*4.2.2. Paracrine factors produced by stromal fibroblasts*

Stromal fibroblasts play a significant role in the development of the mammary gland, not only by creating a complex scaffolding network but also being a source of bioactive compounds. Fibroblasts may also take part in transmission and modulation of signals from superior hypothalamic-pituitary-gonadal axis (HPG). During puberty, mammary fibroblasts surrounding the branching TEBs become activated in response to estrogen and GH released by the ovaries and pituitary gland, respectively [48]. Stromal fibroblasts express growth hormone receptor (GHR) and through secretion of IGF-I may modulate epithelial compartment growth especially in pubertal state [6].

In general, fibroblasts exist in a relatively quiescent state, proliferating slowly and synthesizing only low levels of ECM proteins and matrix metalloproteinases (MMPs) to maintain ECM integrity [48]. During branching morphogenesis, fibroblasts actively synthesize growth factors and proteases. For example, signaling pathways induced by fibroblast growth factors (FGFs) play a major role in the process of mammary placode development [49]. FGFs family contains 18 secreted proteins that can interact with four FGF receptors (FGFRs) having tyrosine kinase activity. These secreted FGFs function as auto- or paracrine factors, but some also show an endocrine function. In addition, there are intracellular FGFs (iFGFs), which are non-signaling proteins serving as cofactors for voltage-gated sodium channels and other molecules [50]. Interaction of FGF ligands with their receptors is regulated by protein or proteoglycan cofactors and by extracellular-binding proteins. The first line of evidence confirming the role of FGF signaling in embryonic stage of mammogenesis came after it was demonstrated that mice lacking either FGF10 or FGFR2b fail to form mammary placodes 1, 2, 3, and 5 [51]. In mouse embryos lacking *Fgf10* gene, an epithelial sprout derived from placode 4 failed to branch, which completely inhibited the formation of a primitive epithelial network in the neonatal mice after birth [51]. In humans, a birth defect known as Poland syndrome, which is characterized by the underdevelopment of the somite-derived pectoral muscle on one side of the body and a corresponding hypoplasia of the overlying breast on the same side, arises from disruption in FGF10 signaling, because *Fgf10*+/− glands show reduced thickening of the ectoderm along the mammary line and subsequent loss of buds 3 and 5 [6]. Furthermore, secreted FGFs are known to stimulate TEBs promoting luminal epithelial cell expansion, ductal branching, and their differentiation into myoepithelial cells. The majority of FGFs is involved in branching process and involution, both of which require ECM rearrangement. In the case of pregnancy, signals through FGFR2-IIIb are essential to stimulate normal lobuloalveolar development [48]. Recent studies revealed that *Spry2* gene, which encodes an inhibitor of signaling via receptor tyrosine kinases, is essential for regulation of both FGF2-based ductal elongation and FGF10-mediated epithelial invasion during normal mammary gland development. For example, loss of *Spry*2 expression results in increased FGF signaling activities, causing more rapid ductal elongation and epithelial invasion, which leads to accelerated epithelial invasion during pubertal branching. Conversely, a decrease of FGF signaling leads to slower than normal ductal elongation and invasion, resulting in stunted epithelial invasion during postnatal branching of the mammary gland [52]. It was also revealed that basal epithelial cells lacking *Fgfr2* gene did not generate an epithelial network due to failure in luminal differentiation, and *Fgfr2−/−* epithelium was unable to undergo ductal branching initiation, which depends on directional epithelial migration [53]. The results of the abovementioned studies demonstrated that distinct types of FGFs stimulate epithelial cells on different levels. FGF2 controls the ductal elongation process, which depends on cell proliferation and expansion, while FGF10 regulates the branch initiation process depended on directional epithelial migration.

and contains less adipose tissue than the fatty mouse mammary stroma. The morphology of the bovine mammary gland resembles that of the human breast, because the mammary epithelium is generally closely associated with fibrous connective tissue, which in this case is

The composition of the mammary stroma largely determines the progression of glandular epithelium development. Attempts to recapitulate human breast epithelial morphogenesis by introducing human MECs into the cleared mammary fat pads of mice were unsuccessful for a long time, due to improper composition of murine stroma comprising mainly adipocytes. Kuperwasser and co-workers used a different approach, creating a model of humanized mouse mammary gland by injecting immortalized human mammary stromal fibroblasts labeled with green fluorescence protein (GFP) into the cleared mice mammary fat pad prior to injection of human breast organoids. Addition of human fibroblasts to the murine fat pad effectively stimulated human MECs proliferation and promoted organization of differentiated acini structures [46]. This experiment pointed to tight stromal-epithelial species affinity [46]. A follow-up study was made, in which human macrophages were also injected. This procedure intensified humanization of the murine fat pad by enhancing fibroblast proliferation and engraftment of the mammary fat pad, thereby providing a larger stromal scaffold for

Stromal fibroblasts play a significant role in the development of the mammary gland, not only by creating a complex scaffolding network but also being a source of bioactive compounds. Fibroblasts may also take part in transmission and modulation of signals from superior hypothalamic-pituitary-gonadal axis (HPG). During puberty, mammary fibroblasts surrounding the branching TEBs become activated in response to estrogen and GH released by the ovaries and pituitary gland, respectively [48]. Stromal fibroblasts express growth hormone receptor (GHR) and through secretion of IGF-I may modulate epithelial compartment growth espe-

In general, fibroblasts exist in a relatively quiescent state, proliferating slowly and synthesizing only low levels of ECM proteins and matrix metalloproteinases (MMPs) to maintain ECM integrity [48]. During branching morphogenesis, fibroblasts actively synthesize growth factors and proteases. For example, signaling pathways induced by fibroblast growth factors (FGFs) play a major role in the process of mammary placode development [49]. FGFs family contains 18 secreted proteins that can interact with four FGF receptors (FGFRs) having tyrosine kinase activity. These secreted FGFs function as auto- or paracrine factors, but some also show an endocrine function. In addition, there are intracellular FGFs (iFGFs), which are non-signaling proteins serving as cofactors for voltage-gated sodium channels and other molecules [50]. Interaction of FGF ligands with their receptors is regulated by protein or proteoglycan cofactors and by extracellular-binding proteins. The first line of evidence confirming the role of FGF signaling in embryonic stage of mammogenesis came after it was demonstrated that mice

*4.2.1. Fibroblast-mammary epithelial cell interactions during mammogenesis*

breast epithelial growth and acini formation [47].

*4.2.2. Paracrine factors produced by stromal fibroblasts*

cially in pubertal state [6].

extensively developed [45].

98 Stromal Cells - Structure, Function, and Therapeutic Implications

Other fibroblast-derived bioactive compounds like TGF-β1, HGF, or stroma cell-derived factor-1 (SDF-1) also known as CXCL12, were shