Development and characterization of breast carcinoma cell lines as in vitro and in vivo models for breast cancer diagnosis and therapy.

To establish a model system for preclinical radioimmunotherapy studies, attempts were made to graft 16 different human breast carcinoma cell lines into BALB/c nu/nu(nude) mice. Nine produced serially transplantable tumors growing at a variable rate, whereas seven failed to do so. Conversely, three new cell lines were established in monolayer culture from transplantable human breast tumors in nude mice. Twelve selected tumors and their corresponding cell lines were characterized for DNA ploidy, % S-phase, and breast epithelial mucin expression by immunohistochemistry and flow cytometry. A wide diversity of these cellular characteristics were found in that each tumor was unique and distinct from the others. DNA ploidy differed among the tumors but was not affected by switching between in vitro to in vivo growth. Some tumors expressed similar levels of the breast mucin both in vitro and in vivo, whereas most expressed lower levels as transplantable tumors. There was a good correlation between immunohistochemical and flow cytometric determination of surface and cytoplasmic mucin expression, and with both techniques estrogen and progesterone receptor positive tumors had significantly higher levels of mucin expression than receptor negative tumors. These 12 transplantable breast tumors, with their corresponding cell lines, provide an excellent model system for testing radioimmunotherapy and other therapeutic reagents because they exhibit diverse phenotypic characteristics that represented a mini-population of breast cancer patients' tumors, allowing assessment of the effect of therapy when confronted with different breast tumors' genotype and phenotype.

Heterotransplantation of human breast carcinomas in nude mice. Correlation between successful heterotransplants, poor prognosis and amplification of the HER-2/neu oncogene.

Four hundred and thirty-three human breast carcinomas and 23 cell lines derived from human breast carcinomas were heterotransplanted in nude mice. Twenty-eight tumors and 13 cell lines took and could be serially transplanted. Their human origin was established by isozyme analysis performed on successive passages. Sixteen primary infiltrating duct-cell carcinomas (PIDC) took, from a total of 262 transplanted (6.1%). This is in striking contrast to the greater than 50% rate of takes of most major cancers of epithelial origin. All 16 PIDC growing in nude mice were highly cellular and lacked desmoplastic hyperplasia. The clinical prognosis of the PIDC patients whose tumors were successfully transplanted was poor. Ten of 16 (63%) died of their disease within 3 years, compared to only 49 (20%) of the 246 PIDC patients whose tumors did not take in nude mice. This could not be attributed to later stage disease of the tumors that took, because only 15% of these patients had 4 or more positive axillary lymph nodes as opposed to 28% of the patients whose tumors did not take. Sixty-four percent of the breast carcinomas growing in nude mice exhibited amplification of the HER-2/neu oncogene which is also correlated with poor prognosis in human breast cancer. It is possible that the nude mouse is more susceptible to a population of highly invasive and lethal breast carcinomas.

Double minutes in the HeLa cell line.

Metaphase preparations of three sublines of the HeLa line showed the presence of double minutes (DM) in varying frequencies. In two sublines (S3 and TCH-3753), the size of the DM was variable, whereas in the Fe-1000 subline, they were uniform. Giemsa banding preparations revealed typical HeLa marker chromosomes in all sublines.

MDA-468, a human breast cancer cell line with a high number of epidermal growth factor (EGF) receptors, has an amplified EGF receptor gene and is growth inhibited by EGF.

Epidermal growth factor (EGF) has been noted to stimulate proliferation of a variety of normal and malignant cells including those of human breast epithelium. We report here that MDA-468, a human breast cancer cell line with a very high number of EGF receptors, is growth-inhibited at EGF concentrations that stimulate most other cells. The basis for the elevated receptor level is EGF receptor gene amplification and over-expression. An MDA-468 clone selected for resistance to EGF-induced growth inhibition shows a number of receptors within the normal range. The results are discussed in relation to a threshold model for EGF-induced growth inhibition.

Expression of endogenous murine leukemia virus in transplantable tumor cells and DNA methylation of the viral sequences.

