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G. F. Pancino, V. Le Doussal, M. H. Mortada, et al. Cancer Res 1989;49:7078-7085. Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/49/24_Part_1/7078
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Characterization and Distribution in Normal and Tumoral Human Tissues of Breast Cancer-associated Antigen Defined by Monoclonal Antibody 7B!0
G. F. Pancino, V. Le Doussal, M. H. Mortada, P. Berthon, E. Osinaga, F. Calvo, and A. Roseto
i .P.R. 43, ( \RS Il!pilal Suini Louis. 75474 Paris Cedex IO <j. F. P., A. R.; Sen-ice d'Anatomopathologie, Centre Ren Hugenin. 92211 Saint Cloud /t'. L. D.J; Division d'lmmunocylologie Applique,f 'niversile tie Technologie de Compiegne, 60206 Compiegne Cedex G.F. P., M. H. M., E. O., A. R./; and Pharmacologie Experimentale, I.VSERM '21)4,Institut d'Hmatologie,Centre Hayem, HpitalSaint Louis, 75475 Paris Cedex K) P. B., F. C.j, France
ABSTRACT
Monoclonal antibody 7B10,raised against the human breast cancer cell line T47D, identifies an antigen found in human breast carcinomas and in normal breast. Western blot and immunoprccipitation studies detected a A/, 76,000 antigen in cytosol, cell membrane, and cell culture super natant* of T47D cells. 7BMIbinding to T47D cell extracts was affected by proteolytic digestion with protease type VI, trypsin, and subtilisin while it was not altered by neuraminidase digestion. Adsorption of breast cancer cell line extracts with concanavalin A reduced 7B,,, immunoreactivity more than 70%. These results suggest that the antigen is a glycoprotein and that the epitope docs not contain sialic acid. 7B" was #, reactive with neither human milk fat globule membrane, nor skimmed milk, nor the milk-derived UHI 100 cell line. Conversely binding was detected in more than 50% of normal breast epithelial cells in culture. 7B," immuno# staining was positive on frozen sections of normal breast and nonmalignant mastopathies in 30 to 90% cells. In frozen sections of other normal tissues, 7Bio immunorcactivity was detected only in colon, apocrine glands of skin, parotid ducts, and luteal phase endometrium, confirming previous data on paraffin sections. Strong, homogeneous immunostaining was observed on frozen sections of intraductal and invasive lobular breast carcinomas (100% of cases), while more heterogeneous staining was found on invasive ductal carcinomas. Colon and rectal carcinomas, one carcinoma of the esophagus, and some cells in serous ovarian carcinomas also showed 7BIOreactivity. Immunoblotting of the 7B,irimmunoreactive fraction isolated by Scpharosc CL-6B chromatography of a breast car cinoma tissue sample extract identified the M, 76,000 antigen, which was also detected in several breast cancer specimens, in colon adenocarcinomas, and in serous ovarian carcinoma fresh tumor extracts. The \t, 76,000 glycoprotein described here represents a breast cancer-associated antigen previously undescribed, mainly expressed in normal breast and breast tumors.
was lost in paraffin sections, while cytoplasmic staining of cancer cells was conserved after fixation procedures. Therefore we decided to study antigen localization further by immunohistochemistry in a panel of frozen human tissues, normal and tumoral, from breast and from other origin. In addition MAb1 7B,o reactivity was analyzed by IIP, immunoperoxidase, ELISA, immunoblotting, and immunoelectron microscopy in HBL 100 cell line, HMFGM, and cultures of normal breast cells. MATERIALS AND METHODS
Monoclonal Antibody: Preparation and Purification. Generation and characterization of the IgGl monoclonal antibody 7BIO has been re ported previously in detail (4). It was produced from a fusion of SP20 murine myeloma cells with spleen lymphocytes of a BALB/c mouse immunized with the T47D breast cancer cell line. The antibody was purified from ascites derived from mice, utilizing the Affi-Gel protein A Maps-2 kit (Bio-Rad), according to manufactur er's instructions. The pH of the eluted fractions was brought to 7 with 1 M Tris buffer, pH 9. Those fractions containing monoclonal antibod ies were then dialyzed overnight against PBS and kept frozen. Monoclonal antibody DF3 (5), used in some experiments, was kindly provided by Oris-France. Cell Lines. The T47D and MCF7 human breast cancer cell lines (6, 7) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 lU/ml penicillin, and 50 ug/m\ streptomycin. HBL 100, a diploid cell line derived from human milk cells and known to contain SV40 (8, 9), was a generous gift of Dr. R. Cassingena (IRSC-CNRS, Villejuif, France), and was cultured under the same conditions. Normal Breast Cultures. Processing for culture of normal human breast cells has been described in detail by Soule et al. (10). Briefly, normal breast tissue from reduction mammoplasty was first dissected with scissors into 2-3-mm-diameter fragments, which were then dis sociated to organoids and single cells with collagenase and hyaluronidase (11). Organoids and cells were washed and centrifuged on FicollHypaque gradient. Dissociated epithelium was first plated for primary culture in 25-cm2 flasks (Corning) in a calcium-free medium, consisting of Dulbecco's modified Eagle's medium-Ham's F-12 (1:1), 5% calcium-depleted horse serum, insulin (10 Mg/ml), Cortisol (5 x 10~" M), penicillin (50 IU/ml), streptomycin (50 ug/m\), cholera toxin (100 ng/ml), epidermal growth factor (20 ng/ml) (Paesel, West Germany), and calcium (1.05 mM). As the cells became confluent the medium was changed to low calcium medium (same as above with 60 MMcalcium), and the cells were further passaged in this medium. HMFGM Preparation. HMFGM isolation was performed as de scribed previously (12). Briefly, human milk from healthy lactating mothers was centrifuged at 3,000 x g for 15 min at 4C. Raw cream was washed three times with 0.9% saline. Washed cream was resuspended in 0.9% saline at 30% v/v and frozen at -80C. After thawing at 37C, the suspension was centrifuged at 100,000 x g for l h and the pellet was used as the HMFGM fraction. The milk serum was centri1The abbreviations used are: MAb, monoclonal antibody; IIP, indirect immunofluorescence; ELISA, enzyme-linked immunosorbent assay; HMFGM. hu man milk fat globule membranes; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; NP40. Nonidet P-40; BSA, bovine serum albumin; p76, A/, 76.000 protein.
