Proc. Natl. Acad. Sci. USAVol. 82, pp. 7409-7413, November 1985Immunology

Monoclonal antibody that defines human myoepithelium(mammary carcinoma/monoclonal antibody-aided prognosis/differentiation)

SHAHNAZ HASHMI DAIRKEE, CARLENE BLAYNEY, HELENE S. SMITH, AND ADELINE J. HACKETTPeralta Cancer Research Institute, 3023 Summit Street, Oakland, CA 94609

Communicated by Donald A. Glaser, July 12, 1985

ABSTRACT We have isolated a mouse monoclonal anti-body that, upon immunohistochemical localization in frozensections, displays specificity for human myoepithelial cells inthe resting mammary gland, sweat glands, and salivary glands.Furthermore, this antibody was strongly and homogeneouslyreactive with frozen sections of 3 of 60 breast carcinomaspecimens. Using immunolocalization techniques in conjunc-tion with polyacrylamide gel electrophoresis, we have deter-mined that the reactivity of this monoclonal antibody is directedtoward a 51,000-dalton keratin polypeptide. The potential usesof this antibody in the prognosis of human mammary carci-noma and in understanding the role of the myoepithelium indevelopment and differentiation are discussed.

Myoepithelial cells are derived from ectoderm and are knownto exhibit both epithelial and mesenchymal characteristics (1,2). They are situated between acinar or ductal luminal cellsand the basal lamina in a number of secretory glands such asbreast, lacrimal, eccrine, and apocrine sweat glands andvarious salivary glands (3).We report here on a monoclonal antibody directed toward

a cytoskeletal keratin that is unique to the myoepithelial or"basal" layer of epithelial cells in human mammary ducts,sweat glands, and salivary glands. Using polyacrylamide gelelectrophoresis and immunoblotting, we have found theapparent molecular weight of this basal epithelium-specifickeratin to be 51,000 daltons. The antibody is generallynonreactive with luminal epithelium, stroma, muscle, fat, andvasculature. Therefore, it appears to be a useful tool instudying the role of the myoepithelium in the normal devel-opment of the breast, during lactation and involution, and inmalignancy and metastasis.

Recently, considerable advances have been made in tumordiagnosis by immunocytochemical typing of intermediatefilaments. For example, carcinomas are characterized byantibodies to cytokeratins; sarcomas of muscle cells, byanti-desmin; nonmuscle sarcomas, by anti-vimentin; andgliomas, by anti-glial fibrillary acidic protein (for review, seeref. 4). The myoepithelium-specific monoclonal antibodydescribed in this report has allowed us to define and char-acterize subclasses of human mammary carcinomas thatwould not be possible to distinguish by conventional tech-niques. The potential application of this antibody in theprognosis of human mammary carcinoma is discussed.

MATERIALS AND METHODSImmunization. BALB/c mice raised under pathogen-free

conditions were injected via subcutaneous or intravenousroutes for 5 weeks at weekly intervals with whole-cellpreparations of a secondary culture of an infiltrating ductalbreast carcinoma. For each inoculation, 106 cells, which hadbeen cultured as previously described (5), were prepared by

scraping the monolayer with a rubber policeman into phos-phate-buffered saline. Splenocytes from immunized animalswere fused with SP2/0 mouse myeloma cells in the presenceof polyethylene glycol (Mr 4000; Merck) by the procedure ofKohler and Milstein (6). Monoclonal antibodies were initiallyscreened by an enzyme-linked immunoassay. The myoepi-thelium-specific monoclonal antibody designated 312C8-1was picked on the basis of its considerably higher opticaldensity with immunogen as compared to skin fibroblasts fromthe same individual.Immunocytochemical Staining. Normal mammary tissues

were obtained primarily from fresh reduction mammoplas-ties. After dissection of apparent fat, the tissues were keptfrozen at -70TC until use. Fresh surgical specimens ofprimary breast carcinoma, salivary glands, and skin associ-ated with either the normal or malignant mammary gland orfrom scalp were also stored frozen until use. The reactivityof the monoclonal antibody with 6-gm unfixed or acetone-fixed frozen sections of tissue was visualized by using anavidin-biotin amplification procedure (Vector Laboratories,Burlingame, CA). The procedure consists of an initial block-ing step with nonimmune serum, incubation with a 1:1000dilution ofan ascites preparation ofthe monoclonal antibody,incubation with biotin-conjugated anti-mouse Ig and thenwith avidin-biotinylated peroxidase conjugate, and finallyprecipitation of colored product by using diaminobenzidine.Sections were counterstained with hemotoxylin and eosin,dehydrated, and mounted in Histomount.

