International workshop on monoclonal antibodies and breast cancer. San Francisco, California; November 8 and 9, 1984.
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Animals↗
Biomedical subjects
R L Ceriani
Publications and source records attributed to R L Ceriani.
Monoclonal antibodies, benign breast disease, and breast cancer. A panel discussion.
Hybridoma technology has now advanced to the point where hundreds of monoclonal antibodies have been produced and are reported to bind to normal and malignant cell surfaces or intracellular organelles. The original hope for tumor-specific antigens has not materialized, but certain antibodies with rather restricted ranges of binding activities have been identified. This panel discusses the use of such monoclonal antibodies in diagnosis, prognosis, and treatment of human breast disease and breast cancer.
Antibodies, Monoclonal↗
Breast cancer markers: comparison between sialyltransferase and human mammary epithelial antigens (HME-Ags) for the detection of human breast tumors grafted in nude mice.
Sialyltransferase (CMP-sialic acid:asialofetuin sialyltransferase) and human mammary epithelial antigens (HME-Ags, cell surface antigens specific to human mammary epithelial cells) were determined in plasma of nude mice grafted with breast and non-breast human tumors to assess their possible usefulness as breast cancer markers. The plasma transferase activity was significantly higher (p less than 0.01) in tumor groups relative to the control. However, no significant difference (p less than 0.05) could be found in the transferase level between breast and non-breast tumor groups, showing the enzyme's lack of specificity for breast cancer. Furthermore, the surgical procedure performed on the control normal healthy group (no tumor), resulted in an important increase of the enzyme level, while HME-Ags remained unchanged. HME-Ags were essentially negative in control as well as non-breast tumor groups. After surgical removal of breast tumors, HME-Ags level dropped drastically to the background level (from 122 to less than 30 ng/ml plasma). These data indicate that HME-Ags are more sensitive and specific than sialyltransferase as markers for human breast tumor, and suggest that HME-Ags may be clinically useful in the early detection of breast cancer as well as in the followup of patients with metastatic breast tumor.
Animals↗
Variability in surface antigen expression of human breast epithelial cells cultured from normal breast, normal tissue peripheral to breast carcinomas, and breast carcinomas.
Single-cell heterogeneity and variability in expression of several surface antigens on human mammary epithelial cells in short-term culture were studied with immunofluorescence techniques, using polyclonal and monoclonal antibodies. The cultures, derived from normal breast, a fibroadenoma, a gynecomastia, normal breast tissue peripheral to breast carcinomas, and breast carcinomas and their metastases, were studied after one passage in vitro. The percentage of positive cells varied considerably from one tissue sample to another in all categories from normal to malignant, although there was an overall trend toward a decreasing percentage of positive cells of malignant tissues. The relative antigen content varied 3- to 8-fold among individual samples of cells from normal, peripheral, and carcinoma tissue, while the mean values in the three categories were similar. The single-cell variability in relative antigen content was considerable in all individual samples of normal, peripheral, and carcinoma tissues, as reflected in the high coefficients of variation. However, the coefficients of variation were significantly higher for cells from carcinoma and peripheral tissues [69 +/- 10% (S.E.) and 75 +/- 9%, respectively] than for cells from normal breast (48 +/- 5%). By analyzing in clonal colonies the appearance of quantitative variants in expression of a specific surface antigen, detected with a monoclonal antibody, the carcinoma cells were found to have a 10-fold higher rate of phenotypic variability (mean, 1.21 X 10(-2)/cell/generation) than did cells from normal breast (mean, 0.119 X 10(-2)) and one gynecomastia (0.045 X 10(-2)). Mammary epithelial cells from apparently "normal" tissue peripheral to a carcinoma had an intermediate rate of phenotypic variability (mean, 0.310 X 10(-2)) that was significantly higher than that of the normal tissue.
Adenofibroma↗
Characterization of cell surface antigens of human mammary epithelial cells with monoclonal antibodies prepared against human milk fat globule.
Hybridomas have been prepared that secrete monoclonal antibodies against three different surface antigens of normal human mammary epithelial cells by fusion of mouse myeloma cells with spleen cells from mice and rats immunized with delipidated human milk fat globules. Using a novel method for molecular weight determination, the three different monoclonal antibodies, BLMRL-HMFG-Mc3, BLMRL-HMFG-McR2, and BLMRL-HMFG-Mc5, were found to identify molecules with apparent molecular weights of 46,000, 70,000, and 400,000 daltons, respectively. The latter is a mucin-like glycoprotein with a high sugar content and has not previously been described as a component of the human milk fat globule or of human mammary epithelial cell membranes. Single-cell quantitation of binding of monoclonal BLMRL-HMFG-Mc5 to three breast tumor cell lines using a Microscope Spectrum Analyzer and indirect immunofluorescence revealed a heterogeneous expression. Further, using a competitive radioimmunoassay, it was found that breast tumor cell lines differed by at least 10-fold in the 400,000-molecular-weight antigen content. None of the three antigens are detectable on several nonbreast cell lines, including normal breast fibroblasts.
Animals↗
Comparison of rates of phenotypic variability in surface antigen expression in normal and cancerous human breast epithelial cells.
A method is described for measuring the rate of phenotypic variability in normal and neoplastic breast epithelial cells. Three groups of normal human mammary epithelial cells were studied, two derived from reduction mammoplasties and one derived from the normal breast tissue of a patient with fibroadenoma. The breast carcinoma cells were all cell lines, four (MCF-7, SKBR-3, MDA-MB-157, and T47D) derived from pleural effusions of patients with breast cancer, and one (BT-20) derived from a primary breast tumor. The heterogeneity and variability in expression of a cell surface glycoprotein with apparent molecular weight of 400,000 were studied at the single-cell level with immunoperoxidase techniques using a specific monoclonal antibody, BLMRL-HMFG-Mc5, to a nonpenetrating glycoprotein. The rate of appearance of quantitative variants in expression of this specific surface antigen (rate of phenotypic variability) was determined in clonal colonies and was found to be severalfold higher in all five breast carcinoma cell lines (mean, 2.23 X 10(-2)/cell/generation) than in the normal breast epithelial cells (mean, 0.36 X 10(-2)/cell/generation). In addition, a considerable quantitative variation in expression of this surface antigen was demonstrated among the cells of each population in both normal and neoplastic breast cells which spread over an 8- to 10-fold range. Furthermore, the quantitative distribution among single cells was not random, for the cells tended to cluster around values that fit a geometric series.
Antigens, Neoplasm↗
Dolichol-bound oligosaccharides and the transfer of distal monosaccharides in the synthesis of glycoproteins by normal and tumor mammary epithelial cells.
The main dolichol diphosphate-bound oligosaccharides present in primary cultures of both normal and tumor mouse mammary epithelial cells had the same size, yielded the same pattern after acetolysis and paper chromatography, had the same number of mannose residues susceptible to alpha-mannosidase degradation, and were composed of the same monosaccharide residues. This is the first demonstration that normal and tumor mammary cells have dolichol diphosphate-bound oligosaccharides with very similar, if not identical, structures. These compounds are intermediates in the synthesis of asparagine-linked oligosaccharides. On the other hand, normal and tumor cells showed differences in the specific activities of the enzymes involved in the transfer of the distal monosaccharides from the sugar nucleotides to glycoproteins. Sialyl- and fucosyltransferases were elevated and galactosyl- and N-acetylglucosaminyltransferases were diminished in mammary tumor cells. The intact tumor cells showed an increased fucosylation of glycoproteins of the asparagine-linkage type.
Animals↗
Circulating human mammary epithelial antigens in breast cancer.
Heterologous specific antisera against human mammary epithelial antigens (HME-Ags), which are present in the human milk fat globule membrane and breast epithelial cells, were used in a solid-phase radioimmunoassay to determine the presence of these antigens in the sera of patients with disseminated cancer of the breast and other organs. Breast cancer patients carry high levels of HME-Ags in their circulation, while patients with disseminated nonbreast cancer, as well as normal female controls, do not. A similar release of HME-Ags in the circulation was shown by us in a model system. To further corroborate these findings, a three-step procedure for the extraction and identification of HME-Ags from the sera was devised. In this analytical procedure, circulating HME-Ags are recovered on a solid phase carrying their corresponding antibody (anti-HME) and radioiodinated in situ. Later, the labeled HME-Ags are released from the solid phase and characterized by NaDodSO4 gel electrophoresis. With this procedure, HME-Ags were isolated from sera of breast cancer patients but not from sera of nonbreast cancer patients or of normal female controls. The extracted HME-Ags had molecular masses of 150,000, 70,000, and 46,000 daltons. To further support these findings, a monoclonal antibody, BLMRL-HMFG-Mc3, directed to the 46,000-dalton HME-Ag was also used to extract its corresponding antigen from sera. Breast cancer patient sera contained such antigen while the sera of the other patients and controls did not. This highly sensitive methodology offers a specific approach to breast cancer diagnosis as well as further insight into the nature of circulating antigens with a view to increasing our understanding of breast cancer biology.
Antigens, Neoplasm↗
Human mammary epithelial antigens (HME-Ags) in the circulation of nude mice implanted with a breast tumor and non-breast tumors.
