Int. J. Cancer: 50,507-513 (1992) 0 1992 Wiley-Liss, Inc.
Publication of the International Union Against Cancer Publication de I Union Internationale Contre le Cancer
EXPRESSION AND CHARACTERIZATION OF TWO DIFFERENTIATION ANTIGENS IN HUMAN STRATIFIED SQUAMOUS EPITHELIA AND CARCINOMAS Jasper J. QUAK',Ad H.G.J. SCHRIJVERS'.~, Joost G.P. BRAKKEE', Hugh D. DAVIS,, Rik J. SCHEPER',Chris J.L.M. MEIJER', Gordon B. SNOW'and Guus A.M.S. VANDONGEN' 'Dept. of OtolaryngologylHead and Neck Surgery and 'Dept. of Pathology, Free University Hospital, Amsterdam, The Netherlands; and 3Centocor,Malvem, PA, USA. Using viable cells of a human squamous-cell carcinoma (SCC) cell line as immunogen, we generated 2 monoclonal antibodies, MAbs K984 and K928, to SCC surface antigens. Immunoperoxidase staining of frozen sections of normal epidermis revealed that MAb K984 reacts with the poorly differentiated basal cells, while MAb K928 is reactive with the more highly differentiated suprabasal cells. A similar complementary reaction pattern of these antibodies was demonstrated in the majority of welldifferentiated human tumors and some moderately differentiated SCCs. In contrast, simultaneous reactivity of MAb K984 and K928 was found for the majority of cells within other welland moderately differentiated SCCs, as well as all poorly differentiated SCCs. Further biochemical characterizationindicated that the antigen recognized by MAb K984 is similar to the one recognized by MAb SF-25. MAb K928 recognizes a 50- to 55-kDa molecule under non-reducing conditions. Antibodies with similar features to MAb K928 have not been described previously. The antigens recognized by MAbs K984 and K928 can be regarded as novel markers associated with cellular maturation in squamous epithelia. The antigen detected by MAb K984 is probably associated with the proliferatingfraction in SCCs.
Squamous-cell carcinoma (SCC) is the most common type of carcinoma of the head and neck, esophagus, lung, cervix, vulva and epidermis. Identification and characterization of antigens associated with SCC can be of help in diagnosis of these neoplasms. Monoclonal antibodies (MAbs) are important as probes to characterize such antigens and as tools to study the function of SCC-associated antigens. When these antigens are localized on the outer surface of the tumor cell, they may also be used for radioimmunolocalization and therapeutic purposes. It has become clear that most tumor-associated antigens are, in fact, differentiation antigens (Damjanov and Knowles, 1983). The number of MAbs to SCC-associated antigens (Boeheim et al., 1985; Kimmel and Carey, 1986; Fernsten et al., 1986; Myoken et al., 1987; Ranken et al., 1987; Samuel et al., 1989; Quak et al., 1990a,b) is still relatively low as compared to that of MAbs directed to other types of tumor. In this study we describe 2 newly developed antibodies which recognize surface antigens associated with squamous differentiation. The antigens are characterized for their expression in normal and neoplastic transformed tissues and some of their biochemical and ultrastructural features are described. MATERIAL AND METHODS
Immunization and hybridoma production Immunization and screening were essentially the same as described previously (Quak et al., 1990~).Briefly, BALBIc mice were injected intraperitoneally with lo7viable cells of the cell line UM-SCC-22A. After 4 weeks, an intrasplenic booster was given under general anesthesia. Three days later the spleen was removed and the dissociated spleen cells were fused with the non-producing myeloma line SP-210. Supernatants of growing hybridomas were screened by ELISA for determination of binding to intact viable tumor cells and for lack of reactivity with red blood cells. Selected antibodies were further screened for reactivity with frozen sections derived
from oral mucosa and a squamous-cell carcinoma from the head and neck.
Zsotypes of the M b s Isotype determination was performed in 96-well ELISA plates which had been coated with affinity-purified rabbit anti-mouse subclass-specific antibodies (IgG,, IgG,,, IgG,,, IgG, and IgM, Miles, Elkart, IN). Tissues and cell lines Neoplastic and non-neoplastic tissues were obtained from surgical procedures. Tissue specimens were snap-frozen and stored in liquid nitrogen until use. The degree of differentiation of SCC was determined by light-microscopic evaluation. Well-differentiated SCCs were characterized by keratin pearl formation, low nucleus/cytoplasm ratio and individual cell keratinization as judged by their eosinophilic cytoplasm. Poorly differentiated SCC lacked keratin pearl formation, and showed a high nucleus/cytoplasm ratio with no or very little individual cell keratinization. Moderately differentiated SCCs had intermediate characteristics. Some tumors which were classified as well-differentiated SCCs by the presence of keratin pearl formation were composed, to a high extent, of poorly differentiated tumor cells as determined by a high nucleus/cytoplasm ratio and individual cell keratinization. Human SCC cell lines of the head and neck were kindly provided by Dr. T. Carey, University of Michigan, Ann Arbor, MI. UM-SCC-l4A, UM-SCC-14B and UM-SCC-14C were derived from 3 separate recurrences of a carcinoma of the floor of the mouth, UM-SCC-22A and UM-SCC-22B originated from a primary hypopharyngeal tumor and its lymph-node metastasis, respectively, while UM-SCC-38 was derived from a primary tumor of a tonsil (tongue base) (Carey, 1985; GrCnman et al., 1991). A 431, derived from a vulvar squamous-cell carcinoma, was a gift from Dr. B. Defize, Hubrecht Laboratory, Utrecht, The Netherlands. Imrnunoperoxidase staining Cryostat sections of frozen tissue blocks were mounted on poly-L-lysine-coated glass slides, air-dried and fixed in acetone for 10 min at room temperature. Immunoperoxidase staining was performed as described by Quak et al. (1990b). The staining patterns of MAb K928 and K984 were compared to those of antibodies directed against CEA (Dako, Copenhagen, Denmark), h4AM-6 (kindly supplied by Dr. J. Hilkens, Amsterdam), 17-1A (Centocor, Malvern, PA), EGFR (BioMakor, Rehovot, Israel), Transferrin-r (OKT-9; Ortho, Raritan, NJ) and the SF-25 antigen (Centocor).
4To whom correspondence and reprint requests should be sent, at the Dept. of Otolaryngology/Head and Neck Surgery, Free University Hospital, P.O. Box 7057, NL-1007 MB Amsterdam, The Netherlands. FAX: +3120 5486688.
Received: May 14, 1991 and in revised form October 4,1991.
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QUAK ET AL.
Irnrnuno-electron-microscopy Cryosections of oral mucosa (10 pm thick) were prepared and mounted on poly-L-lysine-coated glass slides. Sections were immediately fixed with 1% paraformaldehyde and 0.1% glutaraldehyde in phosphate-buffered saline (PBS) for 20 rnin at 4°C. Subsequently, free-aldehyde groups were blocked, and sections were washed in PBS and incubated overnight with undiluted supernatants of MAb K984 or control antibody JSB-1. The latter is directed against the P-glycoprotein associated with multi-drug resistance and not present in oral mucosa (Scheper et al., 1988). Next, sections were incubated with peroxidase-conjugated rabbit anti-mouse immunoglobulins (Dako) for 3 hr and, after subsequent washing in PBS, 3,3'-diaminobenzidine (DAB; Sigma, St Louis, MO) was added. Stained slides were fixed in 1% OsO, in 1 M cacodylate buffer, pH 8.2, and dehydrated in ethanol. Subsequently, the sections were embedded in epoxy resin (EPON 812, Fluka, Buchs, Switzerland) and ultra-thin sections were examined with a Zeiss EM 109 electron microscope.
