Tumour-stromal interactions: Role of the stroma in mammary development
© BioMed Central Ltd 2001
Accepted: 26 February 2001
Published: 22 March 2001
Mammary development depends on branching morphogenesis, namely the bifurcation and extension of ductal growth points (end buds) and secretory lobules into a more or less fatty stroma. Because breast carcinomas are overwhelmingly ductal in origin, this review focuses on stromal influences guiding postnatal ductal development and there is only the briefest account of the role of embryonic stroma (mesenchyme). The stroma as the necessary target for endocrine mammogens and the source of stimulatory growth factors is described and the importance of mammary epithelium-induced modifications of the periductal stroma is emphasized. Evidence is presented that if they are to grow, end buds must condition proximal fatty stroma by recruiting white blood cells as well as inducing stromal cell division and, possibly, estrogen receptors. The induction of a fibrous stromal tunic around the end bud is described and its likely role as a complex ductal morphogen is discussed; a possible role in growth inhibition is also considered. Although the signals governing fibrotic induction, ductal morphogenesis, and growth inhibition are unknown, a role for transforming growth factor-β is highly likely and is discussed. Finally, a need for new conceptual and experimental approaches to understanding stromal-epithelial signaling is discussed.
Keywordsbranching mammary morphogenesis stroma TGF-β
Mammary ducts grow through a complex stroma
By parturition, the mammary stroma comprises multiple cellular and acellular elements. In the mouse, which is the most thoroughly studied model and the focus of this review, adipocytes are the most abundant cell type, followed by fibroblasts, various migratory blood cells, endothelial cells (blood vessels), and nerve cells. Acellular elements include fibrous and non-fibrous collagens, proteoglycans, and glycoproteins, all of which provide mechanical support to the tissue as well as forming a dynamic, developmentally active extracellular matrix/basal lamina complex at the epithelial-stromal boundary (Fig. 1b) . This complex connective tissue is found in the mammary glands of different species with various ratios of fibrous to fatty elements , and it is within this matrix that mammary ducts elongate, arborate, and, finally, terminate growth (Fig. 1a).
At this point in its development, then, the signature features of the gland are the following: (1) ductal as opposed to lobular morphology; (2) large, open spaces between ducts; (3) most active growth focused in end buds; (4) regressed end buds at the edge of the fat pad. A role for the stroma in defining each of these glandular features is supported by experimental evidence that is discussed below.
Stromal signals determine ductal morphology
In seminal experiments by Kratochwil and Sakakura, mammary parenchyma was shown to possess a developmental plasticity that is constrained and directed by the stroma . When Kratochwil cultured a composite of embryonic mammary epithelium and embryonic sub-mandibular (salivary) mesenchyme, the mammary tissue developed salivary gland-like lobules. Extending these experiments in vivo, Sakakura demonstrated that not only embryonic but also adult mammary tissue could respond in this way to salivary mesenchymal signals. Importantly, the instructive properties of the stroma did not extend to cytodifferentiation: in a pregnant host animal, salivary-like mammary transplants synthesized the milk protein α-lactal-bumin. Interestingly, fetal mammary mesenchyme grafted into the adult gland accelerated tumorigenesis, providing an early indication that modifications of stromal signaling could influence the progress of neoplasia.
Open glandular architecture depends on signals from the periductal stroma
The absence of extensive ductal side branching and infilling of interductal spaces is not due to the terminal differentiation of ductal cells. Even the smallest fragment of a duct, when transplanted to stroma devoid of parenchyma, undergoes aggressive growth and can fill a fat pad with a morphologically and functionally complete ductal system. This capacity is attributed to up to three populations of mammary stem or progenitor cells  and is subject to stromal inhibition. When similar fragments are transplanted into a space between existing ducts the graft is maintained but does not grow (Daniel, unpublished data). Normal inhibition of ductal branching must therefore overcome a stromal background that is strongly stimulatory; there is now excellent evidence that transforming growth factor-βs (TGF-βs), acting in part on stromal targets, are responsible.
The rapid and reversible inhibition of end bud growth by experimentally implanted TGF-β1 demonstrates the mammotrophic activity of this growth factor, whereas its normally high concentration in the periductal extracellular matrix and its localized loss over lateral buds strongly implies an action that modulates secondary ductal growth . Studies with transgenic mice overexpressing a constitutively activated form of TGF-β1 , as well as function-ablating mutant TGF-β signaling receptors, were confirmatory. The ectopic expression of TGF-β1 resulted in a significant decrease in lateral branching, and mutant TGF-β receptors expressed in the stroma increased lateral branch infilling . Because the latter are an important site of TGF-β action, normal chronic inhibition of branching must depend, in part, on TGF-β-regulated secondary signals. Recently, hepatocyte growth factor (HGF), which can stimulate the branching of mammary epithelial tubes in vitro and is negatively modulated by TGF-β, has emerged as a candidate secondary signal . In this model, TGF-β inhibits branching through the inhibition of HGF expression in the periductal stroma (reviewed in this issue). However, the protean effects of TGF-βs on mitogenesis and extracellular matrix dynamics in mammary tissue make the assignment of any single, TGF-β-mediated mechanism premature .
Ductal elongation and branching depend on parenchyma-induced modifications of the periductal stroma
End bud growth
It is striking that ductal growth is so exquisitely focused in the end buds. The impression that precisely localized, as opposed to general, signals guide this development is unavoidable. In fact, this impression is correct and epithelium-induced changes develop the growth-promoting potential of the stroma immediately in front of end buds. Exciting new evidence demonstrates that migratory white blood cells, macrophages and eosinophils, are drawn to the vicinity of the end bud by chemoattractants and, surprisingly, prove to be essential for the normal development of end buds . Interestingly, extensive DNA synthesis in the stroma around end buds accompanies this activity, indicating that new stromal cells are not only recruited to the vicinity of the end bud but are also induced by it to proliferate . The absence of stromal DNA synthesis around growth-terminated ducts emphasizes that these inductive signals are growth-related and are not due merely to the presence of epithelium.
Organotypic development depends on two obvious structural modifications of the end bud, its constriction into a tube and its bifurcation. Preceding either, there is focal induction by the end bud of type I collagen-rich connective tissue and extracellular matrix on its flank (Fig. 1b) and in the clefts that indent the tip when two new end buds form (not shown). An active role for collagen in shaping the duct is indicated. In vitro, mammary epithelial cells embedded in collagen gels form narrow tubules that are also seen in vivo when fragments of duct form similar tubules in a bolus of injected type 1 collagen (reviewed in ). Mechanistically, by binding to members of the integrin family of extracellular matrix receptors, collagen can stimulate the formation of actin-cytoskeletal foci that are capable of changing mammary cell shape . Indeed, β1-integrin was localized at the basal surfaces of the end bud epithelium, and function-blocking antibodies against β-integrin, as well as antibodies against laminin, reversibly inhibited end bud development in vivo, while blocking tubulogenesis in vitro .
The molecular signals governing the sites of fibrous induction are largely unknown; however, TGF-βs seem likely to have a role. The experimental release of TGF-β1 in the vicinity of an end bud by plastic implants caused epithelium-dependent induction of a fibrous connective tissue cap over the end bud tip. The molecular composition of this cap reflected that of the fibrous connective tissue on the flank of the end bud and in developing clefts before bifurcation, suggesting that TGF-β1 might be the normal inducer .
More recently, parathyroid-hormone-related protein (PTHrP) has been shown to be crucial for normal ductal development. Transgenic animals overexpressing the peptide show severe impairment of ductal extension and branching . Pertinent to this discussion, PTHrP synthesis is concentrated in the cap cells of end buds and their myoepithelial descendents on the flank, whereas cognate receptors seem to be concentrated in the immediately adjacent fibrous tissue. This indicates a potential role in stromal induction. With this in mind, the fact that TGF-β can positively regulate PTHrP is interesting  because TGF-β is present, often at high levels, in the end bud  (Fig. 3b). These observations suggest that TGF-βs might indirectly cause the induction of the fibrous sheath of end buds and that experiments to investigate whether PTHrP induces fibrosis and whether TGF-β1 normally regulates PTHrP during ductal development would be fruitful.