INTRODUCTION Monoclonal antibodies are powerful tools for detecting and identifying new cellular antigens. Many murine and human monoclonal antibodies have been described which recognize normal and tumoral breast cells (reviewed in Refs. 1 to 3). 7B,o is a murine monoclonal antibody which was raised against the human breast carcinoma cell line T47D (4). It was shown to react strongly with four breast tumor cell lines (T47D, MCF7, ZR-75-1, and HSL51) but not to bind to cancer cell lines of other origin, or with normal fibroblasts and lymphocytes (4). In vivo, it displayed a different reactivity with breast carcinomas compared to normal breast. In this paper we report on further characterization of the 7Bio-reactive antigen, performed by Western blot analysis, immunoprecipitation, filtration chromatography, enzymatic treat ment, and lectin-binding assays. The antigen found in cancer cell lines and in fresh tumors was compared using Western blot. In a previous work, we described a difference in immunostain ing between frozen sections and fixed, paraffin-embedded breast tissues (4). Membrane staining, present in frozen normal breast,
Received 4/14/89; revised 7/13/89; accepted 8/22/89. The costs of publicaci$n 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. 7078
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fuged at 100,000 x g for I h and the supernatant was used as the skimmed milk fraction. Breast Cancer Cell Line Kxtract Preparation. T47D and MCF7 cell flasks were grown to confluence. The cell supernatant was discarded and cells were washed with PBS and harvested by scraping. Cells were then incuhated for 30 min at 4'C in lysis buffer (IO mM Tris-HCl, pH 7.4; 5 HIMKCl; 2 m\t CaCI2; 1 m\i MgCl2: 0.2 M NaCI: l mM PMSF; and 0.5% NP40). The nuclei were removed by centrifugaron (1.000 x g for 10 min) and insoluble material was pelleted by centrifugation at 100,000 x K for 60 min; the 100,000 x g supernatant was used for the experiments. For cell fraction preparation, T47D and MCF7 cells scraped and washed with PBS were resuspended at lO'/ml in hypotonie buffer (10 mM Tris, pH 7.4; 5 nui KCl: 2 m.MCaCU; 1 HIM MgCl,; and 1 mM PMSF) and incubated in ice for 15 min. Cells were subse quently homogenized in a Dounce glass homogenizer (B pestle) and centrifuged at 1,000 x g for 7 min. The supernatant was centrifuged at 4,000 x # for 10 min. The resulting supernatant was then centrifuged at 100,000 x g for l h and the pellet was used as the membraneenriched fraction. The supernatant was used as cytosol fraction. Tissue Extraction. Breast tumors and other tissues were frozen in liquid nitrogen until assay. Tissue nature and the presence of malig nancy were confirmed histologically in cancer patients, and some sec tions were processed for immunostaining. Tissues were then pulverized and homogenized using a hydraulic press (X Press LKB) with 4 volumes of tissues lysis buffer (20 mM Tris-HCl. pH 7.4; 0.1 M NaCI; 5 mM MgCl2; 1% NP40-0.5% sodium deoxycholate; 1 mM PMSF; and 2 Kallikrein inhibitor units/ml bovine aprotonin). The homogenates were centrifuged at 1,000 x g for 10 min 4 C and the resulting supernatants were centrifuged at 100,000 x g for 60 min. The final supernatants were used as tissue extracts and stored at ! 20C. Protein concentration was determined by the method of Lowry et al. (13). Gel Filtration. Gel filtration of breast tumor extracts was performed using Sepharose CL-6B molecular exclusion media (Pharmacia), precalibrated using blue dextran and a calibration protein II kit (Boehringer). Applied samples were developed using as running buffer the tissue lysis buffer without sodium deoxycholate, and eluted fractions were tested for 7B,! i " mmunoreactivity using ELISA. Positive isolates were further examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted. Cell Radiolabeling and Immunoprccipitation. T47D cells were labeled overnight at 37"C in methionine-free minimal essential medium con taining ["SJmethiomne (60 iCi/ml;specific activity, 1154 Ci/mmol; DuPont, NEN Products), 2% fetal calf serum, and glutamine. Cells were then washed to remove free radioactivity, harvested, and extracted with lysis buffer. Immunoprecipitation of radiolabeled cell extract was performed using rabbit anti-mouse IgGl serum-coated protein A-Sepharose. Immunoblotting. HMFGM, T47D extracts, and tissue lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (5 to 15% and 10% acrylamide). Protein transfer to nitrocellulose filter paper (0.45-Mtn pore size) was performed for 3 h at 60 V using the BioRad electroblot system. Electrode buffer was 20 mM Tris (pH 8.3)-192 mM glycinc-20% methanol-0.01% sodium dodecyl sulfate. After trans fer, the nitrocellulose paper was incubated with 5% BSA in 10 mM Tris (pH 7.4)-150 mM NaCl-0.05% Tween 20 for l h at 37C.Overnight incubation with primary MAb (diluted in Tris-NaCl-Tween 20-1% BSA) at 4C was followed by incubation with peroxidase-labeled rabbit anti-mouse serum (Biosys). Visualization of a positive reaction was obtained by incubation with 4-chloro-l-naphthol (Merck). 0.05% in PBS for 10 to 15 min. In all experiments mouse anti-cytomegalovirus monoclonal IgGl (En) (14) was used as negative control and MAb DF3 was used as positive control in experiments on HMFGM and fresh tumor lysates. ELISA. ELISA was performed using an avidin-biotin three-stage method (ABC Vectastain; Vector Laboratories) according to manufac turer's instructions. Enzyme immunoassays were made with T47D and