Tris-HC1 (pH 7.2), then 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM iodoacetamide, 1 pg/ml leupeptin, 1 pg/ml pepstatin and 20 pg/ml trypsin inhibitor were added as protease inhibitors. Nuclear material was pelleted and the clear supernatant put into Eppendorf tubes (all chemicals were obtained from Sigma). The lactoperoxidase-radiolabelled SCC cell lysate (8001,000 1.1) was pre-cleared by incubation with protein-ASepharose CL-4B (Pharmacia) and by subsequent incubation with normal mouse immunoglobulins complexed with proteinA-Sepharose. The pre-cleared lysate (200-250 11.1) was immunoprecipitated with 50 p1 of MAb K984-preformed complex, essentially prepared as described by Schneider et al. (1982) (10% (w/v) in PBS, pH 7.2) by incubation overnight at 4°C. Subsequently, the specific immunoprecipitate was washed 3 times with PBS, resuspended in sample buffer and boiled (for 1 min) for SDS-PAGE (7.5% gel). The gels were dried and autoradiographed by exposure to X-ray film with a Kodak X-Omatic intensifying screen for 18-24 hr at -80°C.
Cell ELISA SCC cell lines growing as monolayers in 96-well ELISA plates were used to determine binding of the antibody to the outer surface of viable tumor cells. Confluent cultures were washed with PBS and incubated with undiluted supernatant of MAbs K984 or K928, for 1 hr at room temperature. In this way only membrane reactivity was detected and no cytoplasmic or nuclear reactivity; MAb K112, which reacts with a 43-kDa antigen present in the nuclei of these cell lines, served as a negative control (Quak et al., 1990~).Subsequently, the plates were washed with PBS+ 0.05% Tween-20 and incubated with peroxidase-conjugated rabbit anti-mouse immunoglobulin (Dako) (1:lOO dilution in PBS) for 1 hr at room temperature and washed again, after which ortho-phenyldiamine (OPD; Sigma) (8 mg/ml) was added as chromogen. Color development was stopped by adding 2 N H,SO,. Absorbance was measured at 492 nm; values are expressed as means of triplicate determinations (Table 111).
SDS-PAGE and irnrnunoblotting SCC cells were grown in monolayer culture until confluence and lysed with the lysis buffer described above. Aliquots of the cell lysate were resuspended in sample buffer containing (final concentration); 5% SDS, 10% 2-mercaptoethanol, 0.0015% bromophenol blue, 15% glycerol and 125 mM Tris-HCI (pH 6.8), and boiled for 1 min. SDS-PAGE was carried out as described by Laemmli (1970), using 5 to 15% linear acrylamide gradient slab gels. Molecular weight markers were obtained from Amersham (RainbowTM,Amersham, Aylesbury, UK). Proteins were transferred from the gel to nitrocellulose with a Multiphor I1 Nova Blot System (LKB, Bromma, Sweden) for semi-dry electrophoretic transfer, using a discontinuous buffer system. After pre-incubation with PBS containing 10% FCS for 30 rnin at room temperature, the nitrocellulose sheets were incubated with 'Z51-labelledMAb K984 or MAb K928 (1 x 10' cpm/ml) for 2 hr at room temperature. Finally, the sheets were autoradiographed by exposure to X-ray film with a Kodak X-Omatic intensifying screen for 18-24 hr at -80°C.
Enzymatic sensitivity Neuraminidase was obtained from Behring (0.5 U/ml, Kbrio cholerae, Behring, Marburg, Germany). Periodate treatment was essentially the same as described by Woodward et al. (1985). Nonidet P-40 (NP-40) lysates were prepared by incubation of whole cells for 30 rnin at 4°C in a 0.01 M Tris-HC1buffer pH 7.8, containing 1% (v/v) NP-40,0.15 M NaCl, 1mM PMSF and 0.02 M trypsin inhibitor. A lectin-bound ELISA (Drouin et al., 1988) was performed by coating the wells of microtiter plates with the lectin wheat-germ agglutinin (Pharmacia, Uppsala, Sweden), followed by incubation with NP-40 lysates and, subsequently, with MAbs. Binding was detected using goat anti-mouse peroxidase complex as described for cell ELISA. Irnrnunoprecipitation assay Cells from SCC cell lines grown as monolayer cultures in 75 cm2culture ff asks were 'ZS-Iodine-labelIedin suspension after detachment with 0.05% EDTA in PBS (10-15 min, room temperature, pH 7.2). Dispersed cells were resuspended in PBS and washed twice. Subsequently, to a 1ml cell-suspension were added; 1 mCi Na'*'I, 50 pl lactoperoxidase [2 mgiml (81 Uimg); Sigma] and 50 p1 0.03% H,O, in PBS. Cells were then incubated for 30 rnin at room temperature and fresh lactoperoxidase and H,O, were added twice after 10 and 20 min, respectively. Labelling was stopped by addition of 100 pl 50 mM ascorbic acid in PBS. Cells were finally washed 3 times with PBS. Radiolabelled SCC cells were lysed (2-4 hr on ice) in lysis buffer containing; 1% Triton-X 100, 150 mM NaCI, 2 mM MgCl,, 2 mM EGTA and 2 mM dithioerythritol in 10 mM
Radioiodination Iodination of MAbs K928 and K984 was performed according to the one-vial method described by Haisma et al. (1986). Briefly, 200 pg of antibody in 0.1 M borate buffer (pH 8.2) were mixed with 1 mCi "'1 in a vial previously coated with 50 pg Iodogen. After 10 min at room temperature, a sample was taken to determine the amount of incorporated iodine. One ml of AG1-X8 resin (BioRad, Richmond, CA) previously mixed with PBS containing 1% BSA was added to absorb unbound iodine. More than 95% of the iodine was bound to the antibody, as revealed by trichloroacetic-acid precipitation. As determined by a modified Lineweaver-Burke plot, the immunoreactive fractions of MAbs K984 and K928 at infinite antigen excess exceeded 80% in all experiments. RESULTS
Selected MAbs Two hybridomas, showing selective antibody binding with frozen sections of SCC, were cloned and subcloned by limiting dilution and designated MAbs K984 and K928. Isotype determination revealed that MAb K984 belongs to the IgG, subclass and MAb K928 is an IgG,, immunoglobulin. Reactivity of MAbs with normal tissues In normal stratified squamous epithelia, MAb K984 reacts exclusively with the basal cells (Fig. 1). Immuno-electronmicroscopic studies revealed that the K984 antigen was present on the cell surface. No reactivity was observed with cytoplas-
MAbs TO SQUAMOUS-CELL DIFFERENTIATION ANTIGENS
509
FIGURE 1- Frozen section of oral mucosa showing reactivity of MAb K984 with the basal cell layer. Immunoperoxidase staining. Bar, 50 Km. mic or nuclear components (Figs. 2,3). Immunohistochemical analysis demonstrated labelling with spermatogonia in testis, the endocrine pancreas and tubular epithelium in the kidney (Table I). Reactivity in these organs was also seen on the surface with little or no cytoplasmic staining. Binding was also observed with Kupffer cells in the liver and histiocytes. In contrast to MAb KY84, MAb KY28 reacts with membranes of suprabasal cells in stratified squamous epithelium (Table I). Basal cells did not show surface reactivity but, instead, slight cytoplasmic staining (Fig. 4). Immuno-electronmicroscopic studies failed to localize the KY28 antigen, probably due to loss of antigenicity of the epitope during fixation. The KY28 antigen was present in pneumocytes in normal lung, duct and acinar cells of salivary glands, bile ducts and canaliculi in liver, oviduct epithelium, all epithelial cells in mammae and urinary bladder and in cells of renal tubuli (type I). Mesenchymal tissues and nervous tissues did not react with MAb KY28, with the exception of peripheral nerves in which Schwann cells stained positive (Table I).
Reactivity of M b s with human carcinomas MAb K984 reacted with a vast majority of the human neoplasms tested. Some tumors, derived from KY84-antigennegative, non-neoplastic tissues, such as colon, lung or mammary gland, also expressed the antigen (Table 11). MAb K928 reacted with even more tumors of the panel tested. However, MAb KY28 showed a more restricted binding, in a sense that it only reacted with tumors derived from antigen-positive tissue (Table 11). Reactivity with SCC of the head and neck Since MAbs KY84 and KY28 apparently recognize differentiation-related antigens in normal squamous-cell epithelium, the reactivity of these antibodies was further investigated with a number of squamous-cell carcinomas of the head and neck in order to study the correlation between antigen expression and the histological degree of differentiation, as determined by light microscopic evaluation. Basically, the 2 following staining patterns could be observed with MAb KY84 on frozen sections. (1) Staining of a single cell layer, e.g. the basal-cell layer in normal squamous epithelium or the most peripheral cell layer in SCC (representing the light-microscopically poorly differentiated cells) (Fig. 5). This staining pattern was seen in 14 out of 22 welldifferentiated SCCs. (2) Staining extending from the most peripheral cell layer to central layers. Multiple or all cell layers reacted with MAb K984 in all poorly (n = 6) differentiated
FIGURE 2 - Ultrastructural localization of the KY84 antigen in cells of oral mucosa. Labelling is found along the cell surface of basal cells. Bar, 1 Wm.
FIGURE3 - Ultrastructural localization of, the K984 antigen. High-power magnification showing the distinct surface labelling. Bar, 200 nm. (Fig. 6) and moderately (n = 12) differentiated SCCs (Fig. 7) and in a minority (8 out of 22) of the well-differentiated SCCs. It should be borne in mind that, in well-differentiated SCC, in which several layers reacted with MAb KY84, these layers were composed of poorly differentiated squamous cells as determined by the lack of individual cell keratinization and a high nucleus/cytoplasm ratio. In the case of MAb KY28, essentially 2 patterns of reactivity were also observed. (1) Reactivity with central cell layers in tumor nests (cells showing features of differentiation as determined by light microscopy), which can be compared with reactivity of suprabasal cells in normal squamous epithelia. (2) Reactivity with central as well as peripheral cell layers (differentiated as well as undifferentiated cells). The first pattern was observed in a proportion of moderately differentiated and most well-differentiated SCCs (Fig. 8). Strikingly, in these tumors a complementary reaction pattern of MAbs KY28 and
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QUAK ET AL.
TABLE 1 - REACTIVITY OF MAb K984 AND MAb K928 WITH NORMAL HUMAN TISSUES AS DETERMINED ON FROZEN SECTIONS BY IMMUNOPEROXIDASE Orrran
K9R4
K928
Oral mucosa basal cells suprabasal cells Epidermis basal cells suprabasal cells sebaceous glands sweat glands Lung trachea bronchus pneumocytes Mammary gland acinar cells duct cells Pancreas acinar cells duct cells islet cells Liver hepatocytes bile caraliculi Kupffer cells Kidney glomerulus Bowman's ca sule tubuli (type 15 Colon, jejunum and duodenum absorptive cells goblet cells Brunner's gland Ovary oocytes oviduct cells Testes spermatogonia Bladder Thyroid follicular cells C-cells Lymph node lymphoid cells histiocytes Peripheral neuron Schwann cells
'+ +, Very strong reactivity-'Slight cytoplasmic reactivity. -'-, No reactivity.'+, Positive staining. For each organ at least 3 specimens were tested.
K984 was seen. The second pattern of MAb K928 staining was observed in some well-differentiated SCCs, in some moderately differentiated SCCs and most poorly differentiated SCCs. Double staining procedures revealed that, within these tumors, the vast majority of cells simultaneously expressed the K928 and K984 antigen, in contrast to the non-transformed keratinocytes, in which this phenomenon was not observed (data not shown). Direct evidence that the epitopes recognized by MAbs K984 and K928 are accessible on the outer surface of viable tumor cells was found in the results of the cell ELISA. We observed good reactivity with all human SCC cell lines tested in this assay, although quantitative differences in reactivity were observed as can be seen in Table 111. Using immunohistochemistry we have also evaluated antibodies directed against CEA, MAM-6, 17-1A, epidermal-growth-
FIGURE 4 - Frozen section of oral rnucosa showing reactivity of MAb K928 with the suprabasal cell layer. Immunoperoxidase staining. Bar, 50 Fm. TABLE I1 - REACTIVITY OF MAb K984 AND MAb K928 WITH HUMAN
CARCINOMAS AS DETERMINED ON FROZEN SECTIONS BY IMMUNOPEROXIDASE
Positive/ tested
Squamous-cell carcinomas head and neck lung Small-cell carcinoma lung Undifferentiated large-cell lung Adeno-carcinomas Lung Breast Colon Ovary Melanomas Lvmuhomas
K984
K928
59/59 616
50159 516
012
212
112
112
416 416 415 116
616
013 012
013 012
616 016 516
factor receptor, transferrin receptor and the recently described SF-25 antigen (Takahashi et al., 1988) on frozen sections of normal squamous epithelium as well as of the same panel of tumors. In contrast to the other antibodies, a striking similarity was found between the reaction patterns of MAb K984 and MAb SF-25 o n both oral mucosa and SCC tissues, indicating recognition of the same antigen (data not shown). Identification of the K984 antigen by immunoprecipitation Since immunoblotting experiments failed to identify the K984 antigen, we investigated the presence of the K984 antigen at the outer cell surface of SCC cells by immunoprecipitation of 'Z51-labelledsurface proteins of UM-SCC-22B cells. Using this technique, we were able to show that MAb K984 recognizes a 90- to 100-kDa surface protein (Fig. 9, lane A). A protein with similar apparent molecular weight was identified upon precipitation with MAb SF-25 (IgG,) (Fig. 9, lane B), but
MAbs TO SQUAMOUS-CELL DIFFERENTIATION ANTIGENS
511
FIGURE 5 - Reactivity of MAb K984 with a verrucous squamous-cellcarcinoma of the maxillary sinus. Note the reactivity with a single basal-cell layer. Bar, 75 pm. FIGURE 6 - Reactivity of MAb K984 with a poorly differentiated SCC of the larynx. Staining can be observed in all layers. Bar, 75 pm. FIGURE 7 - Reactivity of MAb K984 with a moderately differentiated SCC of the floor of the mouth. Binding can be seen in multiple layers. Bar, 75 pm. FIGURE 8 - Reactivity of MAb K928 with a moderately differentiated SCC of the floor of the mouth. Peripheral cells in tumor nests are not bound by the antibody, in contrast to the central layers. Bar, 75 pm. TABLE 111 - REACTIVITY OF MAb K984 AND MAb K928 WITHIN VITRO GROWING HUMAN SCC CELL LINES
UM-SCC- UM-SCC- UM-SCC- UM-SCC- UM-SCC- A 431 14A 14B 14C 22A 22B
MAbK984 MAb K928 MAbK112
1.10 1.90 0.13
1.50 1.40 0.15
1.90 1.00 0.12
1.50 1.30 0.15
1.60 1.30 0.16
1.40 1.50 0.12
Absorbancewas measured at 492 nm. MAb K112 served as a negative control. Standard deviations below 10%.
not with MAb JSB-1 (IgG,) which served as a negative control (Fig. 9, lane C).
Enzymatic sensitivity of the K984 and K928 antigens Results obtained using immunoperoxidase staining of enzymatically or chemically pre-treated tissue sections revealed that, in contrast to the K928 antigen, the K984 antigen is sensitive to periodate, indicating that the K984 antigen determinant is located on a carbohydrate chain. Neuraminidase did not influence the labelling, indicating that no sialic acid residues were part of the epitope. Also, MAb K984 reacted with NP-40 lysates of tumor cells which were bound on wheat-germ agglutin (WGA) in a lectin-bound ELISA, suggest-
ing that MAb K984 recognizes a glycoprotein (data not shown).
ldentijication of the K928 antigen in cultured cell lines by immunoblotting Using immunoblot analysis of 4 different cell lines derived from patients with SCC, we have identified a single band of almost identical molecular weight of 50-55 kDa under nonreducing conditions (Fig. 10, lanes C, F, G, H). Relative differences could be observed between UM-SCC-l4A, UMSCC-14C and UM-SCC-22B, corresponding to the differences as observed with the cell ELISA (Fig. 10, lanes H, F, G, respectively). Upon reduction of the samples the 50- to 55-kDa band could no longer be recognized (Fig. 10, lane B). When total membranes were extracted by NP-40 or Triton X-100, the K928 antigen was found within the detergent-soluble phase (Fig. 10, lanes D, E, respectively), while no antigen could be detected in the detergent-insoluble fractions (data not shown). DISCUSSION
In the present study, 2 MAbs were presented with different reactivity patterns for squamous epithelia and its neoplastic counterpart. MAb K984 recognizes a structure which is, in normal epidermis, expressed on the surface of basal kera-
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FIGURE10 - Immunoblot analysis of cell lysates of squamouscell carcinoma cell lines with radiolabelled MAb K928. Lane A: control cell line HL 60 not reactive with MAb K928; lane B: UM-SCC-14A in the presence of P-mercaptoethanol (reducing conditions); lane C: UM-SCC-38; lane D: UM-SCC-38, NP-40 soluble fraction; lane E: UM-SCC-38 Triton X-100 soluble fraction; lane F: UM-SCC-14B; lane G: UM-SCC-14C and lane H: UM-SCC-14A.
