the Formation of Metastatic Foci.
To examine the possible mechanisms by which PETA-3/CD151 effects metastasis, a time course study of inhibition of experimental metastasis was carried out. As shown in Table 2
, HEp-3 dissemination to the embryonic lungs was markedly inhibited when mAb 1A5 was administered as early as 2 h before or as late as 6 h after inoculation of the tumor cells. In contrast, there was little effect on HEp-3 colonization of the lungs when mAb 1A5 was administered 10 or 20 h after the tumor cells were inoculated. The inability of mAb 1A5 to inhibit lung colonization at the latter time points (when tumor cells would likely have extravasated already) indicates that the antibody does not block metastasis by inhibiting cell growth at the secondary sites. This is consistent with our observations that the anti-PETA-3 mAbs have no effect on HEp-3 growth in vivo or 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 tumor cell migration to selective sites of secondary growth.
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demonstrates that both mAb 50-6 and mAb 1A5 inhibited HEp-3 migration by approximately 45 and 44%, respectively, when compared with controls (P = 0.034).
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, their similar rates of growth in vitro (Fig. 6b)
and similarity in morphological appearance (not shown). The levels of cell surface PETA-3 on the two overexpressing clones, PB6HI and PC3HI, were about 2-fold higher than the HEp-3 cells, whereas the control transfectants, NB11LO and NB17LO, expressed approximately one-quarter to one-half the amount detected on HEp-3 cells (Fig. 6a)
. The growth rates of the four clones were identical (Fig. 6b)
. Migration by the overexpressing clones PB6HI and PC3HI was significantly higher than the underexpressing control transfectants NB11LO and NB17LO (Fig. 6c
; P = 0.001). When mAb 50-6 or mAb 1A5 (50 µg/ml) was added to the chambers, migration of clone PB6HI was significantly reduced (P = 0.034) by 51.6 and 52.8%, respectively, whereas migration of clone PC3HI was significantly reduced (P = 0.007) by 36.7 and 50.6%, respectively, lowering the motility of these cells to the level of control NB11LO cells. Neither mAb 50-6 nor mAb 1A5 inhibited migration of the weakly expressing clones NB11LO (P = 0.729) or NB17LO (P = 0.337), indicating that migration by these cells is mediated by a PETA-3-independent mechanism.
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3ß1 integrin in several cell types (25
, 26)
. Using a monoclonal antibody, mAb 1992, which specifically recognizes the 3ß1 heterodimer, we have detected, by whole-cell ELISA, the 3ß1 integrin on HEp-3 cells (Fig. 7)
at levels equivalent to known positive controls, MDA-MB-231, a human breast carcinoma cell line, and HT1080, a human fibrosarcoma cell line (18)
. A physical interaction between 3ß1 and PETA-3 in HEp-3 cells was demonstrated by the ability of mAb 1992 to coimmunoprecipitate these two molecules from HEp-3 cell lysates. Western blot analysis of immunoprecipitates obtained with mAb 1992 shows a Mr 29,000 protein that immunostains with mAb 1A5 (Fig. 8
, Lane 3). This protein comigrates with the authentic antigen present in the HEp-3 lysates (Fig. 8
, Lane 1) and in immunoprecipitates obtained with mAb 1A5 (Fig. 8
, Lane 2). No PETA-3 signal was observed in control normal mouse IgG "immunoprecipitates" probed with mAb 1A5 (Fig. 8
, Lane 4) or normal mouse IgG (Fig. 8
, Lane 5). The background bands observed in the immunoprecipitated samples are due to the reactivity of the secondary antibody to mouse IgG proteins and IgG fragments as seen in the control lanes.
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3ß1 signal. Weak signals were seen with the control HeLa transfectant (Fig. 7)
. This suggests that PETA-3 may interact with a different integrin in the transfected HeLa cells to effect the observed increase in cell migration (Fig. 6c)
.
PETA-3/CD151 Appears Not to Be Associated with Cell Adhesion.
