Isolation and Characterization of a Monoclonal Antibody Specific for Epithelial Cells

ICANCER RESEARCH 45. 783-790. February 1985|

Anne W. Hamburger,1 Yvonne A. Reid, Barbara Pelle, George E. Milo,2 Inge Noyes, Henry Krakauer, and J. Peter Fuhrer Cell Culture Department, American Type Culture Co/lection, Rockville, Maryland [A. W. H., Y. A. Ft., B. P./,' Comprehensive Cancer Center. Ohio State University, Columbus, Ohio [G. E. M., I. N.Â¡;and the National Institute of Allergy and Infectious Diseases [H. K.Â¡and the National Cancer Institute, Bethesda. Maryland [J. P. F.]

ABSTRACT monoclonal antibodies have been isolated (9, 15, 19, 29), rela tively few monoclonal antibodies specific for epidermal cell sur Monoclonal antibodies were generated to antigens on human face antigens have been described (18). foreskin keratinocytes to identify epithelial-specific molecules. Cell cultures of human epidermis retain epidermal-specific Spleen cells from BALB/c mice, immunized with membrane functions and antigens (10, 11, 20). Thus, these keratinocyte preparations from primary expiants of foreskin epithelial cells, cultures could serve as immunogens in attempts to produce were fused with the NS-1 mouse myeloma line. Hybridoma monoclonal antibodies specific for keratinocyte cell surface an supernatants were screened for the desired immunological reac tigens. We now describe the isolation of hybridomas secreting tivity using enzyme-linked immunosorbant binding assays. Hy- monoclonal antibodies which bind selectively to cultured normal bridomas secreting antibodies reacting with epithelial cells, but keratinocytes and show a high specificity for both cultured and not fibroblasts or lymphocytes, were cloned by limiting dilution, primary epithelial tissues. and two stable clones producing immunoglobulin M K antibodies were selected for study. Evaluation of fixed paraffin-embedded human tissue by an indirect immunoperoxidase technique re MATERIALS AND METHODS vealed that the antibodies bound most strongly to normal strati fied squamous and transitional epithelium, and squamous and Cells and Media. The following human cell lines were obtained from transitional cell carcinomas. Antibodies from the cloned hybri- the American Type Culture Collection, Rockville, MD: B-lymphoblastoid domas also reacted with primary cell cultures of foreskin keratin lines: CCL 213 (Daudi), CCL 114 (RPMI 766); T-lymphoblastoid lines: CCL 119 (CCRF-CEM), CRL 1552 (Molt-3); melanoma: CRL 1424 (G- ocytes, pulmonary epithelium, fetal liver, and amnion cells, but 361), HTB 64 (Malme-64); neuroblastoma: CCL 127 (IMR 32), HTB 11 not with primary cultures of nonepithelial cells. Further testing (SK-N-SH); adrenal adenocarcinoma: CCL 105 (SW 13); carcinoma of by enzyme-linked immunosorbent assays revealed that the an tibodies reacted with some long-term cell lines derived from the pharynx: CCL 138 (Detroit 562); adenocarcinoma of the colon: CCL 220 (Colo 320 DM), CCL 218 (WiDr); adenocarcinoma of the prostate: epithelial tumors. Nonepithelial cell lines were not stained by the HTB 81 (DU 145), CRL 1425 (PC-3); carcinoma of the lung: CCL 185 antibodies. Indirect immunofluorescent studies indicated that (A549, epidermoid carcinoma), HTB 54 (CALU-1, adenocarcinoma); tran staining was confined to the cell surface. These antibodies may sitional cell carcinoma of the bladder: CRL 1473 (HT-1197), HTB-2 (RT- prove useful in studies of differentiation markers of human epi 4); squamous cell carcinoma: CRL 1623 (SCC-15); carcinoma of the thelial cells. pancreas: CRL 1420 (MIA-PaCa-2), CRL 1469 (PANC-1, ductal carci noma); carcinoma of the cervix: CCL 2 (HeLa); renal leiomyoblastoma: CRL 1440 (G-402); Wilms1 tumor: CRL 1441 (G-401); choriocarcinoma: INTRODUCTION CCL 98 (BeWO); adenocarcinoma of the breast: CRL 1500 (ZR 75-1), CRL 1504 (ZR-75-30); foreskin fibroblasts: CRL 1521 (CCD-76), CCL Specific molecular markers which identify cell types in epithelial 109 (Detroit 550); fibrosarcoma: CCL 121 (HT-1080); and Ewing's sar tissue are important for the characterization of cells grown in coma HTB 87 (SK-ES-1 ). A primate cell line from African green monkey culture and for studies of phenotypic changes in carcinogenesis, kidney cells, CCL 70 (CV 1), was also used. The human melanoma cell and they may have applications in clinical diagnosis and organ line WM-9 and a line of normal human lung fibroblasts were obtained transplantation. from the Wistar Institute. A line of epithelial cells derived from normal BALB/c mouse skin (BK-1 ) was obtained from Dr. Stuart Yuspa, National Several proteins have been identified as markers of epithelial Cancer Institute. Primary cultures of human foreskin epithelial cells, lung tissue. Autoantibodies in the serum of patients with bullous fibroblasts, pulmonary epithelia, and epithelial cells from fetal liver were pemphigus and pemphigus vulgaris have been used to detect prepared in the laboratory of Dr. George Milo. Undifferentiated prolifer epidermal-associated antigens and to study epidermal differen ating keratinocytes were prepared as follows (22). Foreskin tissue was tiation (25). Epidermal-specific histocompatibility antigens, be cut into 4-mm pieces in 4 ml of HBSS3 minus calcium and magnesium lieved responsible for immunogenicity of skin allografts, include at pH 7.2 at 21Â°. Tissue pieces were rinsed 3 times, recovered, and the SKA antigens in mice (3) and a non-H-2 alloantigen (epa) placed in 3 ml of prechilled HBSS solution containing 0.125% trypsin identified on murine epidermal cells (26). Keratin, a major differ (Worthington Enzymes, Freehold, NY) and 0.02% EGTA. After 24 hr, the entiation product of epidermal cells, is also considered to be an tissue was removed, and the upper epidermal layers were mechanically epithelial-specific marker (7, 27). While several keratin-specific separated from the dermal tissue and the attached stratum basalis. The lower layer containing the dermis and stratum basalis was placed in 3 1Recipient of support in part from Contract NO102655 from the National Institute ml of HBSS minus calcium and magnesium containing 0.2% trypsin and of Allergy and Infectious Diseases. To whom requests for reprints should be 3 The abbreviations used are: HBSS. Hanks' balanced salt solution; FBS, fetal addressed 2 Recipient of support from Grant NCI RO1CA 25907-04. bovine serum; EGTA, etnyleneglycol bis(/i-aminoethylether)-N,/V,N',W '-tetraacetic Received May 5. 1983: accepted November 5. 1984. acid; ELISA, enzyme-linked immunosorbent assay.