Human mammary epithelial antigens (HME-Ags) obtained from the membrane of the human milk fat globule (HMFG) were tested for their possible role as breast tumor markers. Specific antisera raised against HME-Ags were used to monitor plasma concentrations of these antigens in nude mice implanted with a human breast tumor. The level of plasma HME-Ags, determined by radioimmunoassay, was significantly higher in animals transplanted with a human breast tumor (mean +/- standard error; 687 +/- 184 ng/ml) than those with other types of human tumors (colon carcinoma: 50 +/- 29; lung carcinoma: 82 +/- 78; medulloblastoma: less than 30; and Wilson melanoma: less than 30) and healthy control animals (49 +/- 22). Removal of the breast tumor resulted in a significant drop of HME-Ags level to "background" values, suggesting that animals with the breast tumor did release into the circulation HME-Ags which could be possibly used as breast-tumor markers in breast tumor diagnosis.
Animals↗
Localization of mammary tumors in vivo with 131I-labeled Fab fragments of antibodies against mouse mammary epithelial (MME) antigens.
The Fab fragments of antibodies against cell-type-specific surface antigens of mouse mammary epithelial cells (MME-antigens) were used to localize mammary tumors successfully. The radioiodine-labeled anti-MME (Fab) was injected into mice carrying simulated mammary metastases, and after 24 hours the amount of label per gram of excised tissue was several times greater in the tumor than in liver, brain, lung, or muscle. Kidney showed considerable accumulation of label but this appeared to be nonspecific. Kinetic studies revealed a rapid elimination of labeled Fab in the urine with only 1% of the injected dose remaining in the entire blood pool after 24 hours. Wit a high-purity germanium camera, mammary tumors were clearly located ty the 131I-labeled anti-MME (Fab), and normalization to 99mTc-pertechnetate distribution in the animal increased the specificity. The density of 131I-label was fourfold greater over the mammary tumor than over comparable areas of the mouse. No accumulation of 131I-anti-MME (Fab) was observed in nonmammary tumors nor in mammary tumors when labeled nonspecific Fab was used. An analogous system using an antihuman mammary epithelial antiserum is being developed for localization of breast metastases in humans.
Animals↗
Monoclonal antibodies to epithelium-specific components of the human milk fat globule membrane: production and reaction with cells in culture.
Three hybridomas producing monoclonal antibodies (IgG), reacting with components of the human mammary milk fat globule have been isolated. When tested for binding to a wide range of human cell lines and strains, all three antibodies show negative reactions with fibroblasts, lymphoblastoid cells, and a large number of epithelial cell lines of non-breast origin. Two of the antibodies (1.10.F3 and 3.14.A3) reacted with seven out of eight breast cancer lines tested, and with epithelial cells cultured from human milk. The other antibody (3.15.C3) reacted with only two of the breast cancer cell lines.
Animals↗
Quantitation of human mammary epithelial antigens in cells cultured from normal and cancerous breast tissues.
A sensitive radioimmunoassay technique was developed to quantitate the level of human breast celltype specific antigens on cells from normal breast and from various established cell lines of breast and nonbreast origins. Polyacrylamide gel electrophoresis revealed four major proteinaceous components (150,000; 75,000; 60,000; and 48,000) in human milk fat globule membranes that were used to immunize rabbits in order to elicit antimammary epithelial cell antibody. Antisera obtained were rendered specific by absorptions and were able to recognize three specific mammary epithelial components of the breast epithelial cell. Human mammary epithelial (HME) antigen expression was highest (1290 ng/10(6) cells) in normal breast epithelial cells from primary cultures of normal breasts. Lower levels (range: 955 to 330 ng/10(6) cells) were found in breast epithelial cells from cell lines established from cancerous breast tissue. Cells of nonbreast origins as well as fibroblasts from breast gave much lower values (less than 30 ng/10(6) cells). On treatment, with trypsin, of two breast epithelial cell lines (MDA-MB-157 and MCF-7) 80 to 85% of their HME antigen expression was lost, suggesting that a majority of these breast antigens reside on the cell surface.
Antigens, Surface↗
Analysis of expression of human mammary epithelial antigens in normal and malignant breast cells at the single cell level by flow cytofluorimetry.
The expression of cell-type specific antigens on the surface of human mammary epithelial cells (HME antigens) is analyzed at the single cell level by flow cytofluorimetry. Methods are described to deal with two technical difficulties that have hampered the quantitation of surface antigens on cells from primary cultures by flow cytofluorimetry, namely, cell clumping and the presence of several cell types. By staining the cells simultaneously with two different fluorescent markers: the cell surface HME antigens with fluorescein-tagged antibodies (green fluorescence) and the cell DNA with propidium iodide (red fluorescence), it was possible to determine the expression of HME antigens per unit DNA, analyze their expression on breast cells in mixed populations, and analyze their expression in different phases of the cell cycle. The HME antigens were diminished in malignant breast epithelial cells as compared to their normal counterpart.