A cell line derived from early passage of a transplantable tumor in athymic mice that initially had been injected with human breast adenocarcinoma cells produced a substantial amount of RNA sequences related to the Kirsten murine leukemia virus. The cell line derived from later passage of transplantation of these tumors diminished viral RNA production. Expression of the viral RNA sequences in these cell lines is inversely correlated with the level of viral DNA methylation in the mouse genome.

A retrovirus-producing transformed mouse cell line derived from a human breast adenocarcinoma transplanted in a nude mouse.

A transplantable tumor was established in NIH/Swiss/Nu mice from tissue derived from a human breast adenocarcinoma metastatic to the brain. Cultivation of dispersed cells from the third transplant generation of the tumor produced a rapidly growing, high-density culture of fibroblastlike cells. Chromosome and isozyme assays showed these cells to be of mouse origin. The cells behaved as an established line from initial culture. Cells of the tissue culture line, designated NM-1, produced rapidly growing fibrohistiocytomas in nude mice. Electron microscopy revealed that the cells produced large numbers of type C virus particles. Serological, biochemical, and infectivity assays indicated that the retrovirus produced by NM-1 cells is an ecotropic, infective, murine retrovirus antigenically related to, but distinguishable from, Gross and Moloney viruses. The virus did not transform mouse fibroblasts. The data support the conclusion that mouse stromal cells within the transplanted human tumor had undergone malignant transformation and induction to virus replication. The role of the virus in the malignant transformation remains to be clarified.

Sister chromatid exchanges and proliferation kinetics of human metastatic breast tumor cells lines.

The rate of sister chromatid exchanges (SCEs) and the profiles of proliferation (= number of first, second, and third cycle metaphases distinguishable by their staining patterns after BrDU substitution for one, two, or three rounds of DNA replication) have been estimated in three (resp. five) metastatic human breast tumor cell lines. Line MDA MB 231 exhibited SCE rates equalling the level of normal cells, while line MDA MB 435 exhibited a slight increase, and line MDA MB 468 a more distinct increase of the SCE rate over the baseline frequency of normal cells. By the BRDU labelling technique the lines 231 and 435 were shown to be rather fast proliferating in culture, while the line 468, and even more the lines 330 and 361 showed a much slower proliferation pattern. From these results it was concluded that increased SCE rates are rather a facultative than an obligatory feature of malignant cells, and that the Br-DU-labelling technique might be suitable for gaining additional information on proliferation characteristics of tumor cells lines.

Characteristics of membrane and cytosol forms of the mammary tumor glycoprotein molecule MTGP in human breast carcinoma cell cultures and tumors.

The presence, concentration and selected molecular characteristics of the human mammary carcinoma glycoprotein molecule set MTGP, a trace and apparently tumor-specific molecule, were examined in fifteen cell cultures established from mammary carcinomas, tissue from seven mammary carcinomas and control cultures. Both cytosol and membrane-associated forms of MTGP were analyzed, and each was phenotyped by reference to isoelectric point and buoyant density. All cells or tissues of mammary carcinoma origin contained membrane MTGP, whereas cytosol MTGP was undetectable in cell cultures from half of the mammary carcinomas. Neither membrane nor cytosol MTGP were detectable in cells other than mammary carcinomas. Cytosol MTGP could be assigned to three groups by reference to presence, isoelectric point and buoyant density. Membrane MTGP also exhibited heterogeneity between different tumors and could be assigned to three groups by isoelectric point and buoyant density. Each form of MTGP was homogeneous for a given single tumor or cell culture and retained its phenotypic features with passage and cloning. Four general types of MTGP are proposed, through there may be additional fine heterogeneity that cannot be further resolved at this time. These data provide an initial characterization of the membrane form of MTGP and an integrated characterization that is consistent with the concept that tumor-specific antigens may possess both constant regions by reference to antigens recognized by the antisera and variable structure by reference to physicochemical characteristics.

Cytogenetic analysis on eight human breast tumor cell lines: high frequencies of 1q, 11q and HeLa-like marker chromosomes.