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0.02% EGTA, pH 3.9 to 4.2.' The tissue suspension was reincubated at diaminobenzidine and 0.03% hydrogen peroxide. Sections were coun- 8Â°for an additional 24 hr. The dermal pieces free of the stratum basalis terstained in hematoxylin:eosin. Cytocentrifuge preparations of single- were removed. Freely floating cells from the stratum basalis were re cell suspensions of human tissue or cell lines were fixed in absolute covered by centrifugation at 650 x g. These cells were seeded at 5000 methanol and stained in the same manner. cells/25-sq cm flask in Eagle's minimal essential medium supplemented Immunofluorescence Studies. Indirect immunofluorescence studies with 10% FBS and 0.75 rriM calcium (total free calcium). using monoclonal antibodies were performed on cells growing on glass Keratinocytes were serially passaged by treatment of confluent mon- coverslips or on 5-/Â¿mcryostat sections of human tissue. Cells were olayers with 0.25% trypsin in HBSS:0.02% EDTA at pH 7.2. The cells initially fixed in -20Â° methanol and acetone as described by Sun ef al. were seeded in Eagle's minimal essential medium supplemented with (27) and stained with the appropriate monoclonal antibody and then 10% FBS and 0.75 Ã•TIMcalciumat 106 cells/25 sq cm (1:2). The cells at fluorescein-conjugated goat anti-mouse IgM (Cappel Laboratories, Coch- primary passage exhibited a 75 to 90% attachment efficiency. ranville, PA). Cells were also stained with a monoclonal antibody to All tumor cell lines were grown in monolayer culture as described (2). intermediate filaments (TIB 131) obtained from the American Type Cul The lymphoblastoid lines were grown in RPMI 1640 with 20% FBS. ture Collection or a rabbit anti-human keratin heteroantibody that was Purified human peripheral blood mononuclear cells and bone marrow the generous gift of Dr. T. T. Sun, New York University. In later experi cells were separated by buoyant density centrifugation on Ficoll:Hypaque ments, cells were stained with both antibodies and then fixed in 95% by standard techniques. ethanol:5% glacial acetic acid and mounted in Gelvatol as described (24). The NS-1 cell line (obtained from Mark Lostrum, Fred Hutchinson The cells were viewed using a Zeiss epiilluminated fluorescent micro Cancer Center, Seattle, WA) was grown as a stationary suspension scope. culture in RPMI 1640 with 20% FBS and treated with 10~8 M 8-azagua- Analysis of Monoclonal Antibodies. Antibodies were tested for class nine 3 weeks prior to its use in fusions. Hybridoma cell lines were and subclass by Ouchterlony immunodiffusion using antisera specific for maintained as described for NS-1 cells. the major mouse immunoglobulin classes and for the subclasses of IgG Generation of Hybridoma Cell Lines. BALB/c mice were immunized and light chains. Immunoglobulin class was confirmed by sodium dodecyl by 2 i.p. injections (administered 15 weeks apart) of 100 ^g of membrane- sulfate gel electrophoresis of metabolically labeled antibodies as de enriched preparations (4) of primary cultures of human keratinocytes in scribed (13,16, 23). complete Freund's adjuvant. The third, and final, injection of 100 ÃŸQof membrane protein in phosphate-buffered saline was administered i.v. 3 weeks after the second injection. Three days after the final injection, the RESULTS spleens were excised, and spleen cells were fused with NS-1 cells in the presence of 40% Polyethylene Glycol 4000 as described previously (5). Generation of Monoclonal Antibodies. The immunization Hybrid cells were isolated by growth in hypoxanthineiaminopterin: with membrane preparations of human keratinocytes and sub thymidine selective medium. Ten days after fusion, culture supernatants sequent fusion resulted in the growth of 235 cultures producing were initially screened for antibody activity against primary human kera immunoglobulin. Initially, 113 cultures contained immunoglobulin tinocytes, human foreskin fibroblasts, and a human T-cell line (CCL119). with specificity for the immunizing epithelial cells and not fibro Cells that reacted with the immunizing epidermal cells, but not foreskin blasts or T-lymphocytes. However, only 3 cultures retained fibroblasts or the lymphoid line were cloned 3 times by limiting dilution specificity on continuous passage, while 95 hybridomas stopped (19). Cell lines of interest were frozen in 10% dimethyl sulfoxide and producing antibody, and 15 produced nonspecific antibody. The stored in liquid nitrogen. 