contributed July 1996
last update: June 1998
by
The mammary gland is a unique organ for development and tumorigenesis studies. The uniqueness of the mammary gland resides in several factors. First, the mammary parenchyma experiences the vast majority of its growth postpubertally, thus enabling experiments on development to be performed in the juvenile or adult and presenting opportunities for experimental manipulation of the gland which are not available with other organs. On this characteristic, the fat pad transplantation method was developed which resulted in the elaboration of important concepts in senescence, immortalization and preneoplasia. Second, the accessibility of the gland and the ductal organization provides for means to deliver and localize specific molecules to mammary parenchyma cells, the cells which are the site of origin of neoplastic development. Third, the organ is the target of viral, chemical and physical carcinogens which has resulted in unique and complex models for neoplastic development. Finally, the complexity of hormone and growth factor regulation of mammary gland function provides for a sophisticated approach to the study of hormone action. The purpose of this minireview is to discuss some unique properties of the gland which provides the basis for specialized approaches to study developmental, neoplastic and functional events.
One of the most unique features of mammary gland studies is the ability to transplant mammary cells into their normal microenvironment and assess their growth, differentiation and tumorigenic capabilities. This attribute is based on the mammary epitheliumís low rate of growth prepubertally and the accessibility of the mammary fat pad. This operation is most easily performed in the #4 (inguinal) fat pad in both mice and rats and is identical in both species. This operation, pioneered by DeOme and colleagues provides the basis for a wide variety of experimental approaches.
Mammary cells transplanted into the ìclearedî fat pad breed true. Thus, normal virgin ductal or lobuloalveolar cells will readily take, grow and fill the fat pad with mammary ducts in virgin mice (or differentiate into alveolar cells if the recipient mice become pregnant). In virgin mice or rats, the implanted cells take about 8 weeks to fill the fat pad. The cleared fat pad also serves as a transplant site for preneoplastic and neoplastic mammary cells. Cells from both phenotypic states breed true. The ability to transplant into the fat pad allows one to examine the morphogenic and tumorigenic capabilities of unknown cell populations. This is true for cells removed from the in situ mammary gland and for cells grown in vitro. Thus, the transplantation technique provides the ultimate test for tumorigenic potential. An alternative site for transplantation in the rat is the interscapular fat pad which provides a site of white adipose tissue which does not contain host mammary gland parenchyma. The utility of the transplantation method for examining fundamental problems in mammary morphogenesis and tumorigenesis has been used by numerous investigators over the past 40 years.
DeOme and colleagues demonstrated by transplant studies that there are several different types of hyperplastic lesions in the mouse mammary tumor virus (MMTV)-infected mouse mammary gland but only alveolar hyperplasia, termed the hyperplastic alveolar nodule, was at significantly increased risk for tumor development. This approach provided a direct demonstration of the tumorigenic potential of a population of cells and was the basis for developing the concept of preneoplastic lesions in the mammary gland.
Daniel and colleagues demonstrated directly the pattern of senescence in mammary cells by transplant studies. The experiments showed that the proliferation potential of normal mammary cells declined with serial transplantation and was lost after 5-6 serial transplants. Division potential of the mammary gland was not significantly altered by normal tissue age; rather, the aged host did not support gland growth to the same extent as the young host. The number of prior cell divisions rather than chronological age was the most important determinant for the onset of senescence or lack of division potential. Additionally, they demonstrated that mouse mammary preneoplasias did not senesce and were essentially immortal populations. Theoretically, this same experiment could be performed in the rat or for the human gland, using the nude mouse as recipient, but so far, has not been attempted.
The distinction between progressive and non-progressive neoplasms in the rat was determined by transplantation into cleared fat pads of syngeneic rats. Rivera and Vijayaraghavan reported that the percentage of chemical carcinogen-induced primary mammary tumors in the rat that reproduced tumor outgrowths upon transplantation into the cleared fat pads of syngeneic female rats was remarkably low (only 1 of 9 tumors). The majority of primary tumors produced ductal and hyperplastic outgrowths. In contrast, transplants of ovarian-hormone independent primary mammary tumors bred true and consistently developed progressively growing tumors. This model system was extended by Ethier who combined the analysis of in vitro growth potential and growth factor independence with in vivo transplantation into the interscapular fat pad and demonstrated that tumorous growth was intrinsically related to growth factor independence. Primary tumors which exhibited extended growth in vitro but were dependent upon the growth factors insulin and EGF produced only nonneoplastic mammary outgrowths. In contrast, tumors exhibiting growth factor independence in vitro produced neoplastic outgrowths in vivo.