FIGURE9 - Radioimmunoprecipitation of a cell lysate, prepared from lactoperoxidase radiolabelled UM-SCC-22B cell line. (A) Specific precipitation of a 90- to 100-kDa surface protein by pre-formed complexes of MAb K984 and protein-A-Sepharose CL-4B. (B) Pre-formed complexes of MAb SF-25 precipitated a protein apparently similar in molecular weight to MAb K984. (C) No precipitation is observed with the negative control antibody JSB-1 complexes. Similar typical results were also obtained for the other negative controls; non-complexed protein-A-Sepharose or pre-formed complexes with normal mouse immunoglobulins (not shown). Bars indicate (top to bottom) molecular weight markers of 110,84,47 and 33 kDa, respectively. tinocytes. This was confirmed by immuno-electron-microscopic studies. In squamous-cell carcinoma, MAb K984 reacts with poorly differentiated cells, as determined by light microscopy. The immunohistochemical staining pattern of MAb K984 suggests that the antigen is associated with the proliferative compartment of SCC. Similar antigen expression has also been described for other basal-cell markers (Kimmel and Carey, 1986;Boeheim et al., 1985; Ranken et al., 1987; Defize et a[., 1987). Cowley et af. (1986) described this for the epidermal growth factor receptor (EGFR), speculating that, as a consequence of transformation, cellular differentiation programs controlling the expression of EGFR could be blocked, causing the receptor levels to remain high. MAb KY84 was immunohistochemically compared with MAbs directed against CEA, MAM-6, 17-1A, transferrin receptor, EGF-receptor and the SF-25 antigen for its reactivity with normal epidermis and squamous-cell carcinoma. Of these antibodies, only MAb SF-25, first described by Takahashi et al.
(1988) had a strikingly similar reactivity pattern to MAb K984 on both oral mucosa and SCC. The following findings supported the hypothesis that the antigen defined by MAb K984 might be identical to the SF-25 antigen. (1) The antigenantibody interactions were both disrupted by SDS. (2) An antigen with an apparent molecular weight of 2 100 kDa was precipitated by both MAbs. (3) Both antigens have the features of a glycoprotein. With regard to this last point, the denaturation of the antigen by periodate indicates that the epitope recognized by MAb K984 is located on a carbohydrate chain, and the K984 antigen could be detected in the lectinbound ELISA after lysis of cells with NP-40, suggesting that it is a glycoprotein rather than a glycolipid, since the latter would not be recovered upon treatment with non-ionic detergents. In contrast to MAb KY84, MAb K928 recognizes an antigen on membranes of more differentiated suprabasal cells in normal epidermis. In a proportion of moderately and welldifferentiated SCCs, KY28 antigen expression was also restricted to more highly differentiated cells, located in the central layers of tumor-cell nests. In other tumors, however, not only the more differentiated cells, but also most of the poorly differentiated cells expressed the K928 antigen. MAb K928 reacts with a 50- to 55-kDa molecule under non-reducing conditions. The loss of reactivity under reducing conditions suggests that one or more disulfide bands are directly or indirectly involved in the formation of the epitope recognized by MAb KY28. The strong expression of the KY28 antigen by UM-SCC-14A in comparison to UM-SCC-l4C, as monitored in the cell ELISA, was also seen in immunoblotting. The fact that the antigen was found quantitatively in the detergent phase, upon extraction of total membranes with Triton-X 100 or NP-40, indicates that the antigen is not a keratin polypeptide, since these are found in the NP-40insoluble pool (Fuchs and Green, 1981). The epitope recognized by MAb K928 does not seem to be located on a carbohydrate chain, as shown by periodate treatment.
MAbs TO SQUAMOUS-CELL DIFFERENTIATION ANTIGENS
Although further studies are required to identify the antigens described here, our data indicate that MAbs K984 and K928 recognize surface antigens associated with either proliferating or more differentiated cells in normal squamous epithelia. Similar antigen expression was found in squamouscell carcinoma, although in some of these tumors the K928 antigen was also expressed by poorly differentiated cells. In addition, MAb K984 is able to inhibit the growth of SCC lines in an antibody-concentration-dependent manner (Suh et al., 1990). Together with the strong expression of the K984
513
antigen in the vast majority of SCC, this can make the K984 antibody valuable for radioimmuno-imaging and therapy of SCC. ACKNOWLEDGEMENTS
The authors thank Ms. M. van Walsum for technical assistance, and Dr. W. Blomjous and J. Fritz for their assistance in immuno-electron-microscopic experiments. This work was supported in part by Centocor.
REFERENCES
BOEHEIM,K., SPEAK,J.A., FREI, E. and BERNAL,S.D., SQM-1 antibody defines a surface membrane antigen in squamous carcinoma of the head and neck. Int. J. Cancer, 36,137-142 (1985). CAREY,T.E., Establishment of epidermoid carcinoma cell lines. In: Head and neckcancer, pp. 289-316, J. Wiley, London (1985). COWLEY, G.P., SMITH,J. and GUSTERSON, B., Increased EGF receptors on human squamous carcinoma cell lines. Brit. J. Cancer, 53, 223-229 (1986). DAMJANOV, I. and KNOWLES, B.B., Biology of disease: monoclonal antibodies and tumor-associated antigens. Lab. Invest., 48, 510-525 (1983). DEFIZE,L.H.K., MUMMERY, C.L., MOOLENAAR, W.H. and DELAAT, S.W., Antireceptor antibodies in the study of EGF-receptor interaction. Cell Di$20,87-102 (1987). DROUIN, J., IZAGUIRRE, C.A. and PATENAUDE, P., Quantitation of cell membrane glycoproteins in pathological conditions using a lectinbound enzyme-linked immunosorbent assay (ELISA). J. immunol. Meth., 110,217-223 (1988). FERNSTEN, F.D., PEKNY,K.W., REISFELD,R.A. and WALKER,L.E., Antigens associated with human squamous cell lung carcinoma defined by murine monoclonal antibodies. Cancer Res., 46, 297Cb2977 (1986). FUCHS,E. and GREEN,H., Regulation of terminal differentiation of cultured human keratinocytes by vitamin A. Cell, 25,617-625 (1981). GRENMAN, R.G., CAREY,T.E., MCCLATCHEY, K.D., WAGNER,J.G., PEKKOLA-HEINO, K., SWARTZ, D.R., WOLF,G.T., LACIVITA, L.P., Ho, L., BAKER,S.R., KRAUSE,C.J. and LICHTER,AS., In vitro radiation resistance among cell lines established from patients with squamous cell carcinoma of the head and neck. Cancer, 67,2741-2747 (1991). HAISMA,H.J., HILGERS,J. and ZURAWSKI,V.R., Iodination of monoclonal antibodies for diagnosis and therapy using a convenient one vial method. J. nucl. Med., 27,1890-1895 (1986). KIMMEL,K.A. and CAREY,T.E., Altered expression in squamous carcinoma cells of an orientation-restricted epithelial antigen detected by monoclonal antibody A9. Cancer Res., 46,3614-3623 (1986). LAEMMLI, U.K., Cleavage of structural roteins during the assembly of the head of bacteriophage T4. Nature (lond.), 227,680-685 (1970). MYOKEN, Y., MOROYAMA, T., MIYAUCHI, S., TAKADA, K. and NAMBA, M., Monoclonal antibodies against human oral squamous cell carcinoma reacting with keratin proteins. Cancer, 60,2927-2937 (1987).
QUAK,J.J., BALM,A.J.M., BRAKKEE, J.G.P., SCHEPER,R.J., MEIJER, C.J.L.M. and SNOW,G.B., Production of monoclonal antibodies to squamous cell carcinoma antigens. Arch. Otolaryngol., 116, 181-185 (1990~). QUAK,J.J., BALM,A.J.M., VAN DONGEN,G.A.M.S., BRAKKEE, J.G.P., SCHEPER,R.J., SNOW,G.B. and MEIJER,C.J.L.M., A 22-kd surface antigen recognized by monoclonal antibody E48 is exclusively expressed in stratified squamous and transitional epithelia. Amer. J. Pathol., 136,191-197 (1990b). QUAK,J.J., VAN DONGEN,G.A.M.S., BALM,A.J.M., BRAKEE,J.P.G., SCHEPER,R.J., DE JONGE,L., MEIJER,C.J.L.M. and SNOW,G.B., Identification of a 43 kDa nuclear antigen associated with proliferation by monoclonal antibody K112. Int. J. Cancer, 46,50-55 (1990~). RANKEN, R., WHITE,C.F., GOTTFRIED, T.G., YONKOVICH, S.J., BLAZEK, B.E., MOSS,M.S., FEE, W.E. and LIU, Y.-S.V., Reactivity of monoclonal antibody 17.13 with human squamous cell carcinoma and its application to tumor diagnosis. Cancer Res., 47,5684-5690 (1987). SAMUEL, J., NOUJAIM, A.A., WILLANS, D.J., BREZINSKA, G.S., HAINES, D.M. and LONGENECKER, B.M., A novel marker for basal (stem) cells of mammalian stratified squamous epithelia and squamous cell carcinomas. Cancer Res., 49,2465-2470 (1989). SCHEPER, R.J., BULTE,J.W.M., BRAKKEE, J.G.P., QUAK, J.J., VANDER SCHOOT,E., BALM,A.J.M., MEIJER,C.J.L.M., BROXTERMAN, H.J., KUIPER,C.M., LANKELMA, J. and PINEDO,H.M., Monoclonal antibody JSB-1 detects a highly conserved epitope on the P-glycoprotein associated with multidrug resistance. Int. J. Cancer, 42,389-394 (1988). SCHNEIDER, C., NEWMAN, R.A., SUTHERLAND, D.R., ASSER,U. and GREAVES, M.F., A one-step purification of membrane proteins using a high efficiency immunomatrkJ. biol. Chem., 257,10766-10769 (1982). SUH,Y., QUAK,J.J. and VAN DONGEN, G.A.M.S., Growth inhibitory effect of monoclonal antibody K984 on in vitro cultured head and neck squamous cell carcinoma (HN-SCC) cell lines. Proceedings ofthe world congress of otorhinolaryngology head and neck surgery. Kugler and Ghedini, Amstelveen (1990). TAKAHASHI, H., WILSON,B., OZTURK,M., MOTTE, P., STRAUSS, W., ISSELBACHER, K.J. and WANDS,J.R., In vivo localization of human colon adenocarcinoma by monoclonal antibody binding to a highly expressed cell surface antigen. Cancer Rex, 48,6573-6579 (1988). WOODWARD, M.P., YOUNG,W.W. and BLOODGOOD, R.A., Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. J. immunol. Meth., 78,143-147 (1985).
Publication of the International Union Against Cancer Publication de I Union Internationale Contre le Cancer
EXPRESSION AND CHARACTERIZATION OF TWO DIFFERENTIATION ANTIGENS IN HUMAN STRATIFIED SQUAMOUS EPITHELIA AND CARCINOMAS Jasper J. QUAK',Ad H.G.J. SCHRIJVERS'.~, Joost G.P. BRAKKEE', Hugh D. DAVIS,, Rik J. SCHEPER',Chris J.L.M. MEIJER', Gordon B. SNOW'and Guus A.M.S. VANDONGEN' 'Dept. of OtolaryngologylHead and Neck Surgery and 'Dept. of Pathology, Free University Hospital, Amsterdam, The Netherlands; and 3Centocor,Malvem, PA, USA. Using viable cells of a human squamous-cell carcinoma (SCC) cell line as immunogen, we generated 2 monoclonal antibodies, MAbs K984 and K928, to SCC surface antigens. Immunoperoxidase staining of frozen sections of normal epidermis revealed that MAb K984 reacts with the poorly differentiated basal cells, while MAb K928 is reactive with the more highly differentiated suprabasal cells. A similar complementary reaction pattern of these antibodies was demonstrated in the majority of welldifferentiated human tumors and some moderately differentiated SCCs. In contrast, simultaneous reactivity of MAb K984 and K928 was found for the majority of cells within other welland moderately differentiated SCCs, as well as all poorly differentiated SCCs. Further biochemical characterizationindicated that the antigen recognized by MAb K984 is similar to the one recognized by MAb SF-25. MAb K928 recognizes a 50- to 55-kDa molecule under non-reducing conditions. Antibodies with similar features to MAb K928 have not been described previously. The antigens recognized by MAbs K984 and K928 can be regarded as novel markers associated with cellular maturation in squamous epithelia. The antigen detected by MAb K984 is probably associated with the proliferatingfraction in SCCs.
Squamous-cell carcinoma (SCC) is the most common type of carcinoma of the head and neck, esophagus, lung, cervix, vulva and epidermis. Identification and characterization of antigens associated with SCC can be of help in diagnosis of these neoplasms. Monoclonal antibodies (MAbs) are important as probes to characterize such antigens and as tools to study the function of SCC-associated antigens. When these antigens are localized on the outer surface of the tumor cell, they may also be used for radioimmunolocalization and therapeutic purposes. It has become clear that most tumor-associated antigens are, in fact, differentiation antigens (Damjanov and Knowles, 1983). The number of MAbs to SCC-associated antigens (Boeheim et al., 1985; Kimmel and Carey, 1986; Fernsten et al., 1986; Myoken et al., 1987; Ranken et al., 1987; Samuel et al., 1989; Quak et al., 1990a,b) is still relatively low as compared to that of MAbs directed to other types of tumor. In this study we describe 2 newly developed antibodies which recognize surface antigens associated with squamous differentiation. The antigens are characterized for their expression in normal and neoplastic transformed tissues and some of their biochemical and ultrastructural features are described. MATERIAL AND METHODS
Immunization and hybridoma production Immunization and screening were essentially the same as described previously (Quak et al., 1990~).Briefly, BALBIc mice were injected intraperitoneally with lo7viable cells of the cell line UM-SCC-22A. After 4 weeks, an intrasplenic booster was given under general anesthesia. Three days later the spleen was removed and the dissociated spleen cells were fused with the non-producing myeloma line SP-210. Supernatants of growing hybridomas were screened by ELISA for determination of binding to intact viable tumor cells and for lack of reactivity with red blood cells. Selected antibodies were further screened for reactivity with frozen sections derived
from oral mucosa and a squamous-cell carcinoma from the head and neck.
Zsotypes of the M b s Isotype determination was performed in 96-well ELISA plates which had been coated with affinity-purified rabbit anti-mouse subclass-specific antibodies (IgG,, IgG,,, IgG,,, IgG, and IgM, Miles, Elkart, IN). Tissues and cell lines Neoplastic and non-neoplastic tissues were obtained from surgical procedures. Tissue specimens were snap-frozen and stored in liquid nitrogen until use. The degree of differentiation of SCC was determined by light-microscopic evaluation. Well-differentiated SCCs were characterized by keratin pearl formation, low nucleus/cytoplasm ratio and individual cell keratinization as judged by their eosinophilic cytoplasm. Poorly differentiated SCC lacked keratin pearl formation, and showed a high nucleus/cytoplasm ratio with no or very little individual cell keratinization. Moderately differentiated SCCs had intermediate characteristics. Some tumors which were classified as well-differentiated SCCs by the presence of keratin pearl formation were composed, to a high extent, of poorly differentiated tumor cells as determined by a high nucleus/cytoplasm ratio and individual cell keratinization. Human SCC cell lines of the head and neck were kindly provided by Dr. T. Carey, University of Michigan, Ann Arbor, MI. UM-SCC-l4A, UM-SCC-14B and UM-SCC-14C were derived from 3 separate recurrences of a carcinoma of the floor of the mouth, UM-SCC-22A and UM-SCC-22B originated from a primary hypopharyngeal tumor and its lymph-node metastasis, respectively, while UM-SCC-38 was derived from a primary tumor of a tonsil (tongue base) (Carey, 1985; GrCnman et al., 1991). A 431, derived from a vulvar squamous-cell carcinoma, was a gift from Dr. B. Defize, Hubrecht Laboratory, Utrecht, The Netherlands. Imrnunoperoxidase staining Cryostat sections of frozen tissue blocks were mounted on poly-L-lysine-coated glass slides, air-dried and fixed in acetone for 10 min at room temperature. Immunoperoxidase staining was performed as described by Quak et al. (1990b). The staining patterns of MAb K928 and K984 were compared to those of antibodies directed against CEA (Dako, Copenhagen, Denmark), h4AM-6 (kindly supplied by Dr. J. Hilkens, Amsterdam), 17-1A (Centocor, Malvern, PA), EGFR (BioMakor, Rehovot, Israel), Transferrin-r (OKT-9; Ortho, Raritan, NJ) and the SF-25 antigen (Centocor).