The chromosomal constitution of 8 human breast tumor cell lines has been analyzed by conventional staining and G-banding methods. The stem line number was established in each case. In all cell lines, a large number of marker chromosomes have been identified. In addition to the 1q marker chromosome, previously reported to be present in several breast tumors from this laboratory, we also found marker chromosomes involving the 11q segment in all 8 cases, and markers resembling some of those found in the Hela cells in 6 out of 8 lines. It appears that the primary genetic (and cytogenetic) changes are specific for each type of target cell and are not shared by other neoplasms. Marker chromosomes found in different types of tumors may represent genetic changes associated with cancer progression, which may be the result of a multitude of genetic alterations.

Variations in cell form and cytoskeleton in human breast carcinoma cells in vitro.

Cell form and cytoskeletal organization were investigated in 13 human breast carcinoma cell lines in vitro. Using tubulin antibodies and indirect immunofluorescence to detect the arrangement of cytoplasmic microtubules, three distinct cell phenotypes were recognized: (a) cells with extensive arrays of microtubules (type 1); (b) cells which were diffusely stained with microtubules apparent only near the cell margins (type II intermediate); and (c) cells in which individual microtubules could not be detected and only diffuse fluorescence was apparent (type II diffuse). Type I cells were flattened epithelial-like cells, much like normal mammary epithelial cells, which when stained with actin antibody displayed many brightly fluorescent parallel cables or "stress fibers." Many microtubules and microfilament bundles were observed in type I cells when examined by transmission electron microscopy. Type II cells were more rounded, often grew in multilayered colonies, and displayed fewer microtubules and microfilament bundles when examined by either immunofluorescence or electron microscopy. Type II cells ranged from very small rounded cells with diffuse tubulin and actin immunofluorescence (type II diffuse) to more flattened cells in which microtubules and actin cables were observed near the flattened cell margins (type II intermediate). Since all of the cells were derived initially from malignant metastatic lesions and some were tumorigenic when injected into athymic nude mice, we assume that they remained malignant in vitro. Thus, in human breast carcinoma cells in vitro, it is not possible to associate any specific cell morphology or cytoskeletal phenotype with cancer or metastasis in vivo. Whether or not these same conclusions hold for breast tumor cells in situ remains to be determined.

Autologous and homologous immunofluorescent antibody to established breast cancer cell lines.

Indirect immunofluorescence tests were performed on 14 established human breast cancer cell lines using sera from a variety of subjects. Autologous reactions were studied on 10 cell lines, with positive reactions demonstrable in 8. Tests using sera from a randomly selected population of breast cancer patients showed reactivity in 40 to 66% depending on the target cell line used. Reactivity to other nonbreast cancer cell lines was rare. Several control populations were tested, including normal blood bank donors, persons with benign breast disease, and persons with other forms of cancer; immunofluorescent antibody was detected much less frequently in sera from these populations than those from the breast cancer group. Positive reactions remained in spite of absorption of serum with heterophile antigens, normal human breast tissue, and AB+ red blood cells. Thus established cell lines of human breast cancer possess antigens commonly recognized by sera from breast cancer patients.

Carcinoembryonic antigen levels in malignant pleural fluids obtained from patients with mammary cancer.

The clinical usefulness of the carcinoembryonic antigen test for defining the malignant origin of pleural fluids obtained from patients who have mammary cancer was evaluated. Of 57 malignant pleural fluids obtained from 51 patients who had mammary cancer, 31 (54.4%) demonstrated a significantly elevated carcinoembryonic antigen content: the ratio of pleural fluid to serum carcinoembryonic antigen values ranged from .24 to 3.55; whereas for 14 of 23 patients who had normal levels of both serum and pleural fluid carcinoembryonic antigen, the ratio ranged from 0.25 to 2.30. This study indicates that the pleural fluid carcinoembryonic antigen value may be significantly elevated in only 50-60% of mammary cancer patients having documented malignant pleural fluids, and that the pleural fluid to serum carcinoembryonic antigen ratio is of no value in defining the malignant origin of pleural fluids obtained from these patients.