Ouchterlony Analysis. Serum-free supernatants were con-centrated 10-fold by ammonium sulfate (50%) precipitation,and the class of immunoglobulin present in the concentratewas determined by using commercially available anti-class(IgM) and anti-subclass (IgG1, G2, and G2B) antisera (Miles).

Preparation of Cytoskeletal Extracts. Frozen blocks of afew specimens that had been analyzed previously by im-munocytochemical staining were chosen for the preparationof cytoskeletal extracts. Sections (10 ,um thick) were cut fromthe blocks and extracted by a modification of the procedureof Tseng et al. (7). They were first homogenized in 25 mMTris HCl (pH 7.4) containing 1 mM phenylmethylsulfonylfluoride. The resultant suspensions were centrifuged at10,000 x g for 10 min at 4°C. The supernatants werediscarded, and the residual water-insoluble proteins werethen solubilized by heating at 100°C for 5 min in 1% sodiumdodecyl sulfate/5% 2-mercaptoethanol/25 mM Tris HCl, pH7.4, and designated cytoskeletal extracts.

Gel Electrophoresis and Immunoblotting. One-dimensionalNaDodSO4/PAGE was performed on the cytoskeletal ex-tracts as described by Laemmli (8). Proteins from unstainedpolyacrylamide gels were transferred to nitrocellulose paperby the method of Towbin et al. (9). To visualize the proteinbands and the molecular weight standards, the blot wasstained with Ponceau S for 2 min. The molecular weightstandards were marked on the blot with a ball-point pen tofacilitate estimation of the apparent molecular weight of thetarget antigen. Then the blot was incubated sequentiallythrough all of the steps outlined above for the avidin-biotin-

7409

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Proc. Natl. Acad. Sci. USA 82 (1985)

amplified immunocytochemical staining procedure. The per-oxidase substrate used in this case was 4-chloronaphthol.

RESULTS

The monoclonal antibody described in this report will bereferred to as 312C8-1. Reactivity with 312C8-1 was observedwithout pretreatment of cryostat sections with enzymes orfixatives; however, acetone-fixation of sections mounted onglass slides resulted in improved preservation of the speci-mens during the immunostaining procedure. Immunoglobulinsubclass determination by Ouchterlony analysis has demon-strated this monoclonal antibody to be an IgM.

bImunolocalization of Myoepithelium. Mammary gland.Normal breast tissue was derived for this study from twomain sources-namely, fresh specimens from reductionmammoplasties or normal tissue peripheral to breast carci-noma. In both cases, the material predominantly consisted ofnormal mammary ducts and connective tissue. As is gener-ally believed on the basis of ultrastructural studies (10), innormal, mature, nonlactating human mammary ducts, themyoepithelial cells are situated as a continuous layer betweenthe secretory-type epithelial cells and basal lamina. As shownin Fig. 1, 312C8-1 clearly defines the myoepithelial cells in thenormal human mammary ducts. There was no reactivity withluminal epithelial cells, connective tissue, vasculature, ormusculature. This was the general finding in the mature ductsof 13 different specimens. In the case of smaller ducts andductules in normal breast lobules, the delineation ofmyoepithelium was not as complete as in the mature ductsshown in Fig. 1.

Salivary glands. Cryostat sections from the parotid andsubmandibular glands were examined for reactivity with312C8-1. Fig. 2a shows a continuous layer of 312C8-1-

reactive basal cells, presumably the myoepithelium, in anexcretory duct. Luminal epithelium and stromal elementswere negative. The reactivity of 312C8-1 with acini showeda discontinuous layer of basal cells (Fig. 2b).Sweat glands. The human sweat glands are considered to

be the most differentiated in comparison to those of otheranimals. The basic structure of both eccrine and apocrinesweat glands mainly consists of secretory tubules. The wallsof the secretory tubules are composed ofglandular, secretorycells, and contractile, myoepithelial cells. The glandular cellsare comprised of a simple epithelium and are surrounded bythe myoepithelial cells (11). Fig. 2c shows reactivity of themonoclonal antibody 312C8-1 with only the myoepitheliumor the basal cell layer in the sweat glands of skin.Primary breast carcinomas. A total of 60 breast carcinoma

specimens were studied. The monoclonal antibody 312C8-1was reactive with several of them in two main distributionpatterns. A summary ofthe patterns of reactivity is presentedin Table 1. Epithelial cells in all of the specimens weredesignated as malignant or nonmalignant on the basis ofhistological appearance. The distribution of reactivity overthe tumor population was either homogeneous or heteroge-neous. Three of 60 tumors were strongly and homogeneouslyreactive (Fig. 3a). Thirty-five of 60 tumors had either veryweak or no visible reactivity at all with the antibody.Twenty-two other tumor specimens were found to be quiteheterogeneous in the distribution of the antigen. In several ofthese specimens, the antibody mainly circled some groups ofcells (Fig. 3b). Most often the encircled patches appeared toconsist of malignant cells, but it was difficult to ascertainwhether some tumor cells in these specimens were exhibitingthe antigen or whether the reactivity represented residualmyoepithelial cells. In a few specimens, some tumor nestswere strongly and homogeneously reactive, while neighbor-

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FIG. 1. Mature normal mammary ducts from two reduction mammoplasty specimens showing reactivity with 312C8-1. Arrows indicatemyoepithelial cell layer at the base of the duct. (x400.)

7410 Immunology: Dairkee et al.

Proc. Natl. Acad. Sci. USA 82 (1985) 7411

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FIG. 2. Reactivity of 312C8-1 with salivary gland secretory duct showing continuous myoepithelial layer (a), salivary gland acini showingstrong staining of the basket-like myoepithelial cells (b) and sweat glands in skin displaying intense reactivity of the myoepithelium (c). Arrowsindicate the myoepithelial layer. (x400.)

ing tumor nests were nonreactive. The reactivity in allmalignant specimens also was restricted to tumor cells and allother cell types were nonreactive.

Characterization of 312C8-1-Reactive Antigen: NaDod-S04/PAGE and Immunoblotting. In experiments aimedtoward defining the antigen(s) recognized by the 312C8-1monoclonal antibody, we used cytoskeletal extracts of a

strongly and homogeneously reactive breast carcinoma.Cytoskeletal extracts were preferred for this study becausethe immunoperoxidase localization studies demonstrated afibrillar array, typical of cytoskeletal proteins. As illustratedin Fig. 4, several polypeptides were visualized by theCoomassie blue dye. However, the results of the immuno-blotting experiments indicated a 312C8-1 reactive polypep-tide, most probably a keratin, with an apparent molecularweight of 51,000 in the tumor, which was found to be reactivein frozen sections. Tumors that were nonreactive by im-munolocalization on frozen tissue were also nonreactive inthe immunoblots. Purified proteins such as actin, vimentin,fibronectin, and a-lactalbumin were also nonreactive with312C8-1. We also attempted similar studies on normal breasttissue; not surprisingly, considering the generally low level ofthe myoepithelial component in comparison to other celltypes in the mammary gland, we were unable to detect any312C8-1-reactive moieties by immunoblotting.

DISCUSSIONIn this report, we demonstrate the reactivity of a monoclonalantibody that will be important toward understanding thebiology of the human myoepithelial cell. This monoclonalantibody specifically recognizes a keratin present in themyoepithelium of human mammary, salivary, and sweatglands, while it is unreactive with the luminal epithelium or

other cells in these tissues. Therefore, it also will be useful indefining the role and relationship of the various cytokeratinsduring myoepithelial and luminal epithelial development anddifferentiation. Precedence for such myoepithelium-specific

keratins has been established in the mammary gland ofrodents (12, 13). However, the antibodies used in thesestudies do not crossreact with their human counterparts; andthus, they cannot be applied toward investigations of thehuman myoepithelium.

Studies on the myoepithelial component of various humantissues have been greatly hampered by the lack of suitablemarkers. Previous immunocytochemical studies have uti-lized anti-actin and anti-myosin antibodies to define themyoepithelium in the benign and malignant human mammarygland (14-17). However, these proteins are generally presentin several other cell types and, thus, by themselves, are

inadequate as markers of myoepithelium.Breast carcinomas displayed an interesting variation in

reactivity with the 312C8-1 antibody. The majority of breastcancers were either negative or showed only occasional areasof reactivity, with a pattern of positive cells completelyencircling nests of tumor cells, indicative of residual normalmyoepithelium. Five percent ofthe carcinomas were stronglyand homogeneously reactive over the entire tumor popula-tion, while another 7% displayed strong reactivity with somegroups and absolutely no reactivity with other often neigh-boring groups of tumor cells.A variety of antigens have been used to identify and

separate different cell types of the lymphopoietic lineage andmonoclonal antibodies against differentiation antigens areplaying a significant role in the classification and diagnosis ofheterogeneous groups of tumors such as leukemias (18).Using the leukemias as a model system, we hypothesize thatthe different patterns of reactivity with 312C8-1 observedamong breast carcinomas represent the malignant transfor-mation of cells at different stages of mammary cell differen-tiation. We suggest that the homogeneously reactive tumorsrepresent malignant cells of myoepithelial origin, while thosethat are unreactive represent malignancies of luminal epithe-lial origin. Those tumors with both 312C8-1-positive and-negative nests of malignant cells could have arisen from themalignant transformation of the stem cells themselves, thus

Table 1. Immunolocalization of monoclonal antibody 312C8-1 in frozen sections of breastcarcinoma specimens

Histologic type

Pattern of 312C8-1 Invasive Infiltrating Uncertainreactivity lobular ductal (ductal or lobular) Medullary Total

Strong, homogeneous 0 3 (5%t) 0 0 3 (5%)Strong, heterogeneous 0 4 (7%) 0 0 4 (7%)Circling tumor nests 1 (2%) 16 (27%) 1 (2%) 0 18 (30%)Negative 8 (13%) 22 (37%) 4 (7%) 1 (2%) 35 (58%)

Immunology: Dairkee et al.

Proc. Natl. Acad. Sci. USA 82 (1985)

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FIG. 3. Reactivity of 312C8-1 with mammary carcinoma specimens. (a) Strong, homogeneous reactivity with tumor cells (arrow). (b)Heterogeneous pattern of reactivity encircling tumor nests. Note the lack of reactivity with stromal components in both specimens. (x 180.)

implying a common precursor for the myoepithelial andluminal epithelial lineages. Other studies have also concludedthat luminal epithelial and myoepithelial cells are derivedfrom a common precursor (19). The low frequency of 312C8-1-positive tumors is consistent with their myoepithelial ori-gin, since ultrastructural and immunocytochemical studiesalso suggest that only a small percentage of breast carcino-mas are indeed derived from myoepithelium (17, 20). All ofthese lines ofevidence are consistent with the hypothesis thatthe pattern of reactivity or the lack of reactivity of a tumorwith 312C8-1 antibody indicates its origin either from thecommitted myoepithelial or luminal epithelial cell or from theuncommitted, common stem cell. Since tumors are known toectopically express gene products normally expressed byunrelated cells, and furthermore, since clonal origin for anytumor cannot be assumed, this hypothesis cannot be rigor-ously proven with the data presently available. However, itsmajor contribution is that it provides a conceptual frameworkin which the expression of other mammary antigens can beevaluated toward an effort to test the feasibility of such anhypothesis.Thus far, we have not observed any correlation between

immunostaining pattern, histologic type, and grade of themammary carcinomas examined. However, a follow-up onthe subsequent fate of the patients described above in the

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FIG. 4. NaDodSO4/polyacrylamide gel electrophoresis withCoomassie blue staining of protein bands (lanes 1-3) and immunoblotanalysis of mammary carcinoma specimens (100 ,ug of extract perlane) (lanes 4-6). Lanes: 1, molecular weight markers; 2, 312C8-1-reactive carcinoma; 3, 312C8-1-nonreactive carcinoma; 4, 312C8-1-reactive tumor (note the reactivity of the 51-kDa band); 5, 312C8-1-nonreactive tumor (note the lack of reactivity of the 312C8-1monoclonal antibody). Nonimmune serum used as the negativecontrol in the immunoblotting experiments did not stain any bands ineither 312C8-1-negative or -positive tumors.

7412 Immunology: Dairkee et al.

Proc. Natl. Acad. Sci. USA 82 (1985) 7413

various categories and the period of their survival couldprovide useful information on the prognostic value of the312C8-1 monoclonal antibody. Similarly, this antibody mayalso be useful in immunocytochemical investigations ofadnexal tumors of the skin and of tumors originating fromsalivary glands.

Development of the monoclonal antibody was funded by a grantfrom Xoma Corporation.

1. Cutler, L. S. & Chaudhry, A. P. (1973) J. Morphol. 140,343-354.

2. Franke, W. W., Schmid, E., Frudenstein, C., Appelhans, B.,Osborn, M., Weber, K. & Keenan, T. W. (1980) J. Cell Biol.84, 633-654.

3. Hamperl, H. (1970) Curr. Top. Pathol. 53, 161-220.4. Osborn, M. & Weber, K. (1983) Lab. Invest. 48, 372-394.5. Smith, H. S., Hackett, A. J., Lan, S. & Stampfer, M. R.

(1982) Cancer Chemother. Pharmacol. 6, 237-244.6. Kohler, G. & Milstein, C. (1975) Nature (London) 256,

495-497.7. Tseng, S. C. G., Jarvinen, M. J., Nelson, W. G., Huang, J.,

Woodcock-Mitchell, J. & Sun, T. T. (1982) Cell 30, 361-372.8. Laemmli, U. K. (1970) Nature (London) 277, 680-685.

9. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl.Acad. Sci. USA 76, 4350-4354.

10. Nesland, J. M., Hoie, J. & Johannssen, J. V. (1983) Diagn.Histopathol. 6, 51-67.

11. Kurosumi, K., Shibasaki, S. & Ito, T. (1984) Int. Rev. Cytol.87, 253-329.

12. Asch, B. B., Burstein, N. A., Vidrich, A. & Sun, T. T. (1981)Proc. Natl. Acad. Sci. USA 78, 5643-5647.

13. Allen, R., Dulbecco, R., Syka, P., Bowman, M. & Armstrong,B. (1984) Proc. Natl. Acad. Sci. USA 81, 1203-1207.

14. Gabbiani, G., Csank-Brassert, J., Schneeberger, J. D.,Kapanci, Y., Trencher, P. & Holborow, E. J. (1976) Am. J.Pathol. 83, 457-474.

15. Macartney, J. C., Trevithick, M. A., Kricka, L. & Curran,R. C. (1979) Lab. Invest. 41, 437-445.

16. Bussolati, G., Botta, G. & Gugliotta, P. (1980) Virchows Arch.B 34, 251-259.

17. Gusterson, B. A., Warburton, M. J., Mitchell, D., Ellison, M.,Munro-Neville, A. & Rudland, P. S. (1982) Cancer Res. 42,4763-4770.

18. Greaves, M. & Janossy, G. (1978) Biochim. Biophys. Acta 516,193-230.

19. Rudland, P. S., Ormerod, E. J. & Paterson, F. C. (1980) J. R.Soc. Med. 73, 437-442.

20. Ahmed, A. (1973) J. Pathol. 113, 129-135.

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