Journal of Histochemistry and Cytochemistry, Vol. 46, 127-134, Copyright © 1998 by The Histochemical Society, Inc.


ARTICLE

Immunohistochemical Detection of the MUC1 Gene Product in Human Cancers Grown in scid Mice

Udo Schumachera and Elizabeth Adama
a Human Morphology, University of Southampton, Southampton, United Kingdom

Correspondence to: Udo Schumacher, Human Morphology, Univ. of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK.


  Summary
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

Alterations in mucin expression have been detected in many clinically relevant cancers and, in particular, the polymorphic epithelial mucin, encoded by the MUC1 gene, has attracted considerable attention. We investigated its expression in human breast, colon, ovarian, lung, and skin cancer cells and their metastases grown in severe combined immunodeficient (scid) mice using three different monoclonal antibodies (HMFG-1, HMFG-2, and SM3). Four of five breast cancer cell lines, three of five colon cancer cell lines, two of three small-cell carcinoma of the lung cell lines, and A 431 cells all expressed the MUC1 gene product. Neuraminidase predigestion often enhanced HMFG-1 immunoreactivity, which was more widespread and stronger than SM3 immunoreactivity. A considerable heterogeneity of MUC1 gene product expression was observed in the same tumors grown in different mice. The binding pattern between single-cell/small-cell clusters (up to 10 cells) and larger cell number aggregates varied. The results indicate that the MUC1 gene expression both in primary tumors and metastases is not tightly controlled within a particular tumor cell line. Because of this heterogeneous antigen expression in vivo, it appears impossible to target all metastatic deposits by a single monoclonal antibody directed against the MUC1 gene product. (J Histochem Cytochem 46:127-134, 1998)

Key Words: breast cancer, colon cancer, human, metastasis, microenvironment, mucin, MUC1, polymorphic epithelial mucin, scid mouse, small-cell lung cancer


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Mucins or mucous glycoproteins are large and highly glycosylated proteins. They are generally secreted by epithelial cells, and changes in mucin composition have been detected in a number of diseases, notably in cancer (Strous and Dekker 1992 ). One mucin in particular, MUC1, also named DF3, episialin, CA 15-3, PAS-O, polymorphic epithelial mucin (PEM), or epithelial membrane antigen to list but a few, is unusual because it is not secreted but is an integral membrane protein and contains 50-80% carbohydrate (for review see Patton et al. 1995 ). MUC1 has attracted considerable interest in clinical cancer research because it is widely expressed in breast and ovarian cancers and in some colon and lung cancers. Its glycosylation might be altered in all of these cancers (Taylor-Papadimitriou and Epenetos 1994 ). Because distinct MUC1 changes have been observed in 90% of breast cancer patients, several clincal trials are under way to exploit these changes as the bases for tumor vaccines. This approach is undertaken to elicit an immune response against circulating tumor cells and micrometastases, although it appears unlikely that large primary tumors will be eliminated by this method (Fricker 1997 ). However, these tumor vaccines would work only if all tumor cells of all metastatic deposits were positive for MUC1 and thus would be eliminated by the immune response.

It has proved difficult to model spontaneous metastases of human cancer cells, even in immunodeficient animals. However, recently severe combined immunodeficient (scid) mice have been used for this purpose (Schumacher and Mitchell 1997 ). Human breast and colon cancer cell lines, which were positive for the lectin Helix pomatia agglutinin (HPA), were transplanted into scid mice where, in general, the HPA-positive cell lines metastasized and the HPA-negative cell lines did not (Schumacher and Mitchell 1997 ). This is in agreement with clinical studies in which HPA was of prognostic value in patients with breast (Brooks et al. 1996 ) and colon cancer (Schumacher et al. 1994 ). Because of the same metastatic behavior of the HPA-positive cancer cells in both the patient studies and in the scid mouse experiments, this model system can claim to have a certain degree of clinical relevance regarding those aspects of the metastatic cascade linked to carbohydrate residue expression.