well of 1% gelatin in PBS for blocking free sites on plastic. In all experiments, MAb E,, was used as negative control. In HMFGM assays, MAb DF3 was used as positive control. Knzyme Treatment. Microplates coated with T47D lysate in carbon ate buffer (pH 9.6) were exposed to enzymatic treatment with trypsin (Flow Laboratories; 2.5 mg/ml), subtilisin (Sigma) (I mg/ml), and protease type VI (Sigma) (2 mg/ml) in 10 m\i Tris, pH 8-2 mM CaCl: neuraminidase (Behring), 50 units/ml in 50 mM acetate buffer. pH 5.51 m\i Cadi. Incubation was carried out at 37C for I h. Control plates were incubated with dilution buffers under the same conditions. After treatment, plates were washed and blocked with 1% gelatin in PBS; 100 /u!of 7B,i>(properly diluted) were added to the wells and incubated at room temperature for 1 h. The remaining steps of the assay were the same as for ELISA. In all experiments, MAb En was used as negative control. Live T47D and MCF7 cells were scraped from culture flasks. One half of the cells were treated with trypsin (2.5 mg/ml) in PBS for 30 min at 37C; the other half was used as control and incubated with 1% PBS-BSA. Both treated and untreated cells were incubated for 30 min in ice with fluorescein isothiocyanate-labeled 7Bm MAb washed in 1% PBS-BSA and resuspended in PBS-20% glycerol. I trtin Absorption Test. Crude membrane fraction of MCF7 cell line, solubilized with PBS. 0.5% NP40 (375 ng protein) was added to 600 i of 50% (v/v) suspensions of Sepharose 4B, Sepharose-concanavalin A, Sepharose-lentil lectin and Scpharose-wheat germ agglutinin. After 1 h incubation at 4C (in agitation) the suspensions were centrifuged. Subsequently, serial dilutions of the supernatants were assayed in ELISA for antigen content. Results were expressed as percentage bound as compared to control (nonderivatized Sepharose). Immunofluorescence Staining. An indirect immunofluorescence method on live cell suspensions of T47D, MCF7, and of HBL 100 was performed. The purified antibody was used at a concentration of 20 Mg/ml. The second antibody was a fluorescein isothiocyanate-goat conjugated anti-mouse immunoglobulin from Biosys, France. In some experiments, cells were cytocentrifuged and fixed in acetone for 10 min at 4C. MAb E,.>was used as negative control. Electron Microscopy. T47D, MCF7, and normal breast cells in culture were weakly fixed with 0.1%) glutaraldehyde for 5 min in ice. In other assays, cells were permeabilized with 0.5% Triton X-100 for 5 min after being fixed with 0.5% glutaraldehyde for 5 min. Cells were incubated with 7Bm for 1 h. After a washing with PBS. cells were incubated for another 2 h with 100 n\ of 14 nm or 10 nm colloidal gold-labeled anti-mouse IgG (Jansen Pharmaceuticals) diluted 1:2 in PBS. Cells were then fixed with 1% glutaraldehyde and included in Epon. The sections were analyzed using a Philips EM 301 electron microscope. In all experiments, MAb E" was run as negative control. Immunohistochemistry: Tissue Preparation and Immunostaining Pro cedure. Normal breast cells, after 3 to 4 weeks of culture (second passage) were plated in Labtek multiwell glass slides. After adherence and further growth to subconfluence, the slides were washed 3 times in PBS, fixed in methanol:acetone (2:3, 1:3), and frozen at -80C until stained. Frozen sections of normal and pathological tissues were obtained from blocks embedded in OCT tissue Tek I (Miles) and stored at -80C for 1 to 4 weeks. Tissue sections 4 to 5 nm thick were mounted and fixed in acetone for 5 min at 4"C. All sections were stained using avidin-biotin-peroxidase complex kits (Vector Laboratories) following the manufacturer's guidelines. After blocking with normal horse serum,
sections were incubated with MAb 7Bu, (40 Mg/ml) in bovine serum albumin. 0.1% in PBS, for 1 h at room temperature. Sections were then incubated with biotin-labeled horse anti-mouse immunoglobulin, followed by quenching of endogenous peroxidase activity, performed with 0.5% HiO: in methanol for IO min. Finally, sections were incu bated for 30 min in the avidin-biotin-peroxidase complex. The reaction was revealed with 3-amino-9-ethylcarbazole (Sigma) and slides were MCF7 extracts or HMFGM and skimmed milk as antigens. Microtiter counterstained with Mayer's hemalum and mounted in aqueous me plates (Dynatecs) were coated with antigen in 0.05 M carbonate buffer. dium. For every assay, negative controls using PBS without primarypH 9.6 (60 Mg/ml). at 50 /I/well and dried overnight. Plates were washed 3 times with PBS and incubated for l h at 37C with 200 A/ antibody were included. 7079
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RESULTS Biochemical Characterization of 7BMI-reactive Antigen in Breast Cancer Cell Lines Western blot analysis and immunoprecipitation were used to define the molecular weight of the antigen reactive with MAb TB,,,. Immunoblotting of T47D lysates with 7B,o revealed a M, 76,000 band (Fig. \A, Lane A). The M, 76,000 band was also detected by immunoblotting of cell-free supernatants of T47D cultures (Fig. \A, Lane C). Immunoprecipitation experiments confirmed the 7Bn, reactivity with the M, 76,000 antigen (Fig. \B, Lane A). In order to detect different subcellular localization of the antigen. T47D membrane and cytosol fractions were separated and analyzed using immunoblot and immunoprecip itation. 7B,(, detected the M, 76,000 antigen in both fractions (not shown). To further study the nature of the antigen, the effect of proteases and neuraminidase digestion of T47D cell lysates on 7B,n immunoreactivity was investigated. Protease VI, trypsin, and subtilisin treatments strongly reduced 7B,,, binding in ELISA, while neuraminidase digestion did not modify the immunological reaction (Fig. 2A). In addition, IIF assays on live T47D and MCF7 cells submitted to trypsin treatment confirmed the loss of virtually all immunoreactivity. We use MCF7 membrane lysate for lectin-binding assays. Lectins ab sorbed the 7B,o-reactive antigen at various degrees (Fig. 2B). Concanavalin A retained more than 70% of 7BM, reactivity.
