The mouse has emerged as a primary animal model for human breast cancer because the mammary glands of the two species are very similar in structure and function. In this regard the TDLU and LA have similar morphology. The mouse, infected by MMTV, develops "spontaneous" tumors with specific but limited tumor phenotypes. The advent of genetic manipulation has created transgenic mice that develop hyperplasias and tumors morphologically and cytochemically comparable to lesions in humans. Even experienced pathologists have difficulty distinguishing between lesions from the two species, and the morphological similarities support the utility of the mouse model in understanding human breast cancer. In this essay we review our experience with the histopathology of human and mouse mammary disease by comparing the normal gland with hyperplastic, dysplastic and neoplastic lesions of traditional and transgenic origin.

The effect of mammary gland-specific expression of the polyomavirus middle T antigen was examined by establishing lines of transgenic mice that carry the middle T oncogene under the transcriptional control of the mouse mammary tumor virus promoter/enhancer. By contrast to most transgenic strains carrying activated oncogenes, expression of polyomavirus middle T antigen resulted in the widespread transformation of the mammary epithelium and the rapid production of multifocal mammary adenocarcinomas. Interestingly, the majority of the tumor-bearing transgenic mice developed secondary metastatic tumors in the lung. Taken together, these results suggest that middle T antigen acts as a potent oncogene in the mammary epithelium and that cells that express it possess an enhanced metastatic potential.

Transgenic mice expressing the polyomavirus (PyV) middle T oncogene in the mammary epithelium develop multifocal mammary tumors that metastasize with high frequency. The potent transforming activity of PyV middle T antigen can, in part, be attributed to its ability to associate with and to activate a number of c-Src family tyrosine kinases (c-Src, c-Yes, and Fyn). As a first step toward assessing the role of individual c-Src family tyrosine kinases in PyV middle T antigen-induced mammary tumorigenesis, we have crossed transgenic mice carrying the mouse mammary tumor virus (MMTV)/PyV middle T antigen fusion gene with mice bearing a disrupted c-src proto-oncogene. In contrast to the rapid tumor progression seen in the original MMTV/PyV middle T antigen strains, mice expressing the transgene in the absence of functional c-Src rarely developed mammary tumors. After long latency, these mice did eventually develop abnormal hyperplastic mammary tissue. This growth disturbance was correlated with elevated expression of the PyV middle T antigen and the activation of the PyV middle T antigen-associated c-Yes tyrosine kinase. However, transgenic mice expressing the PyV middle T antigen in the mammary epithelium of wild-type or Yes-deficient mice developed multifocal mammary tumors with comparable kinetics. Taken together, these findings suggest that c-Src tyrosine kinase activity is required for PyV middle T antigen-induced mammary tumorigenesis and also illustrate an in vivo genetic approach to the dissection of mitogenic signal transduction pathways.

Breast cancer is a complex disease. Its aetiology is multifactorial, its period of development can span decades, and its clinical course is highly variable. Evaluation of the role of the immune response in either the development or control of breast cancer is also complex. Nevertheless, there is substantial information that in this disease, the immune response is not a host defence reaction and may even serve to facilitate cancer development. This evidence comes from a variety of sources including clinical-pathological investigations in women that show a correlation between the intensity of lymphocytic infiltration into the tumour mass with poor prognosis, studies in breast cancer patients that demonstrate a similar correlation between delayed hypersensitivity reactivity or in vitro assays of immune reactivity to tumour cell membranes or non-specific antigens and poor prognosis, and analyses of cancer incidence in chronically immunosuppressed, kidney transplant recipients who develop an unexpectedly low incidence of breast cancer. The overall conclusions from these human studies are corroborated by observations in mouse mammary tumour models that also demonstrate immune enhancement of breast cell proliferation in vitro and of breast cancer development in vivo. Potential mechanisms for these effects include production, by inflammatory cell infiltrates, of direct or indirect modulators of breast cell growth, e.g. cytokines, peptide or steroid hormones, enzymes involved in steroid metabolism, as well as of antibodies to growth factors or their receptors. These immune facilitatory mechanisms must be overcome if immune-based therapies are to be applied successfully in breast cancer.

The two models described offer an opportunity to determine relevant genetic alterations during progression from preneoplasia to metastatic disease while avoiding genetic noise among individuals (patients and mice) and independent cancers. The human preneoplastic MCF10AneoT.TGn model allows the identification of altered genes as progression occurs sporadically and affords a method of directly testing the gene affect by introduction into non-expressing variants. The model also will allow testing suspected oncogenes and suppressor genes identified in other cancers. However, its value in the latter studies may be limited because genes may not alter MCF10AneoT phenotypes due to other unidentifiable differences between the MCF10 model and the cancer in which the gene was identified. The mouse model allows similar cause and effect studies to determine at which specific steps metastasis genes might be important. The model also provides an opportunity to determine which host response elements are important deterrents at each step in metastasis and in different organ sites. Together, these models have the potential to elucidate a genetic sequence of progression in breast comparable with that being constructed for colon carcinoma [16].

The purpose of this review was to summarize observations on the type and function of inflammatory infiltrates of mouse mammary tumors and to speculate on the underlying mechanisms and the significance of infiltrates to mammary tumor biology. Although the major conclusion is that much more work is needed, certain themes seem to be emerging. The number of infiltrating cells can be very high but is unrelated to biological behavior of the tumors. What seems to be important is the relative contributions of inflammatory cell subsets. In the case of T-cell subsets and NK cells, the infiltrates from tumors of long-term cell lines so far seem uninformative. The general characteristics are similar to those of infiltrates from rapidly proliferating, normal mammary tissues. These characteristics do not correlate with diverse biological behavior or malignant potential. A more informative model appears to be one in which the development of tumors from preneoplastic tissue can be observed. Here our attention is currently focused on NK cells. By contrast, the correlation between activated TAM and metastatic behavior suggests that our transplantable MMT lines may be biologically relevant in the study of infiltrating macrophages. We are especially interested in the role of TAM in the generation of tumor cell variability. Overall, our data indicate that the host infiltrate is another manifestation of both inter- and intra-tumor heterogeneity and, as such, is not simply a response to, but, rather, a part of the tumor ecosystem. Unraveling the cellular and molecular mechanisms that govern the inflammatory cell component of tumors should provide insight into the types of cellular interactions that result in tumor development and progression.

We have previously reported that tumor-specific cytotoxic T lymphocytes (CTLs) derived from pancreatic and breast cancer patients recognize specific epitopes on the mucin polypeptide core. These CTLs recognize breast and pancreatic tumor cells in a major histocompatibility complex (MHC)-unrestricted fashion, and the lytic activity of these T cells is mediated through the T cell receptor (TCR). To characterize the TCR-mediated MHC-unrestricted CTL function, we used semiquantitative polymerase chain reaction (PCR) and cytofluorometry to analyze the TCR repertoire in CTL lines established from cancer patients and specific for mucin-expressing tumors. We found three TCR Vbeta genes, Vbeta9, Vbeta13.1. and Vbeta17, predominantly expressed in these functional cell lines, established either from one patient by stimulation with various mucin-expressing targets or from different patients. Sequencing of these preferentially used TCR genes unveiled usage of distinct Jbeta and Cbeta but a potentially interesting conservation of certain amino acids in the CDR3 region.