Inhibition of end bud growth
A combined role for TGF-β-induced fibrous stroma in inhibiting end bud growth while guiding morphogenesis has been suggested . Consistent with this hypothesis is the observation that fibrous connective tissue on the flank progressively advances to envelop the tips of end buds that are in the process of stopping growth . Arguing against the matrix as a primary growth inhibitor, however, implanted TGF-β inhibits DNA synthesis up to 12 h before the appearance of the fibrous cap. Furthermore, surprisingly high levels of DNA synthesis can be detected in matrix-ensheathed, growth-quiescent ducts, some quite distant from the end bud (Fig. 2). Although this DNA synthesis might or might not be related to mitosis , it nevertheless demonstrates that growth-stimulatory signals can be quite active in ducts beneath an intact fibrous stromal sheath. Even though it is clear that stromal signals must ultimately inhibit end bud growth [how else can their regression before reaching the limits of the fat pad be explained (Fig. 1a)?], their identity remains unknown (Fig. 3b).
Resolving signaling between epithelium and stroma
During the past decade, classic mammary tissue recombination experiments have been recalled to duty, this time using tissue from genetically engineered mice, and have led to important insights into the stromal origins of ductal mammogenic signals. Much less is known about the epithelial signals that reorganize the periductal stroma and, as I have discussed briefly above, these retrograde signals are crucial to the realization of the morphogenetic and growth-promoting potential of the stroma.
Identifying the relevant epithelial signals and placing them in a proper temporal order with regard to the elicitation of stromal signals and the ensuing morphogenetic events is now a major challenge that will require new conceptual as well as experimental tools. The strong evolutionary conservation of reciprocal, epithelial-stromal signaling in branching morphogenesis, which encompasses the development of branched airways from insects to mammals, for example, suggests that careful study of these systems could provide new ideas pertinent to mammary growth and morphogenesis .
In an earlier review  I suggested that bringing modern molecular methods to bear to investigate the dynamics of gene expression in the stroma and epithelium at obvious growth and morphogenetic inflection points (eg in front of end buds) would be useful. However, this approach does not address the vital issue of the temporal order of signaling, the resolution of which would benefit from a 'time-zero' experimental condition, in which growth-static mammary ducts could be induced to grow in a controlled manner. Although there should be several ways of accomplishing this, simple ductal transplants come first to mind. Through an analysis of the initiation and earliest phases of transplant outgrowth over a finely spaced time-course, it might be possible to obtain an orderly reading of reciprocal epithelial and stromal signals that underlie stromal reorganization and ductal extension.
= estrogen receptor knockout
= hepatocyte growth factor
= parathyroid-hormone-related protein
= transforming growth factor-β.
- Streuli CH, Haslam SZ: Control of mammary gland development and neoplasia by stromal-epithelial interactions and extracellular matrix. J Mamm Gland Biol Neoplasia. 1998, 3: 107-108. 10.1023/A:1018734620748. This article introduces and reviews the contents of an eponymous issue of the Journal of Mammary Gland Biology and Neoplasia. As the title suggests, there are numerous interesting papers related to the subject of the present review.View ArticleGoogle Scholar
- Hovey RC, McFadden TB, Akers RM: Regulation of mammary gland growth and morphogenesis by the mammary fat pad: a species comparison. J Mamm Gland Biol Neoplasia. 1999, 4: 53-68. 10.1023/A:1018704603426.View ArticleGoogle Scholar
- Sakakura T: New aspects of stroma-parenchyma relations in mammary gland differentiation. Int Rev Cytol. 1991, 125: 165-202. This review places the seminal experiments by the author as well as those of K Kratochwil and others in a modern context.PubMedView ArticleGoogle Scholar
- Chepko G, Smith GH: Mammary epithelial stem cells: our current understanding. J Mamm Gland Biol Neoplasia. 1999, 4: 35-52. 10.1023/A:1018752519356.View ArticleGoogle Scholar
- Daniel CW, Robinson S, Silberstein GB: The role of TGFβ in patterning and growth of the mammary ductal tree. J Mamm Gland Biol Neoplasia. 1996, 1: 331-341.View ArticleGoogle Scholar
- Pierce DF, Johnson MD, Matsui Y, Robinson SD, Gold LI, Purchio AF, Daniel CW, Hogan BL, Moses HL: Inhibition of mammary duct development but not alveolar outgrowth during pregnancy in transgenic mice expressing active TGF-β1. Genes Dev. 1993, 7: 2308-2317.PubMedView ArticleGoogle Scholar
- Joseph H, Gorska AE, Sohn P, Moses HL, Serra R: Overexpression of a kinase-deficient transforming growth factor-β type II receptor in mouse mammary stroma results in increased epithelial branching. Mol Biol Cell. 1999, 10: 1221-1234. Prior to this work the conceptual focus of TGFβ action in the mammary gland had been on epithelial, not stromal, targets.PubMedPubMed CentralView ArticleGoogle Scholar
- Kamalati T, Niranjan B, Yant J, Buluwela L: HGF/SF in mammary epithelial growth and morphogenesis: in vitro and in vivo models. J Mamm Gland Biol Neoplasia. 1999, 4: 69-77. 10.1023/A:1018756620265.View ArticleGoogle Scholar
- Gouon-Evans V, Rothenberg ME, Pollard JW: Postnatal mammary gland development requires macrophages and eosinophils. Development. 2000, 127: 2269-2282. This technically elegant study demonstrates a surprising and crucial role for white blood cells in ductal elongation, and is essential reading.PubMedGoogle Scholar
- Berger JJ, Daniel CW: Stromal DNA synthesis is stimulated by young, but not serially aged mouse mammary epithelium. Mech Aging Devel. 1983, 23: 259-264. 10.1016/0047-6374(83)90026-X.View ArticleGoogle Scholar
- Daniel CW, Silberstein GB, Strickland P: Direct action of 17β-estradiol on mouse mammary ducts analyzed by sustained release implants and steroid autoradiography. Cancer Res. 1987, 47: 6052-6057.PubMedGoogle Scholar
- Cunha GR, Young P, Hom YK, Cooke PS, Taylor JA, Lubahn DB: Elucidation of a role for stromal steroid hormone receptors in mammary gland growth and development using tissue recombinations. J Mamm Gland Biol Neoplasia. 1997, 2: 393-402. 10.1023/A:1026303630843.View ArticleGoogle Scholar
- Silberstein GB: Postnatal mammary gland morphogenesis. Microsc Res Technique. 2001, 52: 155-162. 10.1002/1097-0029(20010115)52:2<155::AID-JEMT1001>3.0.CO;2-P. This subject had not been reviewed for more than a decade. Ductal assembly and bifurcation are discussed in the context of new knowledge concerning interactions between classical endocrine mammo-gens and the more recently discovered growth factors.View ArticleGoogle Scholar
- Streuli CH, Edwards GM: Control of normal mammary epithelial phenotype by integrins. J Mamm Gland Biol Neoplasia. 1998, 3: 151-163. 10.1023/A:1018742822565.View ArticleGoogle Scholar
- Klinowska TC, Soriano JV, Edwards GM, Oliver JM, Valentijn AJ, Montesano R, Streuli CH: Laminin and β1 integrins are crucial for normal mammary gland development in the mouse. Dev Biol. 1999, 215: 13-32. 10.1006/dbio.1999.9435.PubMedView ArticleGoogle Scholar
- Silberstein GB, Strickland P, Coleman S, Daniel CW: Epithelium-dependent extracellular matrix synthesis in transforming growth factor-β1-growth-inhibited mouse mammary gland. J Cell Biol. 1990, 110: 2209-2219.PubMedView ArticleGoogle Scholar
- Dunbar ME, Wysolmerski JJ: Parathyroid hormone-related protein: a developmental regulatory molecule necessary for mammary gland development. J Mamm Gland Biol Neoplasia. 1999, 4: 21-34. 10.1023/A:1018700502518.View ArticleGoogle Scholar
- Robinson SD, Silberstein GB, Roberts AB, Flanders KC, Daniel CW: Regulated expression and growth inhibitory effects of transforming growth factor-β isoforms in mouse mammary gland development. Development. 1991, 113: 867-878.PubMedGoogle Scholar
- Silberstein GB, Daniel CW: Glycosaminoglycans in the basal lamina and the extracellular matrix of serially aged mouse mammary ducts. Mech Aging Devel. 1984, 24: 151-162. 10.1016/0047-6374(84)90067-8.View ArticleGoogle Scholar
- Smith GH, Vonderhaar BK: Functional differentiation in mouse mammary gland epithelium is attained through DNA synthesis, inconsequent of mitosis. Dev Biol. 1981, 88: 167-179.PubMedView ArticleGoogle Scholar
- Metzger RJ, Krasnow MA: Genetic control of branching morphogenesis. Science. 1999, 284: 1635-1639. 10.1126/science.284.5420.1635. This outstanding review places molecular regulation of branching morphogenesis in a solid evolutionary context. Focusing on the development of Drosophila trachea and embryonic mouse lung sac, it serves to frame the question of mammary ductal branching in molecular terms.PubMedView ArticleGoogle Scholar