30-
20-
12
16
20
Fig. 2. A. effect of enzymatic digestion of NP40 extracts of T47D cells on 7B,o reactivity. T47D extracts were digested alone (D). or with neuraminidase ii. trypsin (O). protease VI (! ). and subtilisin (A) as described in 'Materials and Methods." After digestion. ELISA was performed using 7Bm. Abscissa, 7B,0 concentrations; ori/mare, absorbance (optical density). B. Icctin absorption of 7B,0 reactive antigen from NP40 extracts of MCF7 membrane fraction. MCF7 mem brane lysates were incubated for l h with Sepharose concanavalin A. Sepharoselentil lectin, Sepharose-wheat germ agglutinin. and Sepharose 4B as control. Nonabsorbed antigen was assayed in ELISA for 7Bm binding. Abscissa, serial dilution of antigen: ordinale, absorbance (optical density). 03,Sepharose; ,wheat germ agglutinin; ! concanavalin . A; O. lentil lectin.
Kd
Kd
_94
76.
76 _*
_67
_69
while a weak reduction was found using wheat germ agglutinin and lentil lectin. Distribution and Characterization of 7B,cl-reactive Antigen in HBL 100 Cell Line, HMFGM, and Skimmed Milk MAb 7B,o binding was tested by IIF on the HBL 100 cell line in comparison with the breast cancer cell lines T47D and MCF7. While T47D and MCF7 cells showed bright staining, no reactivity could be detected with HBL100 cells. HMFGM reactivity with 7BH)was tested by ELISA and immunoblotting and compared to reactivity with MAb DF,. 7B10 did not react with HMFGM, while DF., presented strong reactivity in ELISA, and reactive bands were detected over A/r 330,000 in immunoblot as described by Sekine et al. (15). Immunoelectron microscopy performed on HMFGM did not detect 7B,o stain ing. 7BIOdid not bind to skimmed milk in ELISA. Distribution and Characterization Human Tissues of 7B,,,-reactive Antigen in
_44
_46
_30
_30
-20
_14.3
A B C D B
Fig. 1. A, Western blot analysis of NP40 extracts from T47D cells and of cellfree T47D culture supernatant with 7B,! "Electrophoresis . was run in sodium dodccyl sulfate-1Or( polyacrylamide gel. Immunoblotting was performed as de scribed in "Materials and Methods." p76 was detected in cell lysate (Lane A) and in supernatants (Lane ('). Anti-cytomcgalovirus IgGl IM vvasapplied as negative control (Lanes B and D). B, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates by 7B,0 antibody from NP40 extracts of |J'S|methionine-labelcd T47D cells; 10rr polyacrylamide gel was used. 7B,0 MAb specifically precipitated a M, 76.000 component (Mne A). An IgGl mouse anticytomegalovirus (E,j) MAb was run as negative control (Lane B). Kd. molecular weight in thousands.
Normal Breast and Benign Tumors of the Breast. The expres sion of 7B|0-reactive antigen was tested on normal breast epi thelial cells in culture and on fresh specimens of normal breast biopsy using immunoenzyme staining. In primary cultures of breast epithelium, 50 to 60% of the epithelial cell population, as stained by anti-cytokeratin MAb KL, (Immunotek), was positively stained by 7B10. Reactivity was mainly localized on the cell surface (Fig. 3a). Immunoelectron microscopy with 7B1()confirmed this staining heterogeneity; intense gold stain-
7080
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CHARACTERIZATION
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Fig. 3. 7Bio immunoperoxidase staining of normal human breast or benign breast lesions. Avidin-biotin-peroxidase-Mayer's hematoxylin countcrstain. a. normal breast cells in culture. 7B,0 staining is localized predominantly on the cell membrane. Some cells are not stained, x 400. b, frozen section of normal breast tissues. The staining of epithelial cells is marked on the cell membrane of acinar cells, c, frozen section of adenofibroma of the breast. Note the intense immunostaining of epithelial cells; surrounding connective tissue is negative, x 125. t-. frozen sections of breast adcnosis. Immunostaining shows a heterogeneous pattern. The more proliferarne area is negative, x 250.
ing was observed in some cells, while about 50% of cells showed very poor or no 7B,o reactivity (Fig. 4, A and B), compared to 90% generalized staining of T47D (Fig. 4, C to E) or MCF7 cells. In normal breast tissue (Table 1), positive cells were observed in all specimens, with heterogeneity in staining intensity and number of positive cells varying from 30 to 60% (Fig. 3b). Staining was present in acinar and ductal cells but was stronger in the former. Some diffuse, dusty staining was observed in the cytoplasm. On the cell membrane, staining increased and con sisted of a thick continuous line or irregular dots, marked sometimes at the apical pole. Some myoepithelial cells showed strong staining. Stromal cells, inflammatory cells, and vessels were negative. In benign epithelial proliferation such as epitheliosis and adenosis, staining was heterogeneous (Fig. 3</). In cystic disease, the more marked the apocrine metaplasia, the more the cells were stained by 7BH>. When the cyst epithelium was flat, cells were negative. In adenofibromas (Fig. 3c) and papillomas, epithelial cells showed staining of variable intensity. A diffuse intracytoplasmic and perinuclear staining pattern was observed, with membrane and
apex reinforcement in several cells. Near some carcinomatous ducts, atypical ductal hyperplasia was observed. 7Bi0 positive cells here exhibited a thin membrane or diffuse cytoplasmic staining, lighter than in carcinomatous cells. Breast Carcinomas. All intraductal breast carcinomas tested using immunohistochemistry (Table 1) were strongly stained by MAb 7B,0. Staining was very intense, and involved the cytoplasm and cell membrane (Fig. 5a). The two invasive lob ular carcinomas tested were also strongly stained. Among the 38 invasive ductal carcinomas, some staining heterogeneity was observed, from 100% strongly positive cells to weak staining of some of the tumor cells. Four specimens failed to react with 7B,(i. For positive cells, both membrane and cytoplasmic stain ing were present (Fig. 5b). A mucoid carcinoma showed some reactivity in cells without intracytoplasmic mucin vacuoles, while mucin and secreting malignant cells were not stained. In 6 individual node mtastases of breast carcinoma from individ ual patients, staining was strong (Fig. 5c). Substantial variations from primary tumor staining were not observed. Some patho logical frozen specimens were also analyzed by immunoblotting to characterize the antigen detected in fresh cancer tissue. The
7081
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CHARACTERIZATION
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Fig. 4. Electron microscopy immunostaining with 7B,0 of normal breast cultures in comparison with T47D cells, using colloidal gold (10 mn in A, B; 14 nm in C, D. and ').Preembedding procedure was performed as described in "Materials and Methods." In normal breast cells in culture, staining is heterogeneous. Sparse gold particles are distributed on the cell membrane of some cells (A, arrows); after permeabilization with 0.5% Triton X-100, some cells are strongly stained (B, arrows). while others are negative. On T47D cells fixed with 0.1% glutaraldehyde. gold particles are grouped in clumps on cell membrane (C) and decorate cell pseudopodal structures (D); cytoplasmic localization of gold particles can be observed after T47D cell permeabilization with 0.5% Triton X-100. (E:n. nucleus: c, cytoplasm). A, D, x. 38,000; B, C, x 30.000; E, x 68.000. Table 1 7B,0 immunoperoxidase staining of frozen human mammary tissues diseaseTissueNormalNormal breast and benign breast positive*00010Negative0000 typeIntraductal positive"56201Weakly breastFibrocystic diseaseAdenofibromaPapilloma carci nomaInvasive ductal carci nomaInvasive lobular car cinomaMedullary carcinoma Mucoid carcinoma Metastatic nodeBreast carcinomaTested73821 positive72421 positive01000
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16Strongly 06Weakly
10Negative0400 00
<30% of cells exhibiting intense staining. * 30%of cells stained, or cells exhibiting weak staining reaction.
detergent extract of an invasive ductal carcinoma positively stained by 7BIOin immunohistochemistry was used for filtration gel chromatography to isolate the 7B,0-reactive fraction. Eluted fractions were collected, concentrated, and transblotted. 7B,0
detected the M, 76,000 protein in immunoblot of the total extract and more strongly in the fraction which had been positive in ELISA (Fig. 6/4, Lanes a and b). p76 was detected with MAb 7B,0 in other breast-invasive ductal carcinomas and
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~*1B
Fig. 5. "It,,, immunopcroxidase staining of human breast carcinoma (a-c) and normal colon (</). Avidin-biotin-peroxidase-Maycr's hematoxylin counterstain. a, frozen section of an intraductal carcinoma. Very strong, homogeneous tissue staining involves membrane and cytoplasma. x 400. b. frozen section of an invasive ductal carcinoma. Immunostaining is more heterogeneous than in a and /'. Note the cytoplasmic and membrane localization of staining in positive cells. X 250. r. frozen section of lymph node metastasis. Invasive carcinoma cells are strongly stained, while lymphocytes are negative, x 250.