Because cell adhesion is a prerequisite to cell migration, it is possible that the inhibitory effects of mAbs 50-6 and 1A5 in the chemotaxis assays are related to antibody inhibition of cell attachment. Therefore, to quantitate the role of PETA-3 in cell attachment, HEp-3 cells and the HeLa transfectants were tested for adhesion to various purified matrix proteins in the presence of mAb 50-6, mAb 1A5, or normal mouse IgG. As shown in Fig. 9
, at the 30-min time point, HEp-3 attachment to both FN (Fig. 9a)
and VN (Fig. 9b)
was unaffected by the presence of either of the antimetastatic mAbs. Likewise, attachment of the overexpressing and weakly expressing HeLa transfectants to FN or VN was not blocked by mAb 50-6 or mAb 1A5. There was also no correlation between levels of PETA-3 expression and cell attachment. Similar results were obtained when the adhesion assays were terminated after 15 min (not shown) and when cell attachment was tested on plates coated with laminin, type I collagen, and type IV collagen (not shown). These data suggest that PETA-3 is not functionally involved in cell attachment.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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strongly suggests that its cognate antigen is functionally involved in the metastatic process. Using mAb 50-6 in a modified eukaryotic expression cloning protocol, the full-length cDNA encoding the Mr 29,000 metastasis-associated HEp-3 antigen was isolated, sequenced, and identified as PETA-3/CD 151 (21)
, also known as SFA-1 (27)
. We also demonstrated that PETA-3 is recognized by a previously described metastasis-inhibiting antibody, mAb 1A5 (Fig. 4)
, also generated by subtractive immunization (11)
, but the cognate antigen of which was unknown. The unbiased selection of two independently generated anti-PETA-3 mAbs that block metastasis indicates that the subtractive immunization approach may be a powerful tool to identify functionally important antigens.
PETA-3 is a TM4SF protein. Also known as tetraspanins, members of this protein family are characterized by having four hydrophobic, transmembrane domains, two short cytoplasmic tails, and one small and one large extracellular loop (reviewed in Refs. 22, 23, 24)
. PETA-3 is expressed by a variety of cell types including the basal cells of the epidermis, epithelial cells, skeletal, smooth and cardiac muscle, Schwann cells, platelets, and endothelial cells (28)
. Yáñez-Mó et al. (25)
have shown that PETA-3 expressed by cultured endothelial cells is localized at cell-cell junctions. However, our immunocytochemical analysis of fixed, nonpermeabilized HEp-3 cells show that PETA-3 is distributed over the entire cell surface (Fig. 3a)
. Furthermore, this expression pattern was not restricted to HEp-3 cells because immunostaining over the entire cell surface was also observed on COS-7 transfectants expressing PETA-3 (Fig. 3b)
. Whether there is a functional relevance associated with the different patterns of PETA-3 distribution remains to be determined.
Several TM4SF proteins have been implicated as regulators of cell proliferation. Cell proliferation can be either stimulated (29, 30, 31)
or slowed (32)
in the presence of anti-TM4SF antibodies. Transfection with several tetraspanin family members also retards growth (32, 33, 34, 35)
. Our anti-PETA-3 mAbs 50-6 and 1A5 were shown not to affect in vitro growth of HEp-3 cells (Fig. 2
; Ref. 11
) nor in vivo growth in the primary tumor (Table 1
; Ref. 11
) or at the secondary site (Table 2)
. In addition, the in vitro growth rates of HeLa transfectants overexpressing PETA-3 are no different than control clones (Fig. 6b)
. These results indicate that PETA-3 does not affect tumor cell proliferation but functions specifically in one or more steps in the metastatic process itself.
Metastatic success by tumor cells has been shown to be dependent on initial arrest in the secondary organ (36)
, as well as events that occur after extravasation, such as migration through the stroma to sites of preferred secondary tumor growth (37, 38, 39)
. Our studies on the time course of inhibition of HEp-3 experimental metastasis (Table 2)
suggest that PETA-3 is involved in an early step in the formation of metastatic foci, such as arrest, extravasation, and/or migration into the connective tissue stroma of the secondary organ. Although PETA-3 is known to be expressed on endothelial cells (25
, 28)
, neither mAb 50-6 nor mAb 1A5 react with the endothelium of the chicken embryo, the host in the present metastatic model.7
Thus, the antimetastatic effect of mAb 50-6 and mAb 1A5 is the result of antibody binding to the HEp-3 cells themselves and not to host endothelial cells.