The aim of the present study was to analyze the MUC1 expression in human tumors and their meta-stases when grown in scid mice. The MUC1 expression was assessed using three different monoclonal antibodies (MAbs) named HMFG-1, HMFG-2, and SM3, which have slightly differing but overlapping epitope specificity (PDTR, DTR, PDTRP, respectively). This overlapping specificity is not surprising because similar immunogens were used: human milk fat globule membrane was used as an immunogen for HMFG-1 and HMFG-2 (plus milk epithelial cells), and partially and totally carbohydrate-stripped polymorphic epithelial mucin was used for the production of SM3 (Burchell and Taylor-Papadimitriou 1993 ). In addition to the differing epitopes, carbohydrate residues on the protein core can influence the binding of the antibodies in various ways: (a) HMFG-1 reacts well with the mucin of normal cells, whereas the presence of sialic acid in tumor cells can mask or modify the epitope. (b) The HMFG-2 epitope can be masked by longer carbohydrate side chains on normal mucins, whereas it can be more accessible on shorter glycosylated tumor mucins (Burchell and Taylor-Papadimitriou 1993 ). (c) The binding site of SM3 lies between two glycosylation sites, but flanking oligosaccharide chains can exert an influence on SM3 binding (Brockhausen et al. 1995 ). In addition, SM3 is of particular interest in cancer research because it recognizes a mucin produced by cancer cells but not by normal cells (Girling et al. 1989 ).


  Materials and Methods
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The human breast and colon cancer cell lines were obtained from the European Cell Culture Collection (Porton Down, Salisbury, Wiltshire, UK; for cell lines see Table 1). The classic small-cell lung carcinoma cell line SW2 was established in the laboratory of Dr. S.D. Bernal (Dana Farber Cancer Institute; Boston, MA). The variant small-cell lung carcinoma cell lines OH3 and NCI-N417 were provided by Dr. S. B. Baylin (Johns Hopkins University; Baltimore, MD) and Dr. D. N. Carney (Mater Misericordiae Hospital; Dublin, Ireland), respectively. All cell lines were cultured under standard cell culture conditions.


 
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Table 1. Origin and characteristics of the cell lines used in this study

Pathogen-free male and female BALB/c C57BL/KaIgh-I scid/scid (scid) mice, aged 9-14 weeks, were housed in filter-top cages. The mice were provided with sterile water and food ad libitum and all manipulations were carried out aseptically inside a laminar flow hood. For injection, the tumor cells were harvested by trypsinization, tested for viability, and 5 x 107 viable cells were suspended in 1 ml cell culture medium. Two hundred µl of this suspension was injected SC between the scapulae of each scid mouse for the breast, colon, and lung cancer cell lines and the skin cancer cell line A431. The ovarian carcinomas were transplanted serially by IP injection and the mice were sacrificed when ascites developed. These cell lines have been established in scid mice for over a year in our laboratories (Schumacher et al. 1996b , Schumacher et al. 1997 ).

When subcutaneous tumors developed, mice were sacrificed using a Schedule 2 method. The tumors were dissected out, fixed in neutral buffered formalin, and embedded for routine wax histology. Sections from tumors grown in at least three different animals were used for each tumor cell line. The sections were dewaxed and all antibody incubations were carried out in a Shandon Sequenza Immunostaining Center. The sections were preincubated for 20 min with 0.3% H2O2 in methanol to block endogenous peroxidase activity, washed in PBS followed by a 20-min incubation with 1.5% horse serum in PBS, then a 60-min incubation with the primary mouse MAb HMFG-1, HMFG-2, and SM3, respectively. After washing with PBS, the sections were incubated with biotinylated anti-mouse immunoglobulin (1:200 in PBS) for 60 min and after PBS washing the sections were incubated for 60 min with an avidin-biotin complex labeled with horseradish peroxidase (ABC Kit; Vector Laboratories, Peterborough, UK). Diaminobenzidine hydrochloride/H2O2 was used to localize the binding. The sections were counterstained with hematoxylin and mounted in DPX. To achieve consistency in the immunohistochemical staining, each antibody was incubated with the entire series of sections. Because the binding, particularly of MAb HMFG-1, can be inhibited by a high content of neuraminic acid in the mucin, neuraminidase pretreatment was carried out as described earlier (Plendl et al. 1989 ) because this could enhance its binding (Burchell and Taylor-Papadimitriou 1993 ). All three MAbs were tested for their reactivity with and without pretreatment of the sections with neuraminidase. Wax-embedded HBL100 cells treated with neuraminidase and stained for the lectin peanut agglutinin served as a positive control for the neuraminidase digestion (Schumacher et al. 1996c ).