4To whom correspondence and reprint requests should be sent, at the Dept. of Otolaryngology/Head and Neck Surgery, Free University Hospital, P.O. Box 7057, NL-1007 MB Amsterdam, The Netherlands. FAX: +3120 5486688.
Received: May 14, 1991 and in revised form October 4,1991.
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QUAK ET AL.
Irnrnuno-electron-microscopy Cryosections of oral mucosa (10 pm thick) were prepared and mounted on poly-L-lysine-coated glass slides. Sections were immediately fixed with 1% paraformaldehyde and 0.1% glutaraldehyde in phosphate-buffered saline (PBS) for 20 rnin at 4°C. Subsequently, free-aldehyde groups were blocked, and sections were washed in PBS and incubated overnight with undiluted supernatants of MAb K984 or control antibody JSB-1. The latter is directed against the P-glycoprotein associated with multi-drug resistance and not present in oral mucosa (Scheper et al., 1988). Next, sections were incubated with peroxidase-conjugated rabbit anti-mouse immunoglobulins (Dako) for 3 hr and, after subsequent washing in PBS, 3,3'-diaminobenzidine (DAB; Sigma, St Louis, MO) was added. Stained slides were fixed in 1% OsO, in 1 M cacodylate buffer, pH 8.2, and dehydrated in ethanol. Subsequently, the sections were embedded in epoxy resin (EPON 812, Fluka, Buchs, Switzerland) and ultra-thin sections were examined with a Zeiss EM 109 electron microscope.
Tris-HC1 (pH 7.2), then 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM iodoacetamide, 1 pg/ml leupeptin, 1 pg/ml pepstatin and 20 pg/ml trypsin inhibitor were added as protease inhibitors. Nuclear material was pelleted and the clear supernatant put into Eppendorf tubes (all chemicals were obtained from Sigma). The lactoperoxidase-radiolabelled SCC cell lysate (8001,000 1.1) was pre-cleared by incubation with protein-ASepharose CL-4B (Pharmacia) and by subsequent incubation with normal mouse immunoglobulins complexed with proteinA-Sepharose. The pre-cleared lysate (200-250 11.1) was immunoprecipitated with 50 p1 of MAb K984-preformed complex, essentially prepared as described by Schneider et al. (1982) (10% (w/v) in PBS, pH 7.2) by incubation overnight at 4°C. Subsequently, the specific immunoprecipitate was washed 3 times with PBS, resuspended in sample buffer and boiled (for 1 min) for SDS-PAGE (7.5% gel). The gels were dried and autoradiographed by exposure to X-ray film with a Kodak X-Omatic intensifying screen for 18-24 hr at -80°C.
Cell ELISA SCC cell lines growing as monolayers in 96-well ELISA plates were used to determine binding of the antibody to the outer surface of viable tumor cells. Confluent cultures were washed with PBS and incubated with undiluted supernatant of MAbs K984 or K928, for 1 hr at room temperature. In this way only membrane reactivity was detected and no cytoplasmic or nuclear reactivity; MAb K112, which reacts with a 43-kDa antigen present in the nuclei of these cell lines, served as a negative control (Quak et al., 1990~).Subsequently, the plates were washed with PBS+ 0.05% Tween-20 and incubated with peroxidase-conjugated rabbit anti-mouse immunoglobulin (Dako) (1:lOO dilution in PBS) for 1 hr at room temperature and washed again, after which ortho-phenyldiamine (OPD; Sigma) (8 mg/ml) was added as chromogen. Color development was stopped by adding 2 N H,SO,. Absorbance was measured at 492 nm; values are expressed as means of triplicate determinations (Table 111).
SDS-PAGE and irnrnunoblotting SCC cells were grown in monolayer culture until confluence and lysed with the lysis buffer described above. Aliquots of the cell lysate were resuspended in sample buffer containing (final concentration); 5% SDS, 10% 2-mercaptoethanol, 0.0015% bromophenol blue, 15% glycerol and 125 mM Tris-HCI (pH 6.8), and boiled for 1 min. SDS-PAGE was carried out as described by Laemmli (1970), using 5 to 15% linear acrylamide gradient slab gels. Molecular weight markers were obtained from Amersham (RainbowTM,Amersham, Aylesbury, UK). Proteins were transferred from the gel to nitrocellulose with a Multiphor I1 Nova Blot System (LKB, Bromma, Sweden) for semi-dry electrophoretic transfer, using a discontinuous buffer system. After pre-incubation with PBS containing 10% FCS for 30 rnin at room temperature, the nitrocellulose sheets were incubated with 'Z51-labelledMAb K984 or MAb K928 (1 x 10' cpm/ml) for 2 hr at room temperature. Finally, the sheets were autoradiographed by exposure to X-ray film with a Kodak X-Omatic intensifying screen for 18-24 hr at -80°C.
Enzymatic sensitivity Neuraminidase was obtained from Behring (0.5 U/ml, Kbrio cholerae, Behring, Marburg, Germany). Periodate treatment was essentially the same as described by Woodward et al. (1985). Nonidet P-40 (NP-40) lysates were prepared by incubation of whole cells for 30 rnin at 4°C in a 0.01 M Tris-HC1buffer pH 7.8, containing 1% (v/v) NP-40,0.15 M NaCl, 1mM PMSF and 0.02 M trypsin inhibitor. A lectin-bound ELISA (Drouin et al., 1988) was performed by coating the wells of microtiter plates with the lectin wheat-germ agglutinin (Pharmacia, Uppsala, Sweden), followed by incubation with NP-40 lysates and, subsequently, with MAbs. Binding was detected using goat anti-mouse peroxidase complex as described for cell ELISA. Irnrnunoprecipitation assay Cells from SCC cell lines grown as monolayer cultures in 75 cm2culture ff asks were 'ZS-Iodine-labelIedin suspension after detachment with 0.05% EDTA in PBS (10-15 min, room temperature, pH 7.2). Dispersed cells were resuspended in PBS and washed twice. Subsequently, to a 1ml cell-suspension were added; 1 mCi Na'*'I, 50 pl lactoperoxidase [2 mgiml (81 Uimg); Sigma] and 50 p1 0.03% H,O, in PBS. Cells were then incubated for 30 rnin at room temperature and fresh lactoperoxidase and H,O, were added twice after 10 and 20 min, respectively. Labelling was stopped by addition of 100 pl 50 mM ascorbic acid in PBS. Cells were finally washed 3 times with PBS. Radiolabelled SCC cells were lysed (2-4 hr on ice) in lysis buffer containing; 1% Triton-X 100, 150 mM NaCI, 2 mM MgCl,, 2 mM EGTA and 2 mM dithioerythritol in 10 mM
Radioiodination Iodination of MAbs K928 and K984 was performed according to the one-vial method described by Haisma et al. (1986). Briefly, 200 pg of antibody in 0.1 M borate buffer (pH 8.2) were mixed with 1 mCi "'1 in a vial previously coated with 50 pg Iodogen. After 10 min at room temperature, a sample was taken to determine the amount of incorporated iodine. One ml of AG1-X8 resin (BioRad, Richmond, CA) previously mixed with PBS containing 1% BSA was added to absorb unbound iodine. More than 95% of the iodine was bound to the antibody, as revealed by trichloroacetic-acid precipitation. As determined by a modified Lineweaver-Burke plot, the immunoreactive fractions of MAbs K984 and K928 at infinite antigen excess exceeded 80% in all experiments. RESULTS
Selected MAbs Two hybridomas, showing selective antibody binding with frozen sections of SCC, were cloned and subcloned by limiting dilution and designated MAbs K984 and K928. Isotype determination revealed that MAb K984 belongs to the IgG, subclass and MAb K928 is an IgG,, immunoglobulin. Reactivity of MAbs with normal tissues In normal stratified squamous epithelia, MAb K984 reacts exclusively with the basal cells (Fig. 1). Immuno-electronmicroscopic studies revealed that the K984 antigen was present on the cell surface. No reactivity was observed with cytoplas-
MAbs TO SQUAMOUS-CELL DIFFERENTIATION ANTIGENS
509
FIGURE 1- Frozen section of oral mucosa showing reactivity of MAb K984 with the basal cell layer. Immunoperoxidase staining. Bar, 50 Km. mic or nuclear components (Figs. 2,3). Immunohistochemical analysis demonstrated labelling with spermatogonia in testis, the endocrine pancreas and tubular epithelium in the kidney (Table I). Reactivity in these organs was also seen on the surface with little or no cytoplasmic staining. Binding was also observed with Kupffer cells in the liver and histiocytes. In contrast to MAb KY84, MAb KY28 reacts with membranes of suprabasal cells in stratified squamous epithelium (Table I). Basal cells did not show surface reactivity but, instead, slight cytoplasmic staining (Fig. 4). Immuno-electronmicroscopic studies failed to localize the KY28 antigen, probably due to loss of antigenicity of the epitope during fixation. The KY28 antigen was present in pneumocytes in normal lung, duct and acinar cells of salivary glands, bile ducts and canaliculi in liver, oviduct epithelium, all epithelial cells in mammae and urinary bladder and in cells of renal tubuli (type I). Mesenchymal tissues and nervous tissues did not react with MAb KY28, with the exception of peripheral nerves in which Schwann cells stained positive (Table I).
