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[Cancer Research 59, 3812-3820, August 1, 1999]
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[Cancer Research 59, 3812-3820, August 1, 1999]
© 1999 American Association for Cancer Research

Tumor Biology

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

Jacqueline E. Testa2,,3, Peter C. Brooks4, Jian-Min Lin5 and James P. Quigley3

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


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A monoclonal antibody (mAb), 50-6, generated by subtractive immunization, was found to specifically inhibit in vivo metastasis of a human epidermoid carcinoma cell line, HEp-3. The cDNA of the cognate antigen of mAb 50-6 was isolated by a modified eukaryotic expression cloning protocol from a HEp-3 library. Sequence analysis identified the antigen as PETA-3/CD151, a recently described member of the tetraspanin family of proteins. The cloned antigen was also recognized by a previously described antimetastatic antibody, mAb 1A5. Inhibition of HEp-3 metastasis by the mAbs could not be attributed to any effect of the antibodies on tumor cell growth in vitro or in vivo. Rather, the antibodies appeared to inhibit an early step in the formation of metastatic foci. In a chemotaxis assay, HEp-3 migration was blocked by both antibodies. HeLa cells transfected with and overexpressing PETA-3/CD151 were more migratory than control transfectants expressing little CD151. The increase in HeLa migration was inhibitable by both mAb 50-6 and mAb 1A5. PETA-3 appears not to be involved in cell attachment because adhesion did not correlate with levels of PETA-3 expression and was unaffected by mAb 50-6 or mAb 1A5. The ability of PETA-3 to mediate cell migration suggests a mechanism by which this protein may influence metastasis. These data identify PETA-3/CD151 as the first member of the tetraspanin family to be linked as a positive effector of metastasis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Metastasis is a complex, multistep cascade of cellular events including migration of tumor cells through the surrounding stroma, entry into the circulatory system, and finally arrest, extravasation, and growth at a distant secondary site (reviewed in Refs. 1, 2, 3, 4, 5, 6) . Given the complexity of the metastatic process, it is not surprising that a number of proteins have been associated with tumor cell dissemination including transcription factors, signaling proteins, adhesion molecules, proteases, motility factors, and others (1, 2, 3, 4, 5, 6) . Although nuclear, cytoplasmic, and secreted proteins have been associated with metastatic potential, tumor cell dissemination is executed via the physical interactions of the cancer cell surface with various host tissue elements. Not only is the cell membrane the interface at which cell-cell and cell-substrate contacts are made, it is the portal through which external signals must pass. Activation of cell surface receptors, by mutation or by ligand binding, initiates intracellular signaling cascades that influence expression of genes that promote the malignant phenotype (2 , 3 , 6) . Proteases expressed on or bound to the cell membrane mediate degradation of tissue barriers. Integrins mediate tumor cell motility and transmit environmental cues by virtue of their interactions with different matrix proteins. Our efforts to identify novel metastasis-associated antigens have, therefore, focused on the tumor cell surface. As our model system, we have used a highly metastatic human epidermoid carcinoma cell line, HEp-3, which disseminates to host lung tissue. The distinct characteristics and behavior patterns of this tumor cell line have been described by Ossowski in a series of papers published between 1980 and 1998 (7, 8, 9) . These cells are very aggressive and readily give rise to metastasizing tumors in both the chicken embryo (10, 11, 12) and in the nude mouse model (13 , 14) . As such, these cells should possess distinct surface antigens that are functionally involved in mediating tumor cell dissemination.

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.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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 Type Culture Collection (Rockville, MD). Metastatic human epidermoid carcinoma cells (HEp-3) were obtained from solid tumors serially passaged on the CAMs of chicken embryos (10 , 11) . All cells were maintained as monolayer cultures in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% 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 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) .

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 {alpha}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 {alpha}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.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
mAb 50-6 Recognizes a Mr 29,000 Cell Surface Antigen.
MAb 50-6 was generated by subtractive immunization (11) using intact HEp-3 cells as the immunogen. Localization of the antigen to the cell surface of HEp-3 cells was determined by whole-cell ELISA (not shown) and flow cytometry (Fig. 1)Citation . When compared with cells incubated with normal mouse IgG (Fig. 1a)Citation , the fluorescence intensity signal from live, nonpermeablized HEp-3 cells immunostained with mAb 50-6 (Fig. 1b)Citation is significantly higher. The molecular weight of this cell surface protein was determined by Western blot analysis. As seen in Fig. 1cCitation , mAb 50-6 recognizes a single broad protein band having an apparent molecular weight of Mr 29,000.