Antigens, Surface↗
Epithelial characteristics of five subpopulations of a heterogeneous strain BALB/cfC3H mouse mammary tumor.
We have described previously the isolation and characterization of five distinct subpopulations of tumor cells from a single spontaneous strain BALB/cfC3H mouse mammary tumor (Cancer Res., 38: 3174--3181, 3758--3763, 1978). Subpopulations 68H and 4.10 are polygonal and grow in epithelioid patterns in vitro, whereas subpopulations 66, 67, and 168 are fusiform and grow in lattice or fibroblast-like patterns. Line 4.10 produces tumors with distinctly glandular architecture, whereas the other four subpopulations produce poorly differentiated tumors with mixed epithelial-sarcomatous histological patterns. All five lines were evaluated for epithelial characteristics. Dome formation, characteristic of transporting epithelial cells, could be induced by dexamethasone or dimethyl sulfoxide only in line 4.10 cells. Antibodies to cell type-specific mammary epithelial antigens reacted with each of the subpopulations. All five subpopulations had ultrastructural features of epithelial cells, including desmosomes (all five lines), junctional complexes (68H, 4.10, early-passage 66 and 67 only; poorly defined in 168), and growth in cords demonstrating polarity (68H cells). Less definitive myoepithelial characteristics were also seen in four of the lines, including an incomplete reaction for Na+-K+-ATPase (4.10 cells), hemidesmosome-like junctions (168 and early-passage 66 cells), and pinocytotic vesicles at lower than normal frequency (66, 67, and 168 cells). Thus, none of the lines were distinctly myoepithelial. We conclude that the five subpopulations are epithelial cells that express a spectrum of epithelial characteristics.
Animals↗
Use of human mammary epithelial antigens (HME-Ags) in breast cancer diagnosis.
The use of human mammary epithelial antigens (HME-Ags) was explored in detection of human breast cancers. Specific antibodies were made against HME-Ags, which are normal differentiation antigens of human breast epithelium, and were used in a solid phase radioimmunoassay. Circulating HME-Ags were detected in sera of breast cancer patients with high-tumor load. These antibodies, after radioiodination, were also successfully used to localize human breast tumors grafted in athymic nude mice by whole body radioimaging devices. These results suggest that HME-Ags can be applied to breast cancer diagnosis with higher specificity than those already reported.
Animals↗
Characterization of cells cultured from early lactation milks.
Two major types of cells can be cultured from early lactation human milks: a colony-forming epithelial cell and an adherent nondividing cell referred to as a foam cell. The epithelial cells show a positive reaction with a specific antiserum reactive against membrane components of the milk fat globule, whereas the foam cells do not. The nondividing foam cells are phagocytic and can be killed by silica particles; they produce lysozyme, are resistant to trypsinization, and have Fc receptors. These properties, together with the lack of reaction with antiserum to the milk fat globule membrane, suggest that the foam cells are not terminally differentiated epithelial cells, but tissue macrophages.
Cell Adhesion↗
Junctional intercellular communication pattern of cultured human breast cancer cells.
Junctional intercellular communication between several established human breast cancer cell lines and a variety of mammalian cells has been examined. All the cancer cell lines were found to be either noncommunicators or nonselective communicators. This contrasts with normal human mammary epithelium which shows selectivity in junctional communication. Loss of selectivity in junctional communication appears to be a general feature of cultured human breast cancer cells.
Animals↗
Expression of human mammary epithelial (HME) antigens in primary cultures of normal and abnormal breast tissue.
The expression of human mammary epithelial (HME) antigens was studied in primary cell cultures of normal and abnormal breast tissue and of tissues from other organs. By indirect immunofluorescence it was shown that heterologous anti-HME bound to the cell surface of both suspension and monolayer cultures of human breast epithelial cells from normal breast tissue, normal breast fluid, non-malignant atypical lesions of the female breast, gynecomastia in the male breast, breast carcinomas, and peritoneal and pleural effusions of patients with breast carcinomas. The fibroblast-like cells cultured from either normal, non-malignant atypical, or malignant breast tissue never bound anti-HME, nor did histiobyte-like cells from breast fluid or a sarcoid granuloma of the breast. From a Paget tumor of the breast, two distinguishable epithelial-like colonies appeared in the cultures, only one of which expressed HME-antigens. Anti-HME did not bind to cells cultured from tissues of other organs, whether epithelial-like or fibroblast-like; these organs included thyroid, ovary, endometrium, and skin.
Antigens↗