The estimation of the number of stem cells (clonagens) in the mammary gland was reported by Clifton and Gould. They reported on the successful takes and extent of growth of mammary cells injected into the interscapular fat pad. They injected serially diluted cells into the interscapular fat pad of rats and estimated that mammary epithelial clonogens were present at a frequency of approximately 0.05% in the cell population of the virgin rat mammary gland. Experiments performed in mice have demonstrated that any portion of the mammary tree (i.e., primary duct, tertiary duct), any developmental stage (i.e., virgin, lactating), or any age (i.e., 3wk, 80wk) contains cells capable of repopulating the mammary stroma and undergoing the complete developmental cycle of the parenchyma. These experiments convincingly demonstrate that totipotent stem cells exist throughout the mammary parenchyma tree and are not localized to just the terminal portions of the mammary tree. It is surprising that transplantation analysis has not been extensively incorporated into the variety of transgenic tumor models developed over the past five years. One reason for this neglect rests in the genetic nature of some of the transgenics as some of the models are outbred strains. However, a number of models are inbred. One example of the power of the transplantation method using transgenic animals is the experiment of Smith who investigated the developmental potentials of mouse mammary epithelial cells in WAP-LacZ mice and TGFB1 mice. The results demonstrate that distinct progenitor populations exist for alveolar cells and for ductal cells as well as for multipotent cells. Additionally, increased expression of TGFB1 preferentially affected alveolar-progenitor stem cells. The use of quantitative transplantation analysis along with specific markers for stem cells would provide a more comprehensive understanding of the developmental capabilities of the mammary parenchyma at different stages as well as the effects of specific agents (i.e. TGFb1, oncogenes) in these processes.
The cleared fat pad allows serial transplantation of cell populations. Although established cell lines maintained in cell culture by serial passage have provided a useful means to study the effects of drugs and biological molecules on cell function and growth, this approach is often limited by the absence of tissue interactions. In contrast, the ability to serially transplant mammary cells into the mammary fat pad allows the establishment of stable and immortalized lines of non-neoplastic mammary cells analogous to the established cell lines in vitro.
The uncleared fat pad has also been used as a site for transplantation. Such experiments have demonstrated that tumors but not preneoplasias will grow in the intact fat pad. Using this approach, Faulkin et al. demonstrated that mammary preneoplasias are fully responsive to inhibitory paracrine factors produced by normal mammary ducts. Intercellular contact was not necessary for the ability of normal ductal cells to inhibit the growth of preneoplastic mammary cells. The factors specifying these morphogeneic patterns, such as hox genes, have not been elucidated. Paracrine factors have not been delineated yet, although the experiments of Daniel et al. have seemingly ruled out TGFbís. Faulkin et al. postulated that one of the essential differences between preneoplastic and neoplastic mammary growth was the acquisition of refractoriness to local growth inhibitory factors. Surprisingly, this simple assay has not been utilized to examine the growth potential of cell populations that are debatably neoplastic, i.e. intraductal proliferations in the rat or the hyperplastic ductal lesions in the polyoma transgenic mouse.
A recent technology utilizing transplantation is the transgenic gland as extensively examined by Edwards using a variety of oncogenes; i.e. wnt 1, wnt 4, neu. The transgenic gland takes advantage of the ability to transfer functional genes into primary cultures of mammary epithelial cells via retroviral expression vectors or electroporation and determine the effects of overexpression of the genes of interest on morphogenesis and proliferation by transplanting the transfected (or infected) cells into the cleared mammary fat pad. Such experiments examine the role of genes in the context of a normal tissue environment and can determine the potential role of oncogenes in preneoplastic and neoplastic transformations and the demonstration of oncogenic potential of unknown genes. For example Bera et al. demonstrated that the non-tumorigenic mouse mammary cell line TM3 produced tumors upon transfection with a unknown cDNA isolated from a carcinogen-induced mammary tumor. The mammary gland is a more informative site than subcutaneous heterologous transplantation because it allows evaluation of non-neoplastic as well as neoplastic characteristics embodied in the targeted mammary cells.