3 original cultures producing specific antibodies were cloned 3 ELISA for Antibody Activity. A modification of the ELISA (12) for detecting antibody activity in the culture supernatants was used. Test times. Two clones from one original parent well retained the cells were either grown to confluency on 96-well polystyrene microtiter desired specificity (clones Ep-16 and Ep-17). Both antibodies plates (Costar) or fixed onto plates as suspensions with 0.25% glutaral- were found to be IgM K as determined by immunodiffusion. The dehyde at a final concentration of 2 x 105 cells/well. Cells were incubated metabolically radiolabeled monoclonal antibodies were isolated sequentially with hybridoma culture supernatants, rabbit anti-mouse by immunoprecipitation and analyzed by sodium dodecyl sulfate: immunoglobulin (Miles Laboratories, Kankakee, IL), and peroxidase- gel electrophoresis. Both antibodies showed only one heavy and conjugated goat anti-rabbit IgG (Miles). Substrate (o-phenylenediamine) one light chain (data not shown). The NS-1 light chain was not was added, and absorbance was read at 492 nm in a Multiscan spectra- expressed. photometer (Flow Laboratories, Springfield, VA). All assays were per Reactivity of Monoclonal Antibodies with Long-Term Cell formed in duplicate. Supernatants giving 3 times background values Lines and Primary Cell Cultures. Antibodies produced by clones were considered positive. A monoclonal antibody, generated in this Ep-16 and Ep-17 were titered by ELISA on a battery of long- laboratory that reacts to HLA antigens, was used as a positive control in all assays. Spent medium from the NS-1 parent line was used as the term human cell lines and human primary epithelial cell strains negative control in the binding assays. (Chart 1). As the titration curves were similar, only data from Immunocytochemistry. Autopsy-derived samples of human tissue or clone 16 are presented. Both antibodies bound strongly to the surgical biopsies of normal and malignant tissue were used. The tissues immunizing human epithelial cells to a 1:256 dilution. The slight were fixed in Bouin's solution and embedded in paraffin. Six-/Â¿msections binding to human foreskin fibroblasts (CCL 109, CRL 1521), B- were deparaffinized, and endogenous peroxidase activity was blocked cells (CCL 213), or T-cells (CCL 119) observed at dilutions of with 3% hydrogen peroxide in methanol. Sections were incubated se less than 1:32 was presumed to be nonspecific. quentially with undiluted monoclonal antibody, biotinylated anti-mouse To define the cellular specificities of the antibodies, we exam immunoglobulin (Vector Labs, Burlingame, CA), and avidin-biotinylated ined their binding reactivities to a panel of established cell lines. horseradish peroxidase complex as described (21, 22). Peroxidase activ ity was identified by incubating sections with a solution of 0.1% 3,3'- The results are shown in Chart 2. Both antibodies reacted most strongly with lines derived from squamous keratinizing epithelia ' This unphysiological pH (3.9 to 4.2) preferentially kills the dermal fibroblasts (i.e., a line derived from a squamous pharyngeal tumor, a squa but does not kill (100% survival by trypan blue exclusion) the keratinocytes. Even the continued exposure of the stratum basalis attached to the dermis at 8Â°for an mous cell carcinoma, and transitional cell carcinoma of the additional 24 hr does not kill the keratinocytes. This process selectively produces bladder). The antibodies reacted with some of the tumor lines proliferating keratinocytes from the stratum basalis. derived from simple epithelia (adenocarcinoma of the prostate

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cell lines tested were similar for both monoclonal antibody clones. â€¢ Human Epithelium These similarities suggested that the antibodies may bind the â€¢ CRL1521 same antigen. To test this hypothesis, binding inhibition experi A CCL 109 ments were performed with radiolabeled antibodies (14). In these O CCL 213 studies, the binding of 14C-labeled Ep-17 was reduced 80% by G CCL 119 competitive binding of unlabeled Ep-16 derived from an ascites (data not shown). These results indicated that both antibodies were specific for the same epitope. Subsequent experiments, therefore, were performed with Ep-16 only. Epithelial-specific Staining of Paraffin-embedded Human Tissue. To evaluate the effectiveness of the monoclonal antibody in immunohistochemical staining, Ep-16 was examined by an indirect immunoperoxidase technique on sections of paraffin- embedded fixed human tissue. Ep-16 reacted most strongly with stratified squamous epithelia including neonatal foreskin, adult skin, and esophagus (Table 1). Antibody staining was observed in all layers of the epidermis, including the basal layer, where epithelial stem cells are located (Fig. 1). Staining was not ob served in the dermis. Ep-16 antibody also reacted with hair follicles and sebaceous glands. Hassal's corpuscles in the thy mus, believed to be epithelial cells of endodermal origin (24), stained strongly, while thymic lymphocytes did not bind the antibodies (Fig. 2). The antibodies reacted moderately with distal convoluted and collecting tubules of the kidney, fine ductal system of the pancreas, and pancreatic acinar cells (Table 1). The antibodies bound weakly or not at all to simple or nonsqua- mous epithelia including covering cells of the trachea, bron O 2 4 8 16 32 64 128 256 512 chioles, alveoli of the lung, and colon epithelia (Table 1). Ep-16 bound strongly to sections of transitional cell carcinoma RECIPROCAL DILUTION OF SUPERNATANT of the bladder and squamous cell carcinoma of the tongue (Fig. Chart 1. Binding of monoclonal antibody Ep-16. Serial dilutions of hybridoma 3). Other epithelial-derived tumors, including adenocarcinomas culture media were tested by ELISA assay in duplicate on primary human keratinocytes and cultured cell lines. CRL 1521 and CCL 109 are fibroblast lines derived of the breast (Fig. 4), colon, and ovary, were also stained by the from human neonatal foreskins. CCL 213 is Daudi, a B-lymphoblastoid line, and antibodies. Ep-16 monoclonal antibodies did not react with mus CCL 119 is CCRF-CEM, a T-lymphoblastoid line. cle, connective tissue stroma and fibroblasts, lymphoid cells, blood vessels, nervous tissues, or any other nonepithelial human and breast), but they did not react with lines derived from epithelial tumors of the kidney (Wilms1 tumor), choriocarcinoma, tissues in fixed sections (Table 1). Antigen Localization. Immunoperoxidase staining of fixed adenocarcinoma of the adrenal, colon, prostate, lung (epidermoid tissue sections as well as cytocentrifuge preparations of single- and adenocarcinoma), and pancreas (ductal cell carcinoma). cell suspensions of cell lines (CRL 1500, adenocarcinoma of the Both antibodies failed to react with every nonepithelial-derived breast, CCL 138, squamous pharyngeal tumor, HTB-2, transi cell line tested, including B- and T-lymphoblastoid lines, lines tional cell carcinoma of the bladder) indicated that Ep-16 reactiv of neuroectodermal origin (melanoma, neuroblastoma), and ity was confined to the cell surface (Fig. 5). Ep-16 bound to only Ewing's sarcoma, and did not show binding to any of the a subset of CRL 1500 cells. fibroblast lines (skin, lung, African green monkey kidney) tested. To determine the location of the antigen more precisely, we Marginal binding was observed to a mouse epithelial cell line examined the binding of Ep-16 to epithelial cells by the indirect (BK-1 ) derived from normal BALB/c keratinocytes. immunofluorescent technique. CCL 138 cells or primary human The absence of reactivity of the antibodies to a number of keratinocytes were grown to confluency on glass coverslips, and epithelial tumor cell lines suggested that the antigen expressed the cells were stained in situ. Immunofluorescent staining by originally on these cell lines may have been lost during long-term antibody Ep-16 was confined to the cell surface (Figs. 6 and 8). culture. To confirm the specificity of the antibodies, binding was Staining was improved when cells were treated with antibody determined to primary cultures of normal human epithelial cells prior to fixation. A subset of CCL 138 cells or keratinocytes was and fibroblasts and to freshly isolated human lymphocytes and stained by antibody Ep-16. In contrast, anti-human keratin anti- bone marrow cells. The results of these studies, presented in serum or monoclonal antibodies specific for intermediate fila Chart 3, demonstrate that the antibodies bound only to foreskin ments decorated a network of cytoplasmic filaments in keratin keratinocytes, pulmonary epithelial cells, and epithelial cells de ocytes of CCL 138 cells (Figs. 7 and 9) and uniformly stained all rived from fetal liver and amnion. The antibodies did not bind to cells. short-term cultures of skin fibroblasts, umbilical vein endothelial cells, or freshly isolated human peripheral blood mononuclear DISCUSSION cells or bone marrow cells. With the exception of binding to several strongly binding cell We have produced monoclonal antibodies with specificity for types (Charts 2 and 3), the patterns of reactivity to the epithelial a cell surface antigen displayed on normal human epithelial cells

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_:;i-i-â€¢:-,<â€¢~inrâ€”I'-'-'Ãˆ Wmi pH \a-| FH-i~"'â€¢irm Ã¬mnHuman I"77â€¢~i ! S-, riL [On M-, BK-1KeratinocytesCCI138 CRL1683 HTB-2 HTB73 CRL1435 CRL1500 CRL1504 CCI218 CRL1473 Pharyni Sq.Cell Bladder Prostate Breast Colon Amnion

Positive Epithelial Lines

1.0

HTB81 HTB 54 CCI 185 CRL 1420 CRL 1469 CCL2 1440 CCL98 CCL220 CCL105 Prostate Lung Pancreas Cervii Kidney Chorioca Colon Adrenal

1.0 - Negative Epithelial Lines

r&n rwi CCL 213 CCL 114 CCL 119 CHL 1521 CCL 109 Lung HTB 64 CRL 1424 HTB 11 CCI 127 CCI 121 CCL 70 B Cell T Cell Skin Melanoma Neuroblastoma Sarcoma KidneyFibroblasl Non-epithelial Lines Chart 2. Binding of monoclonal antibodies Ep-16 and -17 to established cell lines. Hybridoma culture media at a dilution of 1:32 were tested in an ELISA assay in duplicate on the cell lines indicated. For each cell type, 3 columns are shown, representing Ep-16 (03),Ep-17 (D), and NS-1 controls (G). Reactions in which A was 3 times control values were considered to be positive. tumors, skin, and esophagus). Hassal's corpuscles of the thy mus, which are histologically similar to epidermis, also stained with the antibodies. The antibodies stained simple epithelia of the intestinal, urinary, and respiratory tracks only weakly. Stain 1.0I!CD<-1^a1n ing was observed only in the ducts of the pancreas and the collecting tubules of the kidney and weakly or not at all in the colon, adrenals, lung, and bronchus. IIH.â€¢â€¢-â€¢:::~1ZIInKeratinocytesThe antigen detected by these antibodies is distinct from other Amnion PulmonaryUverEpithelium Fetal epithelial-specific antigens described previously because of dif (Epithelial)Positive ferences in tissue distribution and cellular localization. Thus, Ep- Cell Strains 16 may define a unique epithelial-specific cell surface antigen. Although the tissue distribution of the antigen(s) detected by Ep- 16 and -17 is similar to that of keratin, evidence indicates the antigen is not a member of the keratin family. For example, a characteristic immunofluorescent pattern of radiating filaments 1.0 - is observed in keratinocytes stained with antibodies to keratin (27). In contrast, fluorescent staining by antibodies Ep-16 and Ep-17 appeared to be confined to the cell surface. Both immunofluorescent and immunoperoxidase staining of Skin Rbroblasts PBL Bone Marrow Endothelium single cells on cytocentrifuge preparations revealed that only a Negative Cell Strains subset of epithelial cells expressed the antigen. Similar hetero Chart 3. Binding of monoclonal antibodies Ep-16 and -17 to primary cell strains. geneity in antigenic expression has been described (4) and may Hybridoma culture media at a dilution of 1:32 were tested in an ELISA assay in be related to transient changes, such as cell cycle status. Alter duplicate on the primary cell strains, freshly isolated human lymphocytes, or bone marrow cells. For each cell type, 3 columns are shown, representing Ep-16 (0), natively, these cell populations may consist of truly heteroge Ep-17 (D), and NS-1 controls (D). Reactions in which A was 3 times control values neous subclones. were considered to be positive. Some epithelial-derived tumors (ovarian, colon, and breast adenocarcinomas) stained more strongly than did the cell lines and on many epithelial-derived tumor cell lines but not on non- derived from them. The reasons for this loss of antigenicity epithelial cells or cell lines. The specificity of the antibodies was during the course of in vitro culture are unknown, but it may be demonstrated not only in long-term cell lines and primary cell due to modulation of gene function (28). strains, but also in paraffin-embedded sections of human tissue. As the monoclonal antibodies described here detect a cell The antibodies were found to react only with epithelial cells in surface antigen, they may prove useful for a variety of basic paraffin-embedded fixed sections. Stratified squamous epithelia studies, (a) The antibodies may be used in the separation of of all types were stained very strongly (including squamous cell epithelial cells from other cell types by panning techniques (18).

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Table 1 different intermediate filament proteins: cell type specific markers on paraffin- Binding of monoclonal antibodies to normal human tissue as determined by embedded human tissue. Lab. Invest., 45: 427-434, 1981. immunoperoxidase studies 2. American Type Culture Collection. Catalogue of Strains. Ed. 4, Vol. 2, pp. 160-176. Rockville. MD: ATCC, 1983. Connective tissue, muscle, blood vessels, and lymphoid tissue present in these 3. Boyse, E. A., and Old, L. J. Loss of skin allograft tolerance by chimeras. sections were not stained by the antibodies. Histological sections of fixed human Transplantation (Baltimore), 6: 619-625,1968 tissue were stained by the monoclonal antibody EP-16 by an indirect immunoper oxidase technique described in "Materials and Methods." Slides were scored on a 4. Colcher, D., Horan-Hand, P., Nutti, M.. and Schlom, J. A. A spectrum of "-" to "+++" basis. monoclonal antibodies reactive with human mammary tumor cells. Proc. Nati. Acad. Sci. USA, 78: 3199-3203, 1981. Tissue Result 5. Cuttita, F., Rosen, S.. Gadzar, A. F., and Minna, J. D. Monoclonal antibodies that demonstrate specificity for several types of human lung cancer. Proc. Stratified squamous epithelia Nati. Acad. Sci. USA, 78: 4591-4594,1981. Human neonatal foreskin 6. Epenetos, A. A., Canti. G.. Taylor-Papadimitrous, J., Curling, M., and Bodmer, Epidermis W. F. Use of two epithelium specific monoclonal antibodies for diagnosis of Dermis Adult skin (3)a malignancy in serous effusions. Lancet, 2: 1004-1006, 1982. 7. Franke, W. W., Schmid, E., Weber, K., and Osborn, M. HeLa cells contain Epidermis intermediate-sized filaments of the prekeratin type. Exp. Cell Res., 778: 95- Hair follicle 109,1979. Sebaceous gland 8. Gabbiani, G., Kapanci, Y., Barazzone, P., and Franke, W. Immunochemical Dermis identification of intermediate sized filaments in human neoplastic cells. Am. J. Esophagus Pathol., 704:206-216, 1981. Squamous cell carcinoma of the tongue (3) 9. Gown. A. M., and Vogel, A. M. Monoclonal antibodies to intermediate filament Squamous cell carcinoma of the skin (1) proteins of human cells: unique and cross-reacting antibodies. J. Cell Biol., 97: Transitional cell carcinoma of the bladder (3) 414-424, 1982. Simple epithelia 10. Hawley-Nelson, P., Sullivan, J. E.. Kung, M., Hennings, H., and Yuspa, S. Kidney (2) Optimized conditions for the growth of human epidermal cells in culture. J. Glomeruli Invest. Dermatol., 75: 176-182, 1980. Collecting tubules 11. Karasek, M. A. In vitro culture of human skin epithelial cells. J. Invest. Proximal tubule Dermatol., 47: 533-540, 1966. Distal tubule 12. Kennett, R. J. Enzyme-linked antibody assay with cells attached to polyvinyl Pancreas chloride plates. In: R. Kennett, J. McKeran, and K. Bechtol (eds.), Monoclonal Ducts Antibodies. Hybridomas: A New Dimension in Biological Analysis, pp. 376- Acini 377. New York: Plenum Press, 1980. Islets 13. Laemmli, U. Cleavage of structural proteins during the assembly of the head Bronchus(2) of bacteriophage T4. Nature (Lond.), 227: 680-685, 1970. Epithelium 14. Lampson, L. A. Binding inhibition using biosynthetically labelled monoclonal Glands antibody. In: R. Kennett, J. McKeran, and K. Bechtol (eds.), Monoclonal Lung alveoli Antibodies. Hybridomas: A New Dimension in Biological Analysis, pp. 395- Adrenal 397. New York: Plenum Press. 1980. Colon (3) 15. Lane, E. B. Monoclonal antibodies provide specific intramolecular markers for the study of epithelial tonofilament organization. J. Cell Biol., 92: 665-673, 1982. 16. Laskey, R. A., and Mills, A. D. Quantitative film detection of 3H and "C in Thymus polyacrylamide gels by fluorography. Eur. J. Biochem., 56: 335-341, 1975. Lymphocytes Epithelium 17. Milo, G. E., Ackerman. A., and Noyes, I. Growth and ultrastructural character Hassal s corpuscles ization of proliferating human keratinocytes in vitro without added extrinsic factors In Vitro (Rockville), 76: 20-30, 1980. Spleen 18. Morhenn, V. B., Wood, G. S., Engleman, E., and Oseroff, A. Selective enrich ment of epidermal cell subpopulations using monoclonal antibodies. J. Invest. Tumors Dermatol., 87: 1275-1315, 1983. Adenocarcinoma of the pancreas (ductal) Adenocarcinoma of the colon 19 Nowinski, R. C., Lostrom, M. E., Tam. M. R., Stone, M. R., and Burnette, W. N. The isolation of hybrid cell lines producing monoclonal antibodies against the p15(E) protein of murine leukemia viruses. Virology, 93: 111 -126, 1979. Adenocarcinoma of the ovary Adenocarcinoma of the breast 20. Rheinwald, J. B., and Green, H. Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature (Lond.), 265: 421-424, 3 Numbers in parentheses, number of different donors. 1977. 21. Schlegel, R., Banks-Schiegel. S.. McLeod, J. A., and Pinkus, G. S. Immuno (b) Studies seeking to determine changes in antigenic patterns peroxidase localization of keratin in human neoplasms. Am. J. Pathol., 707: 41-50, 1980. in different stages of epithelial differentiation, such as described 22. Schlegel, R., Banks-Schlegel. S.. and Pinkus, G. S. Immunohistochemical recently by Stanley ef al. (24,25), may be aided by highly specific localization of keratin in normal human tissues. Lab. Invest., 42: 91-96,1980. 23. Siden. E., Alt, F. W., Shenefeld, L., Sato. V.. and Baltimore, D. Synthesis of monoclonal antibodies, (c) Cell surface glycoproteins are involved immunoglobulin p chain gene products precedes synthesis of light chain during in many important epithelial cell functions, such as cell to cell B-lymphocyte development. Proc Nati. Acad. Sci. USA, 78:1823-1827,1981. 24. Stanley. J. R., Hawley-Nelson, P., Pourier, M.. Katz. S., and Yuspa. S. H. and cell to substrate adhesion, cell recognition, and membrane Detection of pemphigoid antigen, pemphigus antigen, and keratin filaments in transport. Monoclonal antibodies such as those described here cultured human epidermal cells. J. Invest. Dermatol., 75: 183-186,1980. 25. Stanley, J. R.. and Yuspa, S. Specific epidermal protein markers are modulated may aid in understanding regulation of these activities, (d) There during calcium induced terminal differentiation. J. Cell Biol., 96: 1809-1814. is continuing interest in the changes in cell surface proteins that 1983 26. Steinmiller, D.. Tyler, J., and Davis, C. S. Cell-mediated cytotoxicity to non- may be associated with cellular transformation (20). MHC alloantigens on mouse epidermal cells. J. Immunol., 726: 1747-1753, The antibodies may also be useful in clinical diagnosis as they 1981. detect antigens preserved in fixed tissues. Monoclonal and het- 27. Sun, T. T., Sheih, C., and Green, H. Keratin cytoskeleton in epithelial cells of internal organs. Proc. Nati. Acad. Sci. USA. 76: 2813-2817, 1979. eroantibodies directed towards epithelial cells (1, 6, 8, 24) are 28. Taylor-Papodimitrios, J., Peterson, J. A., Arklie, J., Burchell, J., Ariani, R. L., proving useful in distinguishing different types of cancer and in and Bodmer, W. F. Monoclonal antibodies to epithelium specific components determining the cellular origin of poorly differentiated tumors. of the human milk fat globule membrane: production and reaction with cells in culture. Int. J. Cancer, 28: 17-21,1981. 29. Woodcock-Mitchell, J., Eichner, R., Nelson, W. G., and Sun, T. T. Immuno- REFERENCES localization of keratin polypeptides in human epidermis using monoclonal 1. Altmannsberger, M., Osborn, M., Schauer, A., and Weber, K. Antibodies to antibodies. J. Cell Biol.. 95: 580-588. 1982.