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

 
mAb 50-6 Inhibits Spontaneous and Experimental Metastasis of HEp-3 Cells.
To determine whether the Mr 29,000 cell surface protein was involved in HEp-3 dissemination, the effect of mAb 50-6 on spontaneous metastasis was measured using an in vivo model, the chicken embryo 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 1Citation , 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)Citation , nor did it result in a decrease in the size of the primary tumor on the CAM (Table 1)Citation . These results indicate that mAb 50-6 specifically blocks a step(s) in the metastatic cascade.


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

 


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  		Fig. 2. 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 (NM). At 24, 48, and 72 h, the cells in two wells from each culture condition were trypsinized and counted. Data are reported as numbers of cells/well.

 
Eukaryotic Expression Cloning Identifies the Mr 29,000 Metastasis-associated Antigen as PETA-3/CD151.
The antibody inhibition studies demonstrated a functional role for the Mr 29,000 protein in mediating metastasis. To identify the protein, mAb 50-6 was used in a eukaryotic expression cloning strategy to isolate the cDNA from a full-length HEp-3 cDNA library. COS cells were subjected to several rounds of transfection and immunoselection with mAb 50-6. In the final round of the cloning protocol, immunopositive cells showing a strong cell surface signal were selected for isolation of cDNA clones because these cells would more likely harbor full-length inserts (see "Materials and Methods"). Indeed, a clone that directed the synthesis of a cell surface antigen recognized by mAb 50-6 was isolated and sequenced. The open reading frame of this clone encodes a core protein having a predicted molecular mass of 27.8 kDa. There are four distinct hydrophobic domains, and a single N-glycosylation site in a large hydrophilic region that separates the third and fourth hydrophobic domains. Comparison of the sequence of this clone with the National Center for Biotechnology Information database identified the metastasis-associated antigen as PETA-3/CD151, a recently described Member of the tetraspanin family of proteins, which is also known as the TM4SF. The coding sequence was identical to that reported by Fitter et al. (21) . The cell surface distribution of PETA-3 on HEp-3 cells and COS cells transfected with the cloned cDNA was analyzed by immunocytochemistry. On HEp-3 cells incubated with mAb 50-6, there was a strong staining pattern over the entire surface of the cell membrane, including filopods extending from the cell body (Fig. 3a)Citation . The same cell surface staining pattern was seen on COS cells transiently transfected with PETA-3 cDNA (Fig. 3c)Citation . No signal was observed on HEp-3 or PETA-3-transfected COS cells incubated with normal mouse IgG (Fig. 3, b and d)Citation or on control COS transfectants incubated with mAb 50-6 (Fig. 3e)Citation . 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)Citation .



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  		Fig. 3. Immunocytochemical localization of PETA-3 on HEP-3 cells and COS transfectants. Cells were plated onto poly-lysine-coated glass coverslips, fixed 24 h later, and then immunostained with mAb 50-6 or normal mouse IgG. Signals were detected with horseradish peroxidase-conjugated goat anti-mouse IgG and chloronaphthol. All images were photographed with transmitted light. a, HEp-3 cells incubated with mAb 50-6. b, HEp-3 cells incubated with normal mouse IgG. c, COS cells transiently transfected with PETA-3 cDNA and incubated with mAb 50-6. d, COS cells transiently transfected with PETA-3 cDNA and incubated with normal mouse IgG. e, COS transiently transfected with vector (pcDNA I) alone and incubated with mAb 50-6. Bars, 20 µm.

 


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  		Fig. 4. PETA-3/CD151 is recognized by mAb 50-6 and mAb 1A5. COS-7 cells were transiently 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 and analyzed by Western blotting. a, Western blot of HEp-3 cell lysate (10 µg; Lane 1) and lysates from control (COS/pcDNA I; 20 µg; Lane 2) and PETA-3-transfectants (COS/PETA-3; 20 µg; Lane 3) probed with mAb 50-6. b, Western blot of HEp-3 cell lysate (0.5 µg; Lane 1) and lysates from control (COS/pcDNA I; Lane 2; 2.5 µg) and PETA-3 transfectants (COS/PETA-3; Lane 3; 2.5 µg) probed with mAb 1A5. In both panels, the signals were visualized with a peroxidase-conjugated goat anti-mouse IgG by chemiluminescence. Arrow, PETA-3, Mr 29,000. A Mr 25,000 signal is also seen in COS cell lysates probed with both mAb 50-6 and mAb 1A5. Right, molecular weight markers.