Human breast and pancreatic adenocarcinomas are tumors of ductal epithelial cell origin and as such produce and express on their surface polymorphic epithelial cell mucin encoded by the MUC 1 gene. We have previously reported that tumor-specific cytotoxic T cells derived from patients bearing such tumors recognize specific epitopes on the mucin polypeptide core. This recognition was not MHC-restricted because of the highly repetitive sequence of the polypeptide core, which allows simultaneous recognition of many identical epitopes, and cross-linking and aggregation of TCR on mucin-specific T cells. Those studies were performed with limited numbers of tumor cells or allogeneic tumor cell lines. A renewable source of autologous cells presenting this Ag was necessary to further explore mucin-specific immunity. We report here successful establishment and functional analysis of mucin-specific CTL lines and clones derived from breast and pancreatic cancer patients, using either autologous or allogeneic mucin-transfected B cells as Ag. Our results demonstrate that transfection of autologous or allogeneic B cells with mucin confers upon them tumor Ag-presenting ability as well as susceptibility to lysis by mucin-specific CTL. Transfection of APC with this or any other human tumor Ag that may be molecularly defined in the future provides a unique and powerful tool with which to examine the ability of a tumor-associated Ag to stimulate T cell responses.

Systemic treatments for breast carcinoma have improved substantially over the past quarter century. New insights into cancer biology, refinements in biotechnology, and bioengineering of macromolecules hold the promise of even greater reductions in breast and other cancer mortality as a result of biologicals. As exemplified by the clinical results with the monoclonal antibody to HER-2 for antigen-specific passive immunotherapy, biological therapies for breast carcinoma hold substantial promise. The objective of this report is to highlight aspects of preclinical and clinical research on other biologicals for breast carcinoma that also hold potential for improving patient survival. As examples of the potential of cytokines to modulate breast carcinoma cell proliferation and tumor growth, data on cytokines (interferons) with pleiotropic effects and a lymphokine (interleukin-10) acting on T cells and macrophages will be reviewed. HER-2 has promise as a vaccine for active specific immunotherapy; these data will be summarized. Progress on these and other biologicals promises that this will be another modality of therapy resulting in improved survival for patients with both early and metastatic breast carcinoma in the next millennium.

Review Article
No Abstract available

BACKGROUND. Experimental evidence suggests that the growth of a tumor beyond a certain size requires angiogenesis, which may also permit metastasis. To investigate how tumor angiogenesis correlates with metastases in breast carcinoma, we counted microvessels (capillaries and venules) and graded the density of microvessels within the initial invasive carcinomas of 49 patients (30 with metastases and 19 without). METHODS. Using light microscopy, we highlighted the vessels by staining their endothelial cells immunocytochemically for factor VIII. The microvessels were carefully counted (per 200x field), and their density was graded (1 to 4+), in the most active areas of neovascularization, without knowledge of the outcome in the patient, the presence or absence of metastases, or any other pertinent variable. RESULTS. Both microvessel counts and density grades correlated with metastatic disease. The mean (+/- SD) count and grade in the patients with metastases were 101 +/- 49.3 and 2.95 +/- 1.00 vessels, respectively. The corresponding values in the patients without metastases were significantly lower--45 +/- 21.1 and 1.38 +/- 0.82 (P = 0.003 and P less than or equal to 0.001, respectively). For each 10-microvessel increase in the count per 200x field, there was a 1.59-fold increase in the risk of metastasis (95 percent confidence interval, 1.19 to 2.12; P = 0.003). The microvessel count and density grade also correlated with distant metastases. For each 10-microvessel increase in the vessel count per 200x field, there was a 1.17-fold increase in the risk of distant metastasis (95 percent confidence interval, 1.02 to 1.34; P = 0.029). CONCLUSIONS. The number of microvessels per 200x field in the areas of most intensive neovascularization in an invasive breast carcinoma may be an independent predictor of metastatic disease either in axillary lymph nodes or at distant sites (or both). Assessment of tumor angiogenesis may therefore prove valuable in selecting patients with early breast carcinoma for aggressive therapy.

Current technologies can identify subsets of patients who are at greater risk for developing recurrent carcinoma. This article presents conventional and generally accepted pathologic features of breast carcinoma that allow breast carcinoma patients to be placed into low-risk or high-risk categories for recurrence-free or overall survival. Also, the role of flow cytometry, estrogen-progesterone receptor measurement, tumor angiogenesis, and selection oncoprotein expression, such as c-erbB2, are reviewed.