For evaluation of the antibody binding in lung metastases, lungs were serially sectioned and each section adjacent to an H&E-stained section was used for immunohistochemistry, ensuring the detection of small metastatic deposits in the lung.

MCF-7 cells grown in vitro, which are known to express MUC1 (Brockhausen et al. 1995 ), served as a positive control. Omission of the primary antibody served as a negative control.

For semiquantitative assessment of antigen expression, 400-500 epithelial cells were examined and the percentage of positive cells was scored (see van Dam et al. 1991 ). In addition, the immunoreactivity was graded from 0 = no reaction, (+) = very weak staining, + = weak staining, ++ = strong staining, to +++ = very strong staining. Photomicrographs were taken on an Olympus BH2 photomicroscope using Kodak Technical Pan black-and-white film.


  Results
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Breast Cancer
Of the human breast cancer cell lines grown subcutaneously in scid mice, three cell lines, BT20, MCF-7, and T47D, reacted with all three MAbs, whereas HBL 100 and HS578T cells did not (for a summary of the results see Table 2). The reaction product was generally membranous in all three positive cell lines, although positive diffuse intracytoplasmic reactivity and positive intracytoplasmic granules could also be found (Figure 1). After neuraminidase pretreatment of the sections, HBL100 became positive for HMFG-1 (for a summary of neuraminidase pretreatment results see Table 3). A certain degree of heterogeneity in the percentage of positive cells with all three MAbs was observed in MCF-7 and T47D cells grown in different scid mice. This heterogeneity was greatest in BT20 cells, in which it ranged from no reactivity to 40% reactivity with the MAbs HMFG-2 and SM3.



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Figure 1. Reactivity of HMFG-2 with the human breast cancer cell line MCF-7 grown in a scid mouse. Note the heterogeneity of binding and the intense cytoplasmic reactivity of some positive cells (arrowheads). Bar = 40 µm.


 
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Table 2. Reactivity of the anti-MUC1 gene product-specific MAbs HMFG-1, HMFG-2, and SM3 with the human cell lines grown in scid mice (n = 3)a


 
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Table 3. Reactivity of the anti-MUC1 gene product-specific MAbs HMFG-1 and HMFG-2 with the tumor cells without and with neuraminidase pretreatment

In general, neuraminidase pretreatment led to increased binding of SM3 to the connective tissue septae of the tumors and to necrotic areas; this reactivity was regarded as nonspecific. Apart from MCF-7 cells, which became SM3-negative after neuraminidase pretreatment, and T47D, in which the SM3 immunoreactivity was enhanced, no other specific effect of neuraminidase pretreatment for SM3 immunoreactivity was seen.

Spontaneous lung metastases from the primary implant site were observed in the breast cancer cell lines MCF-7 and T47D. In general, the binding pattern of the three MAbs to the metastases was similar to those in the primary tumor, and not all metastatic deposits contained antibody-labeled cells. In addition to HMFG-1-positive MCF-7 metastases, HMFG-1-negative metastases could occasionally be detected within the lungs (up to 80% of the primary tumor cells were HMFG-1-positive). HMFG-2 reacted less frequently with the metastatic cells than HMFG-1. In one larger metastatic deposit, only two of 12 cells were weakly positive. SM3 reacted even less frequently with the lung metastases. In T47D metastases, the lectin binding pattern reflected those in the primary tumor, with HMFG-2 being slightly better at detecting metastases than HMFG-1. SM3 only faintly stained T47D metastatic cells.

Colon Cancer
Without neuraminidase pretreatment, none of the colon cancer cell lines grown in scid mice reacted with any of the three antibodies (Table 2). However, after neuraminidase pretreatment HT29, SW480, and SW620 were immunoreactive for HMFG-1 (Table 3), but this pretreatment had no effect on the binding of the other two antibodies, which remained negative.

Lung Cancer
A weak but distinctive, often granular cytoplasmic reactivity of 10-20% of the tumor cells with HMFG-1 was detected with the human variant small-cell and small-cell lung cancer cell lines OH3 and SW2 (Table 2). The immunoreactivity of the latter could be enhanced by neuraminidase pretreatment (Table 3). The other small-cell lung cancer cell line, NCI-N417, did not react with any of the three antibodies.

Epidermoid Carcinoma
Up to 30% of the A 431 epidermoid carcinoma cells reacted with HMFG-1 but not with the other two MAbs. Neuraminidase pretreatment enhanced HMFG-1 immunoreactivity (Figure 2).



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Figure 2. HMFG-1 immunoreactivity of the human epidermoid cancer cell line A431 grown in a scid mouse without (a) and with (b) prior neuraminidase treatment. Note the considerable increase in the membranous staining. Bars = 40 µm.

Ovarian Cancers
The pattern of reactivity of the three MAbs towards human ovarian carcinomas established in scid mice varied, but HMFG-1 and HMFG-2 immunoreactivity could generally be enhanced by neuraminidase predigestion (see Table 3). In the SoTü 1 cell line, the immunophenotypical expression of the HMFG-1 antigen was relatively constant over this time period (1 year), ranging from 60% to 90% immunoreactivity, and the entire cell membranes were stained. Immunoreactivity for HMFG-1 which was mainly apical, decreased in SoTü 2 from over 80% positive cells in the first passage to a complete absence during later passages. In contrast, HMFG-1 immunoreactivity increased significantly during later scid mouse passages of the tumor cell lines SoTü 3 and SoTü 4. The increased immunoreactivity for HMFG-1 after neuraminidase pretreatment was particularly demonstrable in SoTü 3 cells, in which HMFG-1 reacted with the entire cell membrane of these cells during the early passage of the SoTü 3 cells (Figure 3). A phenotypic change towards an epithelial morphology was noted during later serial passages, which was associated with an apical expression of HMFG-1 immunoreactivity. In SoTü 4 cells the antibodies reacted predominantly with the apical membrane domain.



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Figure 3. HMFG-1 immunoreactivity (a) without and (b) with neuraminidase pretreatment of the human ovarian cancer cells SoTü 3 grown in a scid mouse. Note the considerable increase in staining of the entire cell membrane in b. Bars = 40 µm.

In SoTü 1, HMFG-1 was better suited to detect metastatic deposits than HMFG-2 or SM3, again reflecting the immunoreactivity within the primary tumor (Figure 4). If the primary tumor reacted strongly with HMFG-1 and HMFG-2 in SoTü 2 cells, so did the metastases. Indeed, in SoTü 2 cells the immunohistochemistry was so sensitive that it labeled even small tumor cell aggregates that were overlooked on adjacent HE sections. If most of the SoTü 2 cells were SM3-negative, then so were the metastases. When low or absent immunoreactivity of the primary SoTü 3 tumor (without neuraminidase pretreatment) was observed with all three antibodies, none of the MAbs detected metastatic deposits in the lungs. In SoTü 4, the immunoreactivity for all three antibodies was found almost exclusively in the apical region of the majority of tumor cells. HMFG-1 and HMFG-2 reacted with cell membranes and showed intracytoplasmic immunoreactivity in single/few tumor cell metastatic deposits. The immunoreactivity of both these antibodies with larger metastatic deposits was confined to the apical pattern only. In contrast to the primary tumor, SM3 only very occasionally reacted with SoTü 4 metastatic deposits in the lung.



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Figure 4. Lung metastases of the human ovarian cancer cells SoTü 1; serial sections. (a) HMFG1 immunoreactivity of the majority of the tumor cells within the metastasis. (b) Hematoxylin and eosin staining. (c) HMFG-2 immunoreactivity of a few SoTü 1 cells, predominantly in the apical region of the tumor cells (arrowhead). (d) Absence of SM3 immunoreactivity. Bars = 15 µm.


  Discussion
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The present study was undertaken to demonstrate the presence of the MUC1 gene product in human cancer cell lines grown in vivo in scid mice. The expression of the MUC1 gene product varied considerably between the same established tumor cell lines grown in different scid mice, being particularly evident in the human breast cancer cell line BT20, indicating a considerable heterogeneity in MUC1 gene expression when the cells are grown in vivo. These findings are in agreement with an earlier report in which a similar heterogeneity of mucin-type terminal carbohydrate residue expression was seen in human tumors grown in scid mice compared to the in vitro situation (Schumacher et al. 1996a ). This heterogeneity of expression is therefore not limited to carbohydrate residues of mucins but is also true for the protein backbone of mucins.