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Fig. 1. Neonatal foreskin Staining is present throughout the epidermis, while the dermis is negative. Immunoperoxidase, x 400. Fig. 2. Thymus. Strong staining is present in Hassall's corpuscles. Surrounding lymphoid cells are negative. Immunoperoxidase, x 400.

Fig. 3. Squamous cell carcinoma of the tongue. Strong staining occurs in tumor cells. Immunoperoxidase, x 400. Fig. 4. Adenocarcinomaof the breast metastatic to the pericardium. The nest of tumor cells is strongly stained. Intervening stroma is negative. Immunoperoxidase, x 400.

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Fig. 5. Biotin:avidin:immunoperoxidase staining of a breast carcinoma cell line (CRL 1500) using antibody Ep- 16. Note the cell surface staining, x 400.

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Fig. 6. Indirect immunofluorescentstaining of primary human keratinocytes by antibody Ep-16. The antigen is distributed as points on the cell surface, x 400. Fig. 7. Indirect immunofluorescent staining of primary human keratinocytes by goat anti-human keratin serum. Cytoplasmic keratin filaments are observed (arrows). As the cells were grown on plastic, the resolution of the keratin filaments is not as sharp as that observed for cells grown on glass (17). x 400. Fig. 8. Indirect immunofluorescentstaining of CCL 138 cells by antibody Ep-16. Staining is confined to the cell surface, x 400. Fig. 9. Indirect immunofluorescentstaining of CCL 138 cells by anti-intermediatefilament monoclonalantibodies, x 400.

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Anne W. Hamburger, Yvonne A. Reid, Barbara Pelle, et al.

Cancer Res 1985;45:783-790.

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