Nitric oxide (NO), a potent biological mediator, plays a key role in physiological as well as pathological processes, including inflammation and cancer. The role of NO in tumor biology remains incompletely understood. While a few reports indicate that the presence of NO in tumor cells or their microenvironment is detrimental to tumor cell survival and consequently their metastatic ability, a large body of clinical and experimental data suggest a promoting role of NO in tumor progression and metastasis. We suggest that tumor cells capable of very high levels of NO production die in vivo, and those producing or exposed to lower levels of NO, or capable of resisting NO-mediated injury undergo a clonal selection because of their survival advantage; they also utilize certain NO-mediated mechanisms for promotion of growth, invasion and metastasis. The possible mechanism(s) are: (a) a stimulatory effect on tumor cell invasiveness, (b) a promotion of tumor angiogenesis and blood flow in the tumor neovasculature, and (c) a suppression of host anti-tumor defense. In this review, we discuss these mechanisms on the basis of data derived from experimental models, in particular, a mouse mammary tumor model in which the expression of eNOS by tumor cells is positively correlated with invasive and metastatic abilities. Tumor-derived NO was shown to promote tumor cell invasiveness and angiogenesis. The invasion-stimulating effects of NO were due to an upregulation of matrix metalloproteases and a downregulation of their natural inhibitors. Treatment of tumor-bearing mice with NO-blocking agents reduced the growth and vascularity of primary tumors and their spontaneous metastases. We propose that selected NO-blocking drugs may be useful in treating certain human cancers either as single agents or as a part of combination therapies.

Two new metastatic mouse mammary tumor transplant lines have been established in nude mice. The Met-1 line, with the polyoma virus middle T (PyV-MT) transgene, metastasized with 100% efficiency. The Db-7 line, expressing a PyV-MT transgene mutated at positions 315 and 322, metastasized with 8.8% efficiency. Histology and computer-assisted intravital microscopy demonstrated that internal microcirculation in Met-1 was more complex than Db-7; Met-1 exhibited higher microvessel density and tortuosity (P < 0.0001). These indices of microvascular complexity correlated with the higher Met-1 metastatic rate (P < 0.0001). These two transplantable lines will be useful for investigating the complex relationship between angiogenesis and metastasis.
(not in PubMed)

Development of metastases distant to the primary site of solid tumors marks late stages of tumor progression. Almost all malignant mammary tumors are carcinomas arising from the breast epithelium, but the morphological and molecular alterations in the mammary stroma surrounding the premalignant and the growing tumor contribute to its conversion into neoplastic tissue. Two parameters are critical for initiation of the metastatic process and access of tumor cells to the circulation. These are the ability of tumor cells to invade the basement membrane and the stroma, and the neovascularization of breast tumor tissue. A major site for development of distant metastases is the skeleton. After colonizing the bone, tumor cells promote a cascade of events leading to recruitment of osteoclasts and subsequent osteolytic bone destruction. A ubiquitous theme of neoplastic progression of breast tumors is the overproduction of matrix metalloproteinases. In this review, we summarize the recent insights into the functional consequences of matrix metalloproteinase expression and activation during malignant conversion in the breast, and after bone colonization. The current literature supports the hypothesis that matrix metalloproteinases play a key role in the metastatic expansion of most, if not all, mammary tumors and in the ensuing bone loss.

