Gloria H. Heppner

Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells

The MCF10 series of cell lines was derived from benign breast tissue from a woman with fibrocystic disease. The MCF10 human breast epithelial model system consists of mortal MCF10M and MCF10MS (mortal cells grown in serum-free and serum-containing media, respectively), immortalized but otherwise normal MCF10F and MCF10A lines (free-floating versus growth as attached cells), transformed MCF10AneoT cells transfected with T24 Ha-ras, and premalignant MCF10AT cells with potential for neoplastic progression. The MCF10AT, derived from xenograft-passaged MCF10-AneoT cells, generates carcinomas in ∼25% of xenografts. We now report the derivation of fully malignant MCF10CA1 lines that complete the spectrum of progression from relatively normal breast epithelial cells to breast cancer cells capable of metastasis. MCF10CA1 lines display histologic variations ranging from undifferentiated carcinomas, sometimes with focal squamous differentiation, to well-differentiated adenocarcinomas. At least two metastasize to the lung following injection of cells into the tail vein; one line grows very rapidly in the lung, with animals moribund within 4 weeks, whereas the other requires 15 weeks to reach the same endpoint. In addition to variations in efficiency of tumor production, the MCF10CA1 lines show differences in morphology in culture, anchorage-independent growth, karyotype, and immunocytochemistry profiles. The MCF10 model provides a unique tool for the investigation of molecular changes during progression of human breast neoplasia and the generation of tumor heterogeneity on a common genetic background.

Tumor heterogeneity: biological implications and therapeutic consequences

SummaryIt is now appreciated that cancers can be composed of multiple clonal subpopulations of cancer cells which differ among themselves in many properties, including karyotype, growth rate, ability to metastasize, immunological characteristics, production and expression of markers, and sensitivity to therapeutic modalities. Such tumor heterogeneity has been demonstrated in a wide variety of animal tumors of differing etiology, tissue and cellular origin, and species. It has been shown in autochthonous, as well as transplanted, tumors. Similar results have been reported for human cancers, although much of the evidence that heterogeneity of human cancers, also reflects, at least in part, the existence of clonal subpopulations, is still indirect. Heterogeneity is not a unique property of malignancy. Preneoplastic tumors, as well as normal tissues, are also composed of cellular subpopulations.Proposed mechanisms for the origin of tumor heterogeneity include coalescence of multiple foci of cancer clones and the generation of diverse subpopulations from a single clone. This latter process could be due to genetic errors arising from classical genetic mechanisms or to the production of cellular variants as in normal tissue differentiation. Indeed, certain tumor subpopulations have been shown to produce variants at high frequency. In some cases this frequency can be modified by environmental circumstances. Nontumor cells may also contribute to production of cancer cell variants, perhaps, in the case of infiltrating phagocytic cells, by producing mutagens or by somatic hybridization with cancer cells. Production of tumor cell variants is a dynamic process which can occur at any time.Although tumors are mixed populations of cells, knowledge of the characteristics of individual components is not sufficient to predict the behavior of the whole. Individual cancer subpopulations can interact to affect each others growth, immunogenicity, ability to metastasize, sensitivity to drugs, and clonal stability. The existence of multiple, interactive subpopulations provides a basis for the well-known phenomenon of ‘tumor progression’ in which tumors undergo qualitative changes in characteristics over the course of time. Selection of subpopulations better able to survive changing environmental circumstances allows for such changes as autonomy in regard to endogenous growth regulation, more ‘malignant’ behavior, and loss of response to therapy. Tumor subpopulation interactions may play a regulatory role in this process.Tumor heterogeneity has obvious consequences to the design of effective therapy. It provides one rationale for combination therapies and suggests that initial treatment should be early and comprehensive. The continuing emergence of new clones suggests that treatment which is unsuccessful at one point might be effective later. Assays to predict effective therapy for individual patients need to address the multiplicity of tumor subpopulations and the ability of these subpopulations to influence each other. Subpopulation interactions may also be useful in therapy design, as may be efforts to control the extent of tumor heterogeneity by agents which effect cellular differentiation. Thus, tumor heterogeneity presents both problems and, perhaps, new solutions for control of cancer.

Nontransgenic models of breast cancer

Numerous models have been developed to address key elements in the biology of breast cancer development and progression. No model is ideal, but the most useful are those that reflect the natural history and histopathology of human disease, and allow for basic investigations into underlying cellular and molecular mechanisms. We describe two types of models: those that are directed toward early events in breast cancer development (hyperplastic alveolar nodules [HAN] murine model, MCF10AT human xenograft model); and those that seek to reflect the spectrum of metastatic disease (murine sister cell lines 67, 168, 4T07, 4T1). Collectively, these models provide cell lines that represent all of the sequential stages of progression in breast disease, which can be modified to test the effect of genetic changes.