in a medullary carcinoma (Fig. 6,Lanes a and b\ Fig. 6C, DISCUSSION Lane a). One breast cancer metastasis to the lung showed the Monoclonal antibody 7Bio was generated using the T47D same molecular weight-positive band, while the surrounding breast carcinoma cell line as an immunogen (4). The present normal lung tissue was negative (Fig. 6C, Lanes e and g). investigation was undertaken with two goals. The first was to Tissues of Non-Breast Origin. Most normal tissue investi further characterize the 7Bi0-reactive antigen, its biochemical gated in immunohistochemistry did not react with 7B,0 (Table 2). However, ducts in the parotid gland and apocrine glands of characteristics, and expression in cells in culture and in fresh human tissue specimens. The second was to study the distri the skin showed irregular weak cytoplasmic 7B|0 staining, dif bution of this antigen in normal and tumoral breast and in fuse or in fine dots. In colonie mucosa, the apex of the epithelial cells was also stained, in the form of a thin continuous line other human tissues. Immunoblotting analysis of N P40 extracts of T47D cells and of cell-free culture supernatants identified a surrounding mucous vacuoles. No staining was observed in M, 76,000 antigen. These results have been confirmed by imbasal cells (Fig. 5d). Some glands of the endometrium in the munoprecipitation of [15S]methionine-labeled T47D cell ex luteal phase showed a focal reaction, while glands in the follictracts, showing that 78,precipitated a M, 76,000 antigen, ular phase were negative. In benign tumors, some diffuse cyto plasmic staining was found in an ovarian cystadenoma and in instead of the M, 32,000 protein precipitated in preliminary experiments (4). Further immunoblotting and immunoprecipithe ductal cells of a cystadenolymphoma of the parotid gland. A thyroid follicular adenoma and an amygdaloid cyst were tation of cytosol and membrane fractions of T47D cells detected the same M, 76,000 antigen. The p76 was identified also in negative. In carcinomas tested from sites other than breast, fresh breast carcinoma tissue extracts and the immunoreactive colonie adenocarcinomas, one epidermoid esophagus carci noma, and some cells in two ovarian serous cystadenocarcinofraction was isolated using gel filtration in the same molecular weight range. mas reacted with 7B1().Melanomas and lymphomas were neg ative. One colon and one ovarian serous carcinoma extract each A significant decrease in 7B1(,binding to cancer cell extracts was tested by immunoblot, and the presence of p76 was detected after protease type VI, trypsin, and subtilisin proteolytic treat by 7B, (Fig. 6B, Lane c; Fig. 6C, Lane c). ment and a strong reduction of 7B,0 immunoreactivity with 7083
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CHARACTERIZATION
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B
Fig. 6. Western blot analysis of tumor ex tracts with 7B,0. Tumors were extracted as described in "Materials and Methods." A. 7B,n immunoblotting of a ductal invasive carcinoma crude extract. Flectrophoresis was run in so dium dodecyl sulfate-5 to I5'c gradient polyacrylamide. After gel filtration chromatography. an isolate showing ~It,,, immunoreactivity on ELISA wastransblottedand tested for bind ing with 7B,! " Lane . a, tumor total extract: lane b. 7B,0-positive fraction. The M, 76.000 band detected in Lane a is reinforced in Mne h. The immunoblot with the remaining fractions did not show 7BIUbinding (Lane c). KJ. molecular weight in thousands. B and C. electrophoresis run in sodium dodecyl sulfate-10'"; polyacrylamide. p76 was detected in two breast invasive ductal carcinomas (A, Lanes a and A) and in a colon adenocarcinoma (Lane <) and was com pared to the reactive band in T47D cell extracts (B, Lane ti). In Lane e. Er,. a mouse IgGI was applied to T47D lysate cells as negative con trol. In C, an immunoblot with 7BIOdetected p76 in a breast medullary carcinoma (Lane a). an ovarian cystadenocarcinoma (Lane c), and a breast metastasis to the lung (Lane e). In normal surrounding lung tissue extracts, no reactivity with 7B|0 w'as detected (Laneg). EU IgGI was applied as negative control in breast carcinoma (Lane A), ovarian cystoadenocarcinoma (Lane d). and lung metastasis (Lane f).
Kd
Kd
76_
76 Kd_
76_
abc
MCF7 membrane extracts after concanavalin A absorption strongly suggest the glycoprotein nature of the antigen. Consid ering the frequent presence of sialic acid in antigenic determi nants of breast carcinoma-associated antigens, as mucins rec ognized by MAbs DF, (15), B72-3 (16), or Cal (17), we treated T47D lysate with neuraminidase. No loss of 7Bi0 reactivity was noted, suggesting that sialic acid is not part of the 7B,,,-reactive epitope. Distribution and characterization of the 7B|0-reactive antigen was further evaluated in live cells and fresh tissues, comparing biochemical expression to immunoreactivity on frozen sections. Indeed, from our previous work, we knew that immunostaining in paraffin sections was weaker and more heterogeneous than in frozen sections of tissue specimens, with loss of membrane staining. Partial or total reactivity loss after formalin fixation and paraffin embedding was already reported for other MAbs directed against epithelial cell surface antigens (18, 19). Our present study was performed on milk-secretory cells and HMFGM, cultures of normal breast epithelial cells, normal breast, benign tissue, and malignant breast tumors in order to determine how the antigen was expressed and whether it could be used as a differentiation marker. The work was further extended to other frozen specimens of normal and cancer tissues to evaluate the specificity of 7B]0 reactivity. Milk cells represent a specific state of breast epithelial cell differentiation, and HMFGM are known to carry antigenicity similar to that of the mammary epithelial cell membrane (20). In fact most of the described MAbs against breast tumorassociated antigens are reactive with these components of the lactating mammary gland (Refs. 15 and 21 to 26; reviewed in Ref. 3). No 7B,o reactivity with HMFGM was detected by ELISA, immunoblotting, or electron microscopy. Neither skimmed milk bound 7B,0. Conversely, normal breast cells in culture and in frozen tissue sections showed 7B,0 reactivity, although heterogeneity existed in intensity, number of cells stained, and tissue localization. Some immunostaining hetero geneity was also observed in nonmalignant mammary epithelia.
a b c d e
a b ed e f g
This heterogeneous expression of the antigen, which is present in some normal resting cells and is not expressed in HMFGM and milk, suggests a relationship between expression variations and differentiation stages. In breast carcinoma, the antigen was strongly and homogeneously expressed in the intraductal and lobular carcinomas tested, while in invasive ductal carcinomas, it was heterogeneously detected, with variations in the intensity of 7B10 staining and number of positive cells. While some carcinomas showed intense, generalized 7BIO binding, others were only weakly reactive, and in four specimens no binding was found. These results are in accordance with our previously reported data on breast cancer cell lines, two of seven being poorly reactive with 7B,0 (H466B, MDA-MB231) and one (BT20) being negative. 7B,,i reactivity in normal and nonmalignant tissues of nonbreast origin was quite limited. Cells of nonepithelial origin did not bind 7BI0. In epithelial cells, only ducts in the parotid and in the sweat glands, colonie mucosa, and some glands of the luteal endometrium showed reactivity. These data confirmed previously reported results of immunostaining on paraffin sec tions except an increasing of colon surface epithelium staining. Conversely, some differences in 7B10 reactivity appeared in frozen specimens of cancer tissues, in comparison with paraffin sections. In addition to carcinomas of the breast where the strongest reactivity was observed, a carcinoma of the esophagus and colon carcinomas were stained. Some cells of two ovary serous cystadenocarcinoma were also stained. The p76 antigen detected by 7B|0 in fresh tumor specimens from breast, using immunoblotting, was found also on the colon and ovary carci noma extracts. Conversely, immunoblotting of normal lung tissue surrounding a breast carcinoma metastasis did not show 7B10reactivity. In light of the data concerning its immunocytochemical re activity, MAb 7B,o would be an interesting tool for the detection of breast malignant cells in distal metastasis. In particular the lack of cross-reactivity with bone marrow cells permits its use in the detection of bone marrow metastasis. Cytofluorometry
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CHARACTERIZATION
AND DISTRIBUTION
Table 2 7Bla immunostaininf; of frozen non-hreast human tissues Normal tissues Strongly positive" Colon (3)* Weakly positive' Apocrine glands of skin (2) Parotid ducts (1) Endometrium (luteal phase) (2) Negative Skin (2): epidermis, sebaceous glands Parotid acini (1) Endometrium (3) (follicular phase) Cervix (5) Ovary (3) Thyroid ( I ) Larynx (1) Lung (3) (bronchi, alveoli) Pancreas(1) Liver(2) Gallbladder (1) Striated muscle (2) Smooth muscle (5) Lymph node (4) Bone marrow (4) Benign tumors Weakly positive Ovarian serous cystadenoma ( 1) Parotid cystadenolymphoma (ducts) (I) Negative Thyroid adenoma ( 1) Tonsil cyst (I) Malignant tumors Weakly positive Colon adenocarcinoma (3) Rectal adenocarcinoma (I) Ovary serous cystadenocarcinoma (2) Esophagus epidermoid carcinoma (1/2) Negative Esophagus epidermoid carcinoma (1/2) Stomach adenocarcinoma (poorly differentiated) (1) Pancreas vipoma ( 1) Ovarian undiffcrentiatcd carcinoma (I) Endometrial adenocarcinoma (I) Uterine cervix carcinoma (I) Laryngeal carcinoma ( 1) Lung small cell carcinoma (2) Melanoma (3) Lymphoma (2) 0 30% of cells exhibiting intense staining. * Numbers in parentheses, number of specimens tested. c 30% of cells stained, or cells exhibiting a weak staining reaction.
bodies reactive with breast tumor-associated antigens. Adv. Cancer Res., 43: 143-173. 1985. 2. Frankel, A. E., Boyer. C. M., and Bast. R. C. Inimunobiology of Human Breast Cancer. Immunology of Malignant Diseases, pp. 167-224. United Kingdom: Lancaster. MTP Press. Ltd., 1987. 3. Price, M. R. Breast-cancer-associated antigens defined by monoclonal anti bodies. Subcell. Biochem. Immunol. Aspects, 12: 1-30. 1988. 4. Pancino. G.. Charpin. C"., Calvo. F.. Guillemin, M. C., and Roseto. A. A novel monoclonal antibody (7Bi0) with differential reactivity between human mammary carcinoma and normal breast. Cancer Res.. 47:4444-4452. 1987. Kufe. D.. Inghirami. G.. Abe. M.. Hayes. D.. Justi-V\ heeler. H., and Schlom. J. Differential reactivity of a novel monoclonal antibody (DF3) with human malignant versus benign breast tumors. Hybridoma, 3: 223-232. 1984. Keydar, L.. Chen, I.. Karby. S., Weiss. F. R., Delarea, J.. Rendu, M.. Chaitcik. S., and Brenner, H. J. Establishment and characterization of a cell ITne of human breast carcinoma origin. Eur. J. Cancer. IS: 659-670. 1979. Soule, H. D., Vazquez, J., Long, A., Albert, S.. and Brennan. M. A human cell line (MCF7) from breast carcinoma. J. Biol. Chem., 24S: 6251-6253. 1973. Polanowski. F. P.. Gaffney, E. V.. and Burke. R. E. HBL-100: cell line established from breast milk. In Vitro (Rockville). 12: 328-336. 