 
Brooks et al. (11) had described an antimetastatic mAb, mAb 1A5, which also recognizes a Mr 29,000 HEp-3 cell surface antigen. To determine whether this mAb also recognizes PETA, mAb 1A5 was used to probe Western blots of COS cells transiently transfected with the PETA-3/CD151 cDNA (Fig. 4B)Citation . 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)Citation 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 2Citation , 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 1Citation ; Fig. 2Citation ). 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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  		Table 2 Time course of inhibition of HEp-3 experimental metastasis

 
PETA-3/CD151 Is Involved in Cell Migration in Vitro.
The tetraspanins have been described as molecular facilitators that influence cell adhesion and migration, presumably through their interactions with integrins (reviewed in Refs. 22, 23, 24) , and it is possible that PETA-3/CD151 affects migration of HEp-3 cells. Therefore, the antimetastatic mAbs were tested for their ability to inhibit HEp-3 migration in a chemotaxis assay. Fig. 5Citation 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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  		Fig. 5. Inhibition of HEp-3 migration (chemotaxis) by mAb 50-6 and mAb 1A5. HEp-3 cells were detached from culture plate 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 the migration chambers in AIM-V medium and 50 µg/ml of mAb 50-6, mAb 1A5, or normal mouse IgG. The lower reservoir contained DMEM supplemented with 10% FBS and 50 µg/ml of the appropriate 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 one quadrant of each filter were counted. Experiments were conducted in triplicate; bars, SEM.

 
To further demonstrate the role of PETA-3/CD151 in tumor cell migration, HeLa cells were transfected with the cloned cDNA or with vector alone. Two clones from each group were selected for testing in a chemotaxis assay on the basis of their PETA-3 expression levels (Fig. 6a)Citation , their similar rates of growth in vitro (Fig. 6b)Citation 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)Citation . The growth rates of the four clones were identical (Fig. 6b)Citation . Migration by the overexpressing clones PB6HI and PC3HI was significantly higher than the underexpressing control transfectants NB11LO and NB17LO (Fig. 6cCitation ; 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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  		Fig. 6. Effects of PETA-3/CD151 expression on HeLa cell migration. HeLa cells were cotransfected with the cloned PETA-3/CD151 cDNA and pSV2neo. Controls were cotransfected with pcDNA I and pSV2neo. Two PETA-3 overexpressing clones (PB6HI and PC3HI) and two control clones (NB11LO and NB17LO) were selected. a, the amount of cell surface PETA-3 expressed by the HeLa transfectants and HEp-3 cells was determined by whole-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 were determined by plating 2 x 104 cells into each of two wells in a 24-well plate. At the times indicated, the cells were trypsinized and counted. Results are reported as number of cells per well. c, inhibition of HeLa migration (chemotaxis) by mAb 50-6 and mAb 1A5. HeLa cells were detached from culture plate with Versene (Life Technologies, Inc.), washed twice with serum-free DMEM (Life Technologies, Inc.), and resuspended in AIM-V medium (Life Technologies, Inc.). Cells (2.0 x 104) were added to the upper reservoir of the migration chambers in AIM-V medium and 50 µg/ml of mAb 50-6, mAb 1A5, or normal mouse (NM) IgG. The lower reservoirs contained DMEM supplemented with 10% FBS (HyClone) and 50 µg/ml of the appropriate antibody. After 18 h of incubation, the microporous inserts were fixed and stained, and the cells on the underside of one quadrant of each filter were counted. Experiments were conducted in triplicate; bars, SEM.

 
PETA-3/CD151 has been shown to associate with the {alpha}3ß1 integrin in several cell types (25 , 26) . Using a monoclonal antibody, mAb 1992, which specifically recognizes the {alpha}3ß1 heterodimer, we have detected, by whole-cell ELISA, the {alpha}3ß1 integrin on HEp-3 cells (Fig. 7)Citation 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 {alpha}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. 8Citation , Lane 3). This protein comigrates with the authentic antigen present in the HEp-3 lysates (Fig. 8Citation , Lane 1) and in immunoprecipitates obtained with mAb 1A5 (Fig. 8Citation , Lane 2). No PETA-3 signal was observed in control normal mouse IgG "immunoprecipitates" probed with mAb 1A5 (Fig. 8Citation , Lane 4) or normal mouse IgG (Fig. 8Citation , 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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  		Fig. 7. Whole-cell ELISA of expression of the {alpha}3ß1 integrin heterodimer by equal numbers (2 x 104) of MDA-MB-231 human breast carcinoma cells, HT1080 human fibrosarcoma cells, HEp-3 cells, and the PETA-3 overexpressing HeLa clones (PB6HI and PC3HI) and control HeLa clones (NB11LO and NB17LO). mAb 1992 was used to specifically identify the heterodimer. Data represent the amount of chromogen released in the assay and are expressed as the absorbance (OD) at 405 nm.