Reactivity of M b s with human carcinomas MAb K984 reacted with a vast majority of the human neoplasms tested. Some tumors, derived from KY84-antigennegative, non-neoplastic tissues, such as colon, lung or mammary gland, also expressed the antigen (Table 11). MAb K928 reacted with even more tumors of the panel tested. However, MAb KY28 showed a more restricted binding, in a sense that it only reacted with tumors derived from antigen-positive tissue (Table 11). Reactivity with SCC of the head and neck Since MAbs KY84 and KY28 apparently recognize differentiation-related antigens in normal squamous-cell epithelium, the reactivity of these antibodies was further investigated with a number of squamous-cell carcinomas of the head and neck in order to study the correlation between antigen expression and the histological degree of differentiation, as determined by light microscopic evaluation. Basically, the 2 following staining patterns could be observed with MAb KY84 on frozen sections. (1) Staining of a single cell layer, e.g. the basal-cell layer in normal squamous epithelium or the most peripheral cell layer in SCC (representing the light-microscopically poorly differentiated cells) (Fig. 5). This staining pattern was seen in 14 out of 22 welldifferentiated SCCs. (2) Staining extending from the most peripheral cell layer to central layers. Multiple or all cell layers reacted with MAb K984 in all poorly (n = 6) differentiated
FIGURE 2 - Ultrastructural localization of the KY84 antigen in cells of oral mucosa. Labelling is found along the cell surface of basal cells. Bar, 1 Wm.
FIGURE3 - Ultrastructural localization of, the K984 antigen. High-power magnification showing the distinct surface labelling. Bar, 200 nm. (Fig. 6) and moderately (n = 12) differentiated SCCs (Fig. 7) and in a minority (8 out of 22) of the well-differentiated SCCs. It should be borne in mind that, in well-differentiated SCC, in which several layers reacted with MAb KY84, these layers were composed of poorly differentiated squamous cells as determined by the lack of individual cell keratinization and a high nucleus/cytoplasm ratio. In the case of MAb KY28, essentially 2 patterns of reactivity were also observed. (1) Reactivity with central cell layers in tumor nests (cells showing features of differentiation as determined by light microscopy), which can be compared with reactivity of suprabasal cells in normal squamous epithelia. (2) Reactivity with central as well as peripheral cell layers (differentiated as well as undifferentiated cells). The first pattern was observed in a proportion of moderately differentiated and most well-differentiated SCCs (Fig. 8). Strikingly, in these tumors a complementary reaction pattern of MAbs KY28 and
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QUAK ET AL.
TABLE 1 - REACTIVITY OF MAb K984 AND MAb K928 WITH NORMAL HUMAN TISSUES AS DETERMINED ON FROZEN SECTIONS BY IMMUNOPEROXIDASE Orrran
K9R4
K928
Oral mucosa basal cells suprabasal cells Epidermis basal cells suprabasal cells sebaceous glands sweat glands Lung trachea bronchus pneumocytes Mammary gland acinar cells duct cells Pancreas acinar cells duct cells islet cells Liver hepatocytes bile caraliculi Kupffer cells Kidney glomerulus Bowman's ca sule tubuli (type 15 Colon, jejunum and duodenum absorptive cells goblet cells Brunner's gland Ovary oocytes oviduct cells Testes spermatogonia Bladder Thyroid follicular cells C-cells Lymph node lymphoid cells histiocytes Peripheral neuron Schwann cells
'+ +, Very strong reactivity-'Slight cytoplasmic reactivity. -'-, No reactivity.'+, Positive staining. For each organ at least 3 specimens were tested.
K984 was seen. The second pattern of MAb K928 staining was observed in some well-differentiated SCCs, in some moderately differentiated SCCs and most poorly differentiated SCCs. Double staining procedures revealed that, within these tumors, the vast majority of cells simultaneously expressed the K928 and K984 antigen, in contrast to the non-transformed keratinocytes, in which this phenomenon was not observed (data not shown). Direct evidence that the epitopes recognized by MAbs K984 and K928 are accessible on the outer surface of viable tumor cells was found in the results of the cell ELISA. We observed good reactivity with all human SCC cell lines tested in this assay, although quantitative differences in reactivity were observed as can be seen in Table 111. Using immunohistochemistry we have also evaluated antibodies directed against CEA, MAM-6, 17-1A, epidermal-growth-
FIGURE 4 - Frozen section of oral rnucosa showing reactivity of MAb K928 with the suprabasal cell layer. Immunoperoxidase staining. Bar, 50 Fm. TABLE I1 - REACTIVITY OF MAb K984 AND MAb K928 WITH HUMAN
CARCINOMAS AS DETERMINED ON FROZEN SECTIONS BY IMMUNOPEROXIDASE
Positive/ tested
Squamous-cell carcinomas head and neck lung Small-cell carcinoma lung Undifferentiated large-cell lung Adeno-carcinomas Lung Breast Colon Ovary Melanomas Lvmuhomas
K984
K928
59/59 616
50159 516
012
212
112
112
416 416 415 116
616
013 012
013 012
616 016 516
factor receptor, transferrin receptor and the recently described SF-25 antigen (Takahashi et al., 1988) on frozen sections of normal squamous epithelium as well as of the same panel of tumors. In contrast to the other antibodies, a striking similarity was found between the reaction patterns of MAb K984 and MAb SF-25 o n both oral mucosa and SCC tissues, indicating recognition of the same antigen (data not shown). Identification of the K984 antigen by immunoprecipitation Since immunoblotting experiments failed to identify the K984 antigen, we investigated the presence of the K984 antigen at the outer cell surface of SCC cells by immunoprecipitation of 'Z51-labelledsurface proteins of UM-SCC-22B cells. Using this technique, we were able to show that MAb K984 recognizes a 90- to 100-kDa surface protein (Fig. 9, lane A). A protein with similar apparent molecular weight was identified upon precipitation with MAb SF-25 (IgG,) (Fig. 9, lane B), but
MAbs TO SQUAMOUS-CELL DIFFERENTIATION ANTIGENS
511
FIGURE 5 - Reactivity of MAb K984 with a verrucous squamous-cellcarcinoma of the maxillary sinus. Note the reactivity with a single basal-cell layer. Bar, 75 pm. FIGURE 6 - Reactivity of MAb K984 with a poorly differentiated SCC of the larynx. Staining can be observed in all layers. Bar, 75 pm. FIGURE 7 - Reactivity of MAb K984 with a moderately differentiated SCC of the floor of the mouth. Binding can be seen in multiple layers. Bar, 75 pm. FIGURE 8 - Reactivity of MAb K928 with a moderately differentiated SCC of the floor of the mouth. Peripheral cells in tumor nests are not bound by the antibody, in contrast to the central layers. Bar, 75 pm. TABLE 111 - REACTIVITY OF MAb K984 AND MAb K928 WITHIN VITRO GROWING HUMAN SCC CELL LINES
UM-SCC- UM-SCC- UM-SCC- UM-SCC- UM-SCC- A 431 14A 14B 14C 22A 22B
MAbK984 MAb K928 MAbK112
1.10 1.90 0.13
1.50 1.40 0.15
1.90 1.00 0.12
1.50 1.30 0.15
1.60 1.30 0.16
1.40 1.50 0.12
Absorbancewas measured at 492 nm. MAb K112 served as a negative control. Standard deviations below 10%.