The binding of the three MAbs to the same cell line also differed considerably. These differences can be explained by the fact that the binding regions of the three MAbs to the peptide backbone of the MUC1 gene product overlap and therefore differ slightly. This has consequences for their binding behavior: SM3 binds to an epitope that may be completely masked by carbohydrate side chains of the normal mucin but that may be exposed in the cancer-associated mucin (Taylor-Papadimitriou and Epenetos 1994 ). This binding pattern of SM3 is reflected in this study. The breast cancer cell lines BT20, MCF-7, and T47D all express binding sites for SM3, which is in accord with earlier in vitro studies (Brockhausen et al. 1995 ), whereas HBL 100 cells derived from the milk of a nursing mother (Gaffney 1982 ) do not bind SM3.

The antibody HMFG-2 recognizes an epitope that needs to be flanked by short carbohydrate side chains to be recognized, and these carbohydrate residues are provided by all three cell lines that also bind the other two antibodies. However, if these side chains contain neuraminic acid, this sugar can also inhibit the binding of HMFG-2 in some cell lines, e.g., BT20 and SoTüs 1, 2, and 4. The presence of carbohydrate side chains in these three cell lines is not surprising because earlier in vivo and in vitro studies demonstrated binding sites for several appropriate lectins in these cell lines (Schumacher et al. 1995 , Schumacher et al. 1996a ).

The reactivity of the HMFG-1 antibody is influenced by the presence of neuraminic acid, and if pretreatment with neuraminidase is carried out the binding of this antibody can increase (Taylor-Papadimitriou and Epenetos 1994 ). The effect of neuraminidase pretreatment is particularly noticeable in the three colon cancer cell lines HT29, SW480, and SW620, which reacted with HMFG-1 only after neuraminidase pretreatment.

The MUC1 gene product has been described in colon cancer cells (Hanski et al. 1993 ) and its occurrence was noted in a large proportion of human ovarian cancer cells grown in vitro (McGuckin et al. 1995 ). Both colon and human ovarian cancer xenografts in scid mice expressed the MUC1 gene product. Again, its expression varied among the different ovarian cancer cell lines and passages, which agrees with in vivo observations showing that biological response modifiers such as interferon-{gamma} could significantly increase the MUC1 expression even in ovarian cancer cell lines that exhibited little or no MUC1 expression in the unstimulated state (Clark et al. 1994 ). Consistent patterns of either up- or downregulation of the MUC1 gene product expression during the different passages of the cell lines were not seen, indicating that MUC1 gene expression is not of particular importance at the human-scid mouse interface.

The differential expression of the MUC1 gene product in the primary tumor and at the metastatic sites is particularly interesting in SoTü 4 tumors. In the primary site, all three MAbs reacted almost exclusively with the apical cell membrane of SoTü 4 cells, whereas in small metastatic tumors (single-cell/few-cell aggregates) HMFG-1 and HMFG-2 immunoreactivity was found on all membrane domains. Although the percentage of SM3 immunoreactivity was slightly lower than that of HMFG-1 and HMFG-2, SM3 immunoreactivity was almost completely absent at the metastatic sites. Even in larger metastases in which HMFG-1 and 2 showed apical immunoreactivity, SM3 immunoreactivity was absent or was much less intense than in the primary tumor. Collectively, these findings indicate that the lung microenvironment influences mucin expression and hence differentiation of the tumor cells themselves. This observation is in agreement with recent results obtained in breast cancer cells growing in nude mice, in which it was shown that the extracellular matrix has a considerable influence on the malignant phenotype (Weaver et al. 1997 ).

Our results show that human tumors express the MUC1 gene product at both the implantation and the metastatic site. Because of the heterogeneous expression of the MUC1 gene product within the primary tumor and particularly in the metastatic tumor deposits, any of the three antibodies used alone would not be sufficient to target all the metastatic cells and, as a result, anti-MUC1 antibodies may in the end prove useless for cancer therapeutic applications.


  Acknowledgments

The antibodies were the kind gift of Dr J. Taylor-Papadimitriou, Imperial Cancer Research Fund, London, UK. We thank Ms J. Norman and Ms C. Gradidge for technical assistance.

Received for publication January 27, 1997; accepted July 22, 1997.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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