1976. Car#nde Fromcntel. C., Nardeux. P. C., Soussi, T., Lavalle. C., Estrade, S.. Carloni. G.. Chandrasekaran. K.. and Cassingena. R. Epithelial HBL-100 cell line derived from milk of an apparently healthy woman harbours SV40 genetic information. Exp. Cell Res.. IMI: 83-94. 1985. Soule, H. O., and McGrath. C. M. Simplified method for passage and longterm growth of human mammary epithelial cells. In Vitro (Rockville), 22:612, 1986. Stampfer, R. C., Hallowes, R. C., and Hackett, A. J. Growth of normal human mammary cells in culture. In Vitro (Rockville), 16: 415-425, 1980. Keenan, T. W., Moire. D. J.. Olson, D. E., Yunghans. W. N., and Patton. S. Biochemical and morphological comparison of plasma membrane and milk fat globule membrane from bovine mammary gland. J. Cell Biol., 44: 80-93. 1970. Lowry, O. H., Rosebrough, N. J.. Farr, A. L.. and Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem.. 193: 265-275, 1951. Colimon, R., Mazeron, M. C., Roseto, A., and Perol, Y. Monoclonal anti bodies. A new approach to the diagnosis of cytomegalovirus infections. Birth Defects Orig. Art. Ser.. 20: 390-391. 1984. Sekine. H., Ohno, !.. and Kufe, D. W. Purification and characterization of a high molecular weight glycoprotein detectable in human milk and breast carcinomas. J. Immunol., 135: 3610-3615, 1985. Johnson. V. G., Schlom. J.. Paterson, A. J.. Bennett, J.. Magnani. J. L.. and Colcher. D. Analysis of a human tumor-associated glycoprotein (TAG-72) identified by monoclonal antibody B72.3. Cancer Res.. 46: 850-857, 1986. Bramwell. M. E., Bhavanandan. V. P., Wiseman, G., and Harris. H. Structure and function of the Ca antigen. Br. J. Cancer. 48: 177-183, 1983. Momburg. F.. Moldenhauer, G., Hammerbry. G. J.. and Mller,P. Immunohistochemical study of the expression of a A/r 34,000 human epitheliumspecific surface glycoprotein in normal and malignant tissues. Cancer Res., 47: 2883-2891, 1987. Dempsey, P. J., De Kretser. A., Brown. R. W., Whittehead, R. H., and Jose, D. G. A monoclonal antibody CIBrl7 recognizes a myoepithelium-specific antigen in human mammary gland. Int. J. Cancer, 37: 857-866. 1986. Ceriani, R. L., Thompson, K., Peterson, J. A., and Abraham, S. Surface differentiation antigens of human mammary epithelial cells carried on the human milk fat globule. Proc. Nati. Acad. Sci. USA, 74: 582-586. 1977. Arklie. J.. Taylor-Papadimitriou. J.. Bodmer. W.. Egan, M.. and Millis. R. Differentiation antigens expressed by epithelial cells in the lactating breast are also detectable in breast cancers. Int. J. Cancer, 28: 23-29. 1981. Edwards, P. A. W., and Brooks, I. M. Antigcnic subsets of human breast epithelial cells distinguished by monoclonal antibodies. J. Histochem. Cytochem.,32: 531-537, 1984. Ceriani, R. L., Peterson, J. A., Lee, J. Y., Moneada, R.. and Blank. E. W. Characterization of cell surface antigens of human mammary epithelial cells with monoclonal antibodies prepared against human milk fat globule. So matic Cell Genet., 9: 415-427. 1983. Hilkens, J., Buijs, F., Hilgers, J., Hageman, P., Calafat, J., Sonnenberg, A., and Van der Valk, M. Monoclonal antibodies against human milk fat globule membranes detecting differentiation antigens of the mammary gland and its tumors. Int. J. Cancer, 34: 197-206, 1984. Edwards, P. A. W., and Foster, C. S. Monoclonal antibodies that bind to normal and neoplastic breast epithelial cells distinguish subsets of normal breast epithelial cells. In: Monoclonal Antibodies and Cancer, pp. 285-292. New York: Academic Press. 1983. Price. M. R.. Edwards. S., Owainati. A.. Bullock. J. E.. Ferry. B.. Robins. R. A., and Baldwin. R. W. Multiple epitopes on a human breast-carcinomaassociated antigen. Int. J. Cancer. 36: 567-574, 1985. Miglierina, R.. Aubre, B.. Pancino. G., Calvo, F., and Roseto, A. Detection of rare metastatic breast cancer cells in the bone marrow by flow cytometry using monoclonal antibody 7B,0 in an experimental model. J. Tumor Marker Oncol., 4: 45-53, 1989.
5. 6. 7. 8. 9.
10.
11. 12.
studies revealed its ability to detect rare T47D cells in an experimental model of a mixture of living T47D cells and normal human bone marrow cells (27). We are now extending this model to bone marrow biopsies from breast cancer patients. The differing reactivities observed in various physiological and pathological states of the mammary gland also suggest that this antigen could be involved in differentiation stages. Further studies are in progress to evaluate this aspect, as well as the purification of the 7B,-reactiveantigen. ACKNOWLEDGMENTS
We gratefully thank Dr. J. Feries and Jean-Philippe Barque for helpful discussion, Laurent Joron for his previous help in antibody purification, O. Champy and M. Velut for technical assistance, and C. Guilbert for typing the manuscript.
22. 23.
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26. 27.
REFERENCES
I. Schlom, J., Colcher, D., Horan Hand, P., Greiner, J., Wunderlich, D., Weeks, M., Fisher, P. B., Noguchi, P., Petska, S., and Kufe. D. Monoclonal anti
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