not with MAb JSB-1 (IgG,) which served as a negative control (Fig. 9, lane C).
Enzymatic sensitivity of the K984 and K928 antigens Results obtained using immunoperoxidase staining of enzymatically or chemically pre-treated tissue sections revealed that, in contrast to the K928 antigen, the K984 antigen is sensitive to periodate, indicating that the K984 antigen determinant is located on a carbohydrate chain. Neuraminidase did not influence the labelling, indicating that no sialic acid residues were part of the epitope. Also, MAb K984 reacted with NP-40 lysates of tumor cells which were bound on wheat-germ agglutin (WGA) in a lectin-bound ELISA, suggest-
ing that MAb K984 recognizes a glycoprotein (data not shown).
ldentijication of the K928 antigen in cultured cell lines by immunoblotting Using immunoblot analysis of 4 different cell lines derived from patients with SCC, we have identified a single band of almost identical molecular weight of 50-55 kDa under nonreducing conditions (Fig. 10, lanes C, F, G, H). Relative differences could be observed between UM-SCC-l4A, UMSCC-14C and UM-SCC-22B, corresponding to the differences as observed with the cell ELISA (Fig. 10, lanes H, F, G, respectively). Upon reduction of the samples the 50- to 55-kDa band could no longer be recognized (Fig. 10, lane B). When total membranes were extracted by NP-40 or Triton X-100, the K928 antigen was found within the detergent-soluble phase (Fig. 10, lanes D, E, respectively), while no antigen could be detected in the detergent-insoluble fractions (data not shown). DISCUSSION
In the present study, 2 MAbs were presented with different reactivity patterns for squamous epithelia and its neoplastic counterpart. MAb K984 recognizes a structure which is, in normal epidermis, expressed on the surface of basal kera-
5 12
FIGURE10 - Immunoblot analysis of cell lysates of squamouscell carcinoma cell lines with radiolabelled MAb K928. Lane A: control cell line HL 60 not reactive with MAb K928; lane B: UM-SCC-14A in the presence of P-mercaptoethanol (reducing conditions); lane C: UM-SCC-38; lane D: UM-SCC-38, NP-40 soluble fraction; lane E: UM-SCC-38 Triton X-100 soluble fraction; lane F: UM-SCC-14B; lane G: UM-SCC-14C and lane H: UM-SCC-14A.
FIGURE9 - Radioimmunoprecipitation of a cell lysate, prepared from lactoperoxidase radiolabelled UM-SCC-22B cell line. (A) Specific precipitation of a 90- to 100-kDa surface protein by pre-formed complexes of MAb K984 and protein-A-Sepharose CL-4B. (B) Pre-formed complexes of MAb SF-25 precipitated a protein apparently similar in molecular weight to MAb K984. (C) No precipitation is observed with the negative control antibody JSB-1 complexes. Similar typical results were also obtained for the other negative controls; non-complexed protein-A-Sepharose or pre-formed complexes with normal mouse immunoglobulins (not shown). Bars indicate (top to bottom) molecular weight markers of 110,84,47 and 33 kDa, respectively. tinocytes. This was confirmed by immuno-electron-microscopic studies. In squamous-cell carcinoma, MAb K984 reacts with poorly differentiated cells, as determined by light microscopy. The immunohistochemical staining pattern of MAb K984 suggests that the antigen is associated with the proliferative compartment of SCC. Similar antigen expression has also been described for other basal-cell markers (Kimmel and Carey, 1986;Boeheim et al., 1985; Ranken et al., 1987; Defize et a[., 1987). Cowley et af. (1986) described this for the epidermal growth factor receptor (EGFR), speculating that, as a consequence of transformation, cellular differentiation programs controlling the expression of EGFR could be blocked, causing the receptor levels to remain high. MAb KY84 was immunohistochemically compared with MAbs directed against CEA, MAM-6, 17-1A, transferrin receptor, EGF-receptor and the SF-25 antigen for its reactivity with normal epidermis and squamous-cell carcinoma. Of these antibodies, only MAb SF-25, first described by Takahashi et al.
(1988) had a strikingly similar reactivity pattern to MAb K984 on both oral mucosa and SCC. The following findings supported the hypothesis that the antigen defined by MAb K984 might be identical to the SF-25 antigen. (1) The antigenantibody interactions were both disrupted by SDS. (2) An antigen with an apparent molecular weight of 2 100 kDa was precipitated by both MAbs. (3) Both antigens have the features of a glycoprotein. With regard to this last point, the denaturation of the antigen by periodate indicates that the epitope recognized by MAb K984 is located on a carbohydrate chain, and the K984 antigen could be detected in the lectinbound ELISA after lysis of cells with NP-40, suggesting that it is a glycoprotein rather than a glycolipid, since the latter would not be recovered upon treatment with non-ionic detergents. In contrast to MAb KY84, MAb K928 recognizes an antigen on membranes of more differentiated suprabasal cells in normal epidermis. In a proportion of moderately and welldifferentiated SCCs, KY28 antigen expression was also restricted to more highly differentiated cells, located in the central layers of tumor-cell nests. In other tumors, however, not only the more differentiated cells, but also most of the poorly differentiated cells expressed the K928 antigen. MAb K928 reacts with a 50- to 55-kDa molecule under non-reducing conditions. The loss of reactivity under reducing conditions suggests that one or more disulfide bands are directly or indirectly involved in the formation of the epitope recognized by MAb KY28. The strong expression of the KY28 antigen by UM-SCC-14A in comparison to UM-SCC-l4C, as monitored in the cell ELISA, was also seen in immunoblotting. The fact that the antigen was found quantitatively in the detergent phase, upon extraction of total membranes with Triton-X 100 or NP-40, indicates that the antigen is not a keratin polypeptide, since these are found in the NP-40insoluble pool (Fuchs and Green, 1981). The epitope recognized by MAb K928 does not seem to be located on a carbohydrate chain, as shown by periodate treatment.
MAbs TO SQUAMOUS-CELL DIFFERENTIATION ANTIGENS
Although further studies are required to identify the antigens described here, our data indicate that MAbs K984 and K928 recognize surface antigens associated with either proliferating or more differentiated cells in normal squamous epithelia. Similar antigen expression was found in squamouscell carcinoma, although in some of these tumors the K928 antigen was also expressed by poorly differentiated cells. In addition, MAb K984 is able to inhibit the growth of SCC lines in an antibody-concentration-dependent manner (Suh et al., 1990). Together with the strong expression of the K984
513
antigen in the vast majority of SCC, this can make the K984 antibody valuable for radioimmuno-imaging and therapy of SCC. ACKNOWLEDGEMENTS
The authors thank Ms. M. van Walsum for technical assistance, and Dr. W. Blomjous and J. Fritz for their assistance in immuno-electron-microscopic experiments. This work was supported in part by Centocor.
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