Tumor invasion of tissue elements is one hallmark of the malignant phenotype and is dependent on the ability of tumor cells to transiently adhere to various matrix proteins and to migrate into the surrounding stroma. TM4SF proteins are known to associate with other tetraspanins, integrins, and potential signaling molecules and are believed to facilitate the formation and stabilization of these macromolecular complexes and thus influence a number of cellular functions including migration and adhesion (Refs. 25
, 33
, and 40, 41, 42, 43, 44
; reviewed in Refs. 22, 23, 24
). In the present report, several experiments demonstrate a positive role for PETA-3 in mediating cell migration: (a) we were able to inhibit HEp-3 chemotaxis with mAb 50-6 and mAb 1A5 (Fig. 5)
; (b) we showed that HeLa cells transfected with and overexpressing PETA-3 were more migratory than control transfectants (Fig. 6c)
; and (c) the increase in motility by PETA-3-transfected HeLa clones was inhibitable by both mAb 50-6 and mAb 1A5 (Fig. 6c)
. The results of our antibody inhibition studies are consistent with recent observations that random migration of endothelial cells (25)
and polymorphonuclear chemotaxis (26)
are sensitive to inhibition by anti-PETA-3 mAbs. The mechanism by which PETA-3 influences migration of these cells is apparently related to the association of this tetraspanin with the 3ß1 integrin (25
, 26)
and potential signaling molecules (26)
. In the present study, we have demonstrated, by coimmunoprecipitation, a physical association between PETA-3 and 3ß1 in HEp-3 cells (Fig. 8)
. However, there is little or no detectable 3ß1 expressed on our HeLa transfectants (Fig. 7)
. TM4SF proteins can interact with several different integrin molecules, primarily those in the ß1 class (22, 23, 24)
, and it is possible that PETA-3 expressed by the HeLa cells associates with another integrin to effect the observed increase in motility.
The effects of PETA-3 on migration of HEp-3 and HeLa cells appear not to be related to changes in cell adhesion. We found no correlation between levels of cell surface PETA-3 expression and adhesion to wells coated with the ß1 substrates FN (Fig. 9a)
, LN, collagen type I, or collagen type IV (not shown) or the ß5 substrate VN (Fig. 9b)
. In addition, there was no significant difference in adhesion when cells were plated onto these same substrates in the presence of mAb 50-6 or mAb 1A5 (Fig. 9
and data not shown). Yáñez-Mó et al. (25)
found that endothelial cell adhesion to FN, LN, and collagen type I increased slightly but significantly in the presence of their anti-PETA-3 mAbs. The reason for the differences between the results of this latter study and our observations remains to be determined.
Several members of the tetraspanin family of proteins have been associated with the metastatic phenotype, but these associations have been, for the most part, negative. KAI-1/CD82 expression suppressed experimental metastasis of rat prostate tumor cells (45) , decreased motility and invasion of colon carcinoma cells (44) , and decreased invasion and metastasis of mouse melanoma cells (46) . Likewise, experimental metastasis of mouse melanoma was reduced in cells expressing motility-related protein (MRP)-1/CD9 (33) . In addition, (over)expression of CD9 slowed growth and blocked migration of CHO cells, and human lung adenocarcinoma and myeloma cells (33) . CD63 expression also resulted in decreased in vivo growth of human melanoma cells (34) and NIH3T3 cells (35) and blocked experimental metastasis of human melanoma cells (34) . Claas et al. (32) recently cloned the rat homologue of CO-029, a tetraspanin that appears to affect metastasis by altering the homing pattern of tumor cells. Transfection of BSp73AS cells, a weakly metastatic rat pancreatic adenocarcinoma cell line, with the homologue shifted the metastatic burden from the lymph nodes to the lungs and resulted in an increased survival rate of animals inoculated with the transfectants. A monoclonal antibody to the CO-029 homologue partially reduced a consumptive coagulopathy associated with expression of this protein; however, the effect of the antibody on metastatic dissemination was not reported (32) . In contrast to the reports cited above, we have, in the present study, exploited the techniques of subtractive immunization and eukaryotic expression cloning to detect, clone, and identify PETA-3/CD151 as a metastasis-associated antigen that appears to contribute positively to the metastatic phenotype. PETA-3 does not affect tumor cell proliferation but rather appears to be specifically involved in an early step in the formation of secondary metastatic lesions. The ability of PETA-3 to mediate tumor cell migration provides a possible mechanism for the role of this protein in effecting metastatic dissemination. Our studies identify PETA-3 as the first member of the tetraspanin family of proteins to be linked as a positive effector of metastasis.
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FOOTNOTES |
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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 Vascular Biology, 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: jtesta{at}scripps.edu
3 Present address: Department of Vascular Biology, VB-1, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037.
4 Present address: Department of Biochemistry and Molecular Biology, Norris Cancer Center, Topping Tower, Room 5409, 1441 Eastlake Avenue, University of Southern California, Los Angeles, CA 90033.
5 Present address: Matrix Pharmaceuticals, Inc., 34700 Campus Drive, Fremont, CA 94555.
6 The abbreviations used are: mAb, monoclonal antibody; CAM, chorioallantoic membrane; FBS, fetal bovine serum; FN, fibronectin; LN, laminin; TM4SF, transmembrane 4 superfamily; VN, vitronectin.
7 Quigley, unpublished results.
Received 1/14/99. Accepted 6/ 2/99.
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