A serologic study of cultured breast cancer cell lines: lack of antibody response to tumour specific membrane antigens in patients.

Humoral antibodies to tumour associated membrane antigens of cultured human breast cancer cell lines were studied using the immune adherence (IA) test. Sera from 353 post-operative breast cancer patients and from twenty-five patients immunized by allogeneic breast cancer cells were tested against the MDA-MB-436 cell line. Fifty-five (15.6%) sera samples from the non-vaccinated group and 131 (77.3%) of 168 sera samples from the immunotherapy group were IA-positive to this cell line after absorption with bovine erythrocytes to exclude antibody to heterologous membrane antigens (HM Ag). Forty-five of the 55 positive-sera from the non-immunized group and 113 of the 131 positive sera from the immunized group became IA-negative after further absorption with lymphoblastoid cells autologous to MDA-MB-436. Subsequently, the twenty-eight positive sera remaining sere tested for oncofetal antigens (OFA). After absorption with OFA rich tissues (fetal brain and M14 melanoma cells), no reactivity remained in the sera samples. In order to identify antibodies specific to breast cancer antigens, the 129 sera samples from non-immunized patients were tested against four other breast cancer cell lines; MDA-MB-157, MDA-MB-231, MCF-7 and UCLASO-B1. Four sera which reacted to more than three of the cell lines were identified. The reactivity of three of the four was due to anti-OFA antibody. The last serum sample was reactive to anti-HLA antibodies. These results indicate that sera of patients with breast cancer contain antibodies to OFA, but do not detect breast histologic type specific antigens as tested by IA using five breast cancer cultured cell lines.

Mutually exclusive genetic signatures of human breast tumor cell lines with a common chromosomal marker.

Seventeen recently established human breast carcinoma cell lines of metastatic origin (MDA series), HeLa cells, and MCF-7 (a well-established breast carcinoma cell line) were studied by starch gel electrophoresis for allozymic differences at 17 enzyme loci. Ten loci proved to be informative in establishing unique genetic signatures for all of the lines with the exception of MDA-134 and -309, which had the same genetic signatures. The probability of these latter two lines being of independent origin and finding their similar genetic signatures by chance is 0.07. These studies enable us to conclude that the chromosomal marker shown to be common to these breast carcinoma cell lines of metastatic origin is not present because of cross-contamination of the lines with other long-term lines or each other.

A human breast adenocarcinoma with chromosome and isoenzyme markers similar to those of the HeLa line.

Pleural effusion was obtained from a 51-year-old black woman who had breast adenocarcinoma and had received chemotherapy and radiation therapy after a radical mastectomy. Cytogenetic and isoenzymic analyses of the cells were performed within a few hours of obtaining the sample. Similar analyses were also done with a cell line established from this effusion. The stemline chromosome number was 35, one of the lowest in human neoplasms. In addition to a marker chromosome involving 1q, which is common in human breast tumors, we found several other marker chromosomes whose G-banding patterns were similar to some of the typical HeLa markers. Genetic signature analysis of 15 isoenzyme loci revealed that 13 were identical to those of HeLa. Both HeLa and the cell line described here express glucose-6-phosphate dehydrogenase type a, yet they were derived from heterozygotic individuals (ab). Our data indicate the necessity to extensive cytogenetic and biochemical analysis before conclusions are made that cell lines are actually intercell-line contaminants.

Long-term human breast carcinoma cell lines of metastatic origin: preliminary characterization.

Nineteen human breast carcinoma cell lines have been established as continuous cultures during the past 6 years in our laboratory. This preliminary report is designed to list the lines by their designated code numbers (MDA-MB) and present a brief summary of their morphological, cytogenetic and biochemical characteristics. Sixteen of our lines were obtained from pleural effusions, two from brain metastases, and one from pericardial fluid. All lines have been shown to be distinct entities and are uncontaminated by HeLa cells or each other. A lq marker chromosome is present in all but one of the lines examined.