An intact basement membrane is essential for the proper function, differentiation and morphology of many epithelial cells. The disruption or remodeling of the basement membrane occurs during normal development as well as in the disease state. To examine the importance of basement membrane during development in vivo, we altered the matrix metalloproteinase and tissue inhibitor of metalloproteinases balance in mammary gland. Inhibition of matrix metalloproteinase synthesis by glucocorticoids or implants or transgenic overexpression of tissue inhibitor of metalloproteinases -1 delays matrix degradation and the involution process after weaning. The mammary glands from transgenic mice that inappropriately express auto-activating isoforms of stromelysin-1 are both functionally and morphologically altered throughout development. Transgenic mammary glands have supernumerary branches, and show precocious development of alveoli that express beta-casein expression and undergo unscheduled apoptosis during pregnancy. This is accompanied by progressive development of an altered stroma, which becomes fibrotic after postweaning involution, and by development of neoplasias. These data suggest that metalloproteinases and disruption of the basement membrane may play key roles in branching morphogenesis of mammary gland, cell cycle, apoptosis, and stromal fibrosis as well as in induction and progression of breast cancer.

Reciprocal interactions between epithelium and mesenchyme mediate crucial aspects of embryonic development and direct the coordinated organogenesis, correct spatial orientation, and the timely expression of functional activity consistent with physiological demands. The mesenchymal equivalent in the adult organism is the stroma, i.e., the loose connective tissue that is separated from the epithelial compartment by an intact basement membrane. In carcinomas, the cellular organization is dramatically changed, and the stroma is extensively modified. The basement membrane is penetrated in a process of degradation and/or decreased synthesis, and direct contact between tumor cells and the surrounding stroma coincides with neovascularization, inflammatory cell influx, and extensive remodeling of extracellular matrix. In this review, we highlight our current knowledge of tumor cell stromal interactions in the mammary gland with particular emphasis on cellular origins and functional phenotypes. We focus both on normal mammary gland and breast tumors and on culture systems developed to dissect individual aspects of cell-cell and cell-extracellular matrix interactions.

Extracellular matrix-degrading matrix metalloproteinases (MMPs) are invariably upregulated in epithelial cancers and are key agonists in angiogenesis, invasion and metastasis. Yet most MMPs are secreted not by the cancer cells themselves, but by stromal cells within and around the tumor mass. Because the stromal environment can influence tumor formation, and because MMPs can alter this environment, MMPs may also contribute to the initial stages of cancer development. Several recent studies in MMP-overexpressing and MMP-deficient mice support this possibility, but have required carcinogens or pre-existing oncogenic mutations to initiate tumorigenesis. Here we review the spontaneous development of premalignant and malignant lesions in the mammary glands of transgenic mice that express an autoactivating form of MMP-3/stromelysin-1 under the control of the whey acidic protein gene promoter. These changes were absent in nontransgenic littermates and were quenched by co-expression of a human tissue inhibitor of metalloproteinases-1 (TIMP-1) transgene. Thus by altering the cellular microenvironment, stromelysin-1 can act as a natural tumor promoter and enhance cancer susceptibility.

Matrix metalloproteinases (MMPs) orchestrate tissue remodeling and play diverse roles during organ development. They are produced excessively during the course of various pathological conditions, including solid tumors. An important function of MMPs during tumor progression is to provide the proteolytic activity that is necessary both for tumor cells to invade extracellular matrix (ECM) and for neovascularization of tumor tissue by endothelial cells. Recently, independent studies in transgenic animals suggest that MMPs may, in addition, promote very early stages of tumor progression. To investigate this possibility further, we have analyzed the consequences of MMP overexpression in functionally normal and nontumorigenic mouse mammary epithelial cells in culture. Our observations demonstrate that the MMP stromelysin-1 (SL-1) triggers an epigenetic molecular program in mammary epithelial cells that results in a number of phenotypic alterations that eventually culminate in the generation of a malignant tumor-cell phenotype.

Alterations in mammary gland structure and function are associated with changes in the expression of members of the matrix metalloproteinase (MMP)3 family of enzymes. In this review, the evidence for a role for specific MMPs in mammary gland development and cellular differentiation, proliferation, and apoptosis is discussed. In addition, MMP expression is altered during the development and progression of preneoplastic and neoplastic breast lesions. The expression of MMP family members in human breast cancer is described, and studies with mouse model systems addressing the role of MMPS in the initiation, growth, invasion, and metastasis of breast neoplasms are reviewed.