 


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  		Fig. 8. PETA-3 expressed by HEp-3 cells associates with the {alpha}3ß1 integrin heterodimer. HEp-3 cell lysates were incubated with mAb 1A5 (Lane 2), mAb 1992 (specific for the {alpha}3ß1 heterodimer; Lane 3), or normal mouse IgG (NM; Lane 4). Immune complexes were collected on GammaBind Plus Sepharose, resolved by SDS-PAGE, and transferred to nitrocellulose. The blot was incubated with unlabeled mAb 1A5 as a primary antibody, followed by horseradish peroxidase-conjugated goat anti-mouse IgG. Signals were visualized by chemiluminescence. As a control, a NM "immunoprecipitate" was probed with NM IgG and peroxidase-conjugated goat anti-mouse IgG (Lane 5). A sample of the original HEp-3 lysate is shown in Lane 1. *, PETA-3 signal. Left, molecular weight markers (in thousands).

 
When the PETA-3-transfected HeLa clones were analyzed by whole-cell ELISA, there was no detectable {alpha}3ß1 signal. Weak signals were seen with the control HeLa transfectant (Fig. 7)Citation . 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)Citation .

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. 9Citation , at the 30-min time point, HEp-3 attachment to both FN (Fig. 9a)Citation and VN (Fig. 9b)Citation 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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  		Fig. 9. Effects of mAb 50-6, mAb 1A5, and PETA-3 expression on cell adhesion to various matrix proteins. HEp-3 cells, PETA-3-overexpressing HeLa clones (PB6HI and PC3HI) and control HeLa clones (NB11LO and NB17LO) were added to the wells of 96-well plates coated with either fibronectin (a) or vitronectin (b) in the presence of 50 µg/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 expressed as the absorbance (OD) at 595 nm of the incorporated crystal violet dye and are representative of two separate experiments conducted in triplicate; bars, SEM.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The metastatic process has been correlated with expression of a wide variety of cellular proteins, including adhesion molecules, growth factors, motility factors, proteases, transcription factors, and signaling molecules. However, a correlative association does not necessarily imply that a protein is functionally relevant to tumor cell metastasis. In the present study, mAb 50-6 was used to immunologically target a cell surface antigen that is mechanistically involved in tumor cell dissemination. This mAb was generated by subtractive immunization against a highly metastatic human epidermoid carcinoma cell line, HEp-3, without bias as to the nature or identity of the antigen. The ability of mAb 50-6 to block metastasis specifically (Table 1)Citation 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)Citation , 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)Citation . 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)Citation . 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. 2Citation ; Ref. 11 ) nor in vivo growth in the primary tumor (Table 1Citation ; Ref. 11 ) or at the secondary site (Table 2)Citation . In addition, the in vitro growth rates of HeLa transfectants overexpressing PETA-3 are no different than control clones (Fig. 6b)Citation . 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)Citation 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)Citation ; (b) we showed that HeLa cells transfected with and overexpressing PETA-3 were more migratory than control transfectants (Fig. 6c)Citation ; and (c) the increase in motility by PETA-3-transfected HeLa clones was inhibitable by both mAb 50-6 and mAb 1A5 (Fig. 6c)Citation . 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 {alpha}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 {alpha}3ß1 in HEp-3 cells (Fig. 8)Citation . However, there is little or no detectable {alpha}3ß1 expressed on our HeLa transfectants (Fig. 7)Citation . 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)Citation , LN, collagen type I, or collagen type IV (not shown) or the ß5 substrate VN (Fig. 9b)Citation . 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. 9Citation 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.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 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. Back

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 Back

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

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. Back

5 Present address: Matrix Pharmaceuticals, Inc., 34700 Campus Drive, Fremont, CA 94555. Back

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. Back

7 Quigley, unpublished results. Back

Received 1/14/99. Accepted 6/ 2/99.


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 DISCUSSION
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Cancer Res., May 1, 2008; 68(9): 3204 - 3213.
[Abstract] [Full Text] [PDF]