Clinical and pharmacological implications of cancer cell differentiation and heterogeneity

Abstract Various aspects and interrelationships of intra-neoplastic diversity have been investigated at two levels: (1) differences among multiple subpopulations of tumor cells at any point in time (heterogeneity) and (2) variations among these subpopulations as a result of their maturation with time (differentiation). The importance of tumor cell heterogeneity and differentiation in neoplasia has been studied using cancer cells obtained from murine mammary tumors and rhabdomyosarcoma, as well as from patients with carcinoma of the colon. Several cancer cell lines derived from these tumors have been cloned and characterized. Neoplastic cell differentiation has been induced using a polar solvent, N,N-dimethylformamide (DMF); the differentiation is evidenced by morphological maturation, conversion of tumor cell markers and cell culture characteristics to those consistent with a more benign phenotype, and loss of tumorigenicity. Striking morphological and biological heterogeneity has been observed in neoplastic subpopulations isolated from a single tumor, including marked variability in growth potential, surface antigens, and sensitivity to chemotherapeutic agents. The biological and pharmacological significance of these findings is discussed from the standpoint of their profound implications for clinical therapy.

Cellular interactions in metastasis

The metastatic cascade is a sequence of events that must be completed for metastases to be established. The realization that tumors are heterogeneous, consisting of many different subpopulations differing in many characteristics, and the belief that there are selective events in the metastatic process have led several laboratories to isolate and characterize variants with both high and low metastatic potential. Typically, the highly metastatic variants have been able to form distant metastases when implanted into the subcutis. Such lines have been popular for studies of metastatic mechanisms and anti-metastatic therapy, but they may be atypical examples, and thus not the best experimental models. Recent studies indicate that normal tissue influences metastasis such that many tumors metastasize only if placed in the orthotopic site. Furthermore, some cells that do not metastasize individually are able to do so in conjunction with other variant subpopulations. Thus, mixtures of tumor cells in the tissue of origin can express a more malignant character. We review possible mechanisms for such influential interactions, as well as the role of cellular interactions in generating heterogeneity and stabilizing tumor characteristics.

FACS Quantitation of Leucine Aminopeptidase and Acid Phosphatase on Tumor‐Associated Macrophages From Metastatic and Nonmetastatic Mouse Mammary Tumors

Macrophages were isolated by adherence from tumors produced by a number of murine mammary carcinoma lines and were examined by fluorescence‐activated cell sorting for quantitation of leucine aminopeptidase and acid phosphatase. The tumors included three lines, 66, 67, and 168, which were originally derived from a single, spontaneously arising tumor in a BALB/cfC3H mouse and two other lines, D2A1 and D2F2, which were derived from a single tumor arising from the transplantable hyperplastic alveolar nodule line, D2. These five lines differ from one another in a number of characteristics, including the ability to metastasize spontaneously to the lung from subcutaneous implants and to form experimental metastases in lungs following intravenous injection. Line 67 is nonmetastatic under both circumstances, whereas lines 66, D2A1, and D2F2 are metastatic under the same conditions. Intermediate to these is line 168, which is nonmetastatic from the subcutaneous site but capable of colonizing the lung with an efficiency similar to 66 when injected IV. Tumor‐associated macrophages (TAM) from lines 66, D2A1, and D2F2 contained the greatest amounts of leucine aminopeptidase (LAP) and those from line 67 the least, with TAM from 168 being intermediate. Conversely, the TAM from line 67 had the greatest amounts of acid phosphatase (APTase) and those from line 168 the least. In addition to differences among tumors in enzyme levels of the adherent TAM, the precentages of TAM that were adherent were also different among the tumors. Only 12% of TAM from line 67 were recovered in the adherent fraction as opposed to 35–38% of TAM from lines 66 and 168.

Significance of three-dimensional growth patterns of mammary tissues in collagen gels.

SummaryFive tumor cell lines that originated from a single mouse mammary adenocarcinoma, normal mammary tissue, a preneoplastic alveolar nodule line, and a tumor developing spontaneously from that preneoplastic line were used to study the different three-dimensional growth patterns that mammary tissues produce in collagen gel. We describe five different outgrowth morphologies, one of which may represent normal stromal tissue and infiltrating cells. One type was produced both by tumors and by normal mammary gland tissue in an age-related fashion, i.e. more outgrowths of this type were produced by mammary tissue from older mice. The outgrowth patterns for the tumor cell lines were not related to morphology in monolayer culture. Certain of the tumor lines, including one variant-producing line, produced multiple outgrowth patterns. Our results indicate that this cultivation technique may be a useful method for studying the heterogeneity of mammary tumors and may facilitate the isolation of mammary tumor subpopulations.

Cellular and Molecular Biology of Mammary Cancer

Mammary Epithelial Markers and Cell Lineages of Mammary Neoplasia.- Changes in Antigen Patterns During Development of the Mouse Mammary Gland: Implications for Tumorigenesis.- Stem Cells in Mammary Development and Cancer.- Structural Components as Markers of Differentiation and Neoplastic Progression in Mammary Epithelial Cells.- Chromosomes in Breast Cancer.- Extra cellular Matrix and Cell-Cell Interaction.- Epithelium - Mesenchyme Interaction in the Fetal Mammary Gland.- Extracellular Matrix Effects on Mammary Cell Behavior.- Extracellular Matrix: Structure, Biosynthesis, and Role in Mammary Differentiation.- Adipocyte, Preadipocyte and Mammary Epithelial Cell Interaction.- Interaction of Mammary Tumor Subpopulations.- Hormones and Growth Factors.- Rodent Models to Examine In Vivo Hormonal Regulation of Mammary Gland Tumorigenesis.- Primary Culture Systems for Mammary Biology Studies.- Prolactin Effects and Regulation of its Receptors in Human Mammary Tumor Cells.- Regulation of Growth and Secretion of Growth Factors by 17 Beta-Estradiol and V-RASH Oncogene in Human Mammary Cell Lines.- Growth Factor Production by Mammary Tumor Cells.- Prostaglandins in Breast Cancer.- MMTV and Gene Expression.- Exogenous and Endogenous Mouse Mammary Tumor Viruses: Replication and Cell Transformation.- On the Mechanism of Carcinogenesis by Mouse Mammary Tumor Virus.- Hormonal Control of Mouse Mammary Tumor Virus Transcription.- Overt and Cryptic Functions of the Mouse Mammary Tumor Virus Long Terminal Repeat DNA Sequence.- Transformation.- Cell Biology of Mouse Mammary Carcinogenesis in Organ Culture.- Involvement of Oncogenes in Carcinogenesis.- Role of Differentiation on Transformation of Human Breast Epithelial Cells.- Growth and Transformation of Human Mammary Epithelial Cells in Culture.- Cellular Manifestations of Human Breast Cancer.- Cytokinetics of Mammary Tumor Models and the Effect of Therapeutic Intervention.- Current Concepts of Selenium and Mammary Tumorigenesis.- Dietary Retinoids and the Chemoprevention of Mammary Gland Tumorigenesis.

Reactive oxygen-mediated damage to murine mammary tumor cells

We have shown, in a preliminary report, that macrophages can induce strand breaks in the DNA of co-cultured tumor cells (Chong et al., 1988). The present study is designed to determine if oxygen-centered species generated by the cell-free enzyme-substrate combination of hypoxanthine and xanthine oxidase can induce similar lesions and to identify the specific mediator(s). We report that co-incubation of murine mammary tumor cell lines with hypoxanthine and xanthine oxidase leads to the induction of DNA-strand breaks as determined by fluorescence analysis of DNA unwinding (FADU) assay or alkaline elution techniques. This damage is preventable by catalase which removes hydrogen peroxide but no protection is provided by agents to remove or prevent the formation of superoxide anion (superoxide dismutase), or hydroxyl radical (mannitol or the iron chelator o-phenanthroline). Likewise, cyclooxygenase or lipoxygenase inhibitors of arachidonate metabolism (indomethacin, nordihydroguaiaretic acid, caffeic acid) or bromophenacyl bromide do not alter the degree of DNA scission. Treatment with higher doses of oxygen species leads to significant toxicity as determined by evaluation of cell growth potential or colony-forming ability. Again, toxicity is prevented only by the presence of catalase. Tumor cells are able to rejoin strand breaks at lower, less toxic doses. When comparing different tumor cell subpopulations at various stages of progression, i.e., metastatic vs. nonmetastatic, for sensitivity to hydrogen peroxide-induced strand breakage, we found that at lower concentrations (less than 5 microM) metastatic populations are sensitive whereas nonmetastatic populations exhibit no significant breakage. At higher concentrations of hydrogen peroxide, all lines were sensitive, suggesting that a lower threshold of sensitivity may exist for more progressed tumor cell lines.

Facs analysis of tumor associated macrophage replication differences between metastatic and nonmetastatic murine mammary tumors

The proliferation activity of adherent, tumor‐associated macrophages (TAM) from three related murine mammary carcinoma lines was measured by fluorescence‐activated cell sorter (FACS) analysis of the incorporation of 5′‐Bromo‐2′deoxyuridine (BrdU) in vivo during a 1 hr period prior to tumor removal. The tumor lines examined were line 67 which is nonmetastatic following either subcutaneous (SC) or intravenous (IV) injection, line 168 which colonizes the lung after IV injection but not SC, and line 66 which can colonize the lung from either site.