The accessibility of the mammary fat pad would seemingly provide a normal and successful transplant site for heterologous transplantation of breast tumors. However, the poor transplantability of rat and human mammary carcinomas into the mammary fat pad of athymic nude mice is an unexplained result and part of a puzzling paradox. Experiments by Welsch et al. have shown that the normal mammary epithelium of the rat, but not carcinomas, is easily transplanted and grows well in the athymic nude mice. The poor acceptance rate of tumors (<15%) was not enhanced by hormonal stimulation of the host. A similar poor success rate also is observed when human mammary carcinomas are transplanted into athymic nude mice. The inability of heterologously transplanted mammary carcinomas to grow in nude mice is unexplained but may find an answer in the recent experiments by Mehta et al. They demonstrated that the success rates of transplantation of human tumors was increased to 50% by the coinjection of Matrigel, a mixture of extracellular matrix molecules. Matrigel enhanced both tumor growth and metastasis. These experiments suggest that heterologous transplantation of tumors into nude mice is possible and may provide the basis for an explanation of the paradox of normal-tumor cell heterologous transplantation. Further, the use of Matrigel might also facilitate the successful takes of murine in vitro cell lines which have lost the capability to grow in vivo. Gould and coworkers devised an elegant approach to gene therapy utilizing the unique morphology of the mammary gland to introduce oncogenes directly to mammary cells in situ by microinjection. Basically, the gene of interest, under control of the MMTV LTR promoter, was subcloned into a retroviral vector and introduced at high concentration directly to the mammary epithelial cells by injecting the primary ducts of each gland directly through the nipple. The investigators simply exploited the anatomy of the gland and combined gene therapy technology and ingenuity to develop a new model for mammary tumorigenesis using the activated ras and neu genes. Conceptually, any gene could be introduced to mammary cells in this manner.
The practical demonstration of the in situ gene therapy methodology raises the issue of whether similar techniques could be applied to the human breast. In the human breast, the epithelial cells of interest are on the luminal surface of the ducts and lobules and are therefore accessible. Furthermore, the primary ducts are relatively large and accessible. Finally, with modern imaging techniques, specific areas of the breast can be readily localized and defined. If one considers that early lesions; i.e. ductal carcinoma in situ, and stage 1 carcinomas, are being detected more frequently, might not another therapeutic option be in situ gene therapy?
The postpubertal development and the accessibility of the mammary gland provide opportunities to conduct in vivo experiments examining the role of specific molecules in growth and development. Daniel and coworkers took advantage of the two factors to introduce growth factors locally to the mammary gland. They developed an ingenious delivery vehicle capable of delivering hormones and growth factors to local regions of the growing gland over several days. They mixed defined amounts of growth factors with a ethylene/vinyl acetate copolymer (EVac) to make a hard pellet and surgically implanted small pieces (<1 mg) of the pellet directly in front of the growing ductal tree. Using this method and examining both the morphology of the gland and DNA labeling indices, the investigators demonstrated that factors like cholera toxin and estradiol-17b stimulated growth of the ductal epithelium, deoxycorticosterone acetate stimulated lobuloalveolar differentiation and TGFb¢s inhibited ductal cell growth. This elegant method can be combined with morphometric analysis of the gland or electron microscopic observations to determine the response of specific cell or tissue types to the administered factors.
Recently, confocal microscopy has been applied to the developing mammary gland. Confocal microscopy combines the best elements of the two methods discussed above. It provides in a single specimen cytological analysis of cell structure and function within a visual three-dimensional framework. Confocal microscopy can be successfully combined with cytokinetic as well as immunocytochemical probes to provide spatial-temporal information. The application of intraduct microinjections using vital dyes imparted a high degree of resolution to the microdissection. The cellular detail and intercellular relationships are delineated with high resolution. Confocal microscopy allows optical sectioning of tissue slices into sections as thin as 0.3mM. Serial images can be recorded on a computer using a digital camera and quantitatively analyzed or reprocessed into two or three-dimensional images. The main limitation in application of confocal microscopy is the high cost of the microscopy hardware. The analysis is dependent on sophisticated software component as well as excellent quality optics and personnel specifically trained to use the equipment. However, even with these limitations, confocal microscopy can provide unique insights into organ growth and function. The mammary gland is wonderfully suited for confocal microscopy as one can microdissect specific portions of the mammary parenchyma.
Mammary cancer in rodents shows a pathogenesis similar to that in humans. Mammary cancer is the result of multiple alterations and therefore progresses through multiple stages. The mammary gland is a particularly attractive organ in which to examine the basic events occurring in multi-stage carcinogenesis because of its accessibility, its experimental manipulation and the presence of well-defined rodent models. The most striking similarities are in the overall pathogenesis and the site of origin of the majority of the breast cancers. Breast cancer arises in humans and in chemical carcinogen-treated mice and rats from ductal cells altered in the terminal portions of the mammary tree. The pathogenesis involves an initial intraductal epithelial hyperplasia which progresses through cellular atypia and occlusion of the duct. The most extreme atypical hyperplasia is referred to as neoplasia in rodents and intraductal carcinoma in situ in humans, although the latter term is technically a misnomer. Neoplasia is followed by locally invasive carcinoma and subsequent metastasis to lung (rodents, humans), and bone and liver (humans). If one compares chemical-carcinogen induced preinvasive and locally invasive lesions in the rodent with lesions in the human breast, the similarities are striking with respect to histological and cytological atypia. The emphasis on the viral etiology in mouse mammary cancer has obscured the close parallel of chemical carcinogen-induced lesions in the mouse to those of the rat and human. The new transgenic mouse models offer the opportunity to build upon the past research by combining the power of transgenic technology to explore the relevant biological effects of specific oncogenes and tumor suppressor genes using transplantation methodology. This has been utilized in part by investigators such as Smith and Edwards; however, it could be used effectively to study pathogenesis and progression in other transgenic models. Hopefully, such studies will be designed into future uses of transgenic mouse models.
Medina, D. (1996) The mammary gland: A unique organ for the study of Development and Tumorigenesis. J Mammary Gland Biol. Neoplasia, 1:5-19.
Smith, G.H. (1996) Experimental mammary epithelial morphogenesis in an in vivo model: Evidence for distinct cellular progeni tors of the ductal and lobular phenotype. Breast Cancer Res. Treatment, 39:21-31.
Daniel, C.W. (1972). Aging in cells during serial propagation in vivo. Adv. in Gerontological Res., 4:167-200.
Ethier, S.P. and Cundiff, K.C. (1987). Importance of extended growth potential and growth factor independence on in vivo neoplastic potential of primary rat mammary carcinoma cells. Cancer Res., 47:5316-5322.
Clifton, K.H. and Gould, M.N. (1985). Clonagen transplantation assay of mammary and thyroid epithelial cells. In C.S. Potten and J.H. Hendry (eds.), Cell Clones, Churchill Livingstone, Inc., New York, pp 128-138.
Kordon, E.C., McKnight, R.A., Jhappan, C., Henninghausen, L., Merlino, G. and Smith, G.H. (1995). Ectopic TGFb1 expression in the secretory mammary epithelium induces early senescence of the epithelial stem cell population. Dev. Biol., 168:47-61.
Wang, B., Kennan, W.S., Yasukawa-Barnes, J., Lindstrom, M.J. and Gould, M.N. (1991). Carcinoma induction following direct in situ transfer of v-Ha-ras into rat mammary epithelial cells using replication-defective retrovirus vectors. Cancer Res., 51:2642-2648.
Coleman, S. and Daniel, C.W. (1990). Inhibition of mouse mammary ductal morphogenesis and down-regulation of the EGF receptor by epidermal growth factor. Dev. Biol., 137:425-433.
Wellings, S.R., Jensen, M.M., and Marteum, R.G. (1975). An atlas of subgross pathology of the human breast with special reference to possible precancerous lesions. J. Natl. Cancer Inst., 55:231-274.
Welsch, C.W., OíConnor, D.H., Aylsworth, C.F. and Sheffield, L.G. (1987). Normal but not carcinomatous primary rat mammary epithelium readily transplanted to and maintained in the athymic nude mouse. J. Natl. Cancer Inst., 78:557-565.
Mehta, R. R., Graves, J.M., Hart, G.D., Shilkaites, A. and Das Gupta, T.K. (1993). Growth and metastasis of human breast carcinomas with Matrigel in athymic mice. Breast Cancer Res. and Treatment, 25:65-71.
Edwards, P.A.W., Abram, C.L., and Bradbury, J.M. (1996). Genetic manipulation of mammary epithelium by transplantation. J. Mammary Gland Biol. Neoplasia, 1:75-89.
Bera, T.K., Guzman, R.C., Miyamoto, S., Panda, D.K., Sasaki, M., Hanyu, K., Enami, J. and Nandi, S. (1994). Identification of a mammary transforming gene (MAT1) associated with mouse mammary carcinogenesis. Proc. Natl. Acad. Sci. USA 91:9789-9793.
Keywords
mammary development, mammary development, mammary development, mammary development
Submitted by: Daniel Medina, Ph.D.
Baylor College of Medicine
Cell Biology
One Baylor Plaza
Houston, Texas 77030
USA
Phone: (713) 798-4483
Fax: (713) 790-0545
E-mail: dmedina@bcm.tmc.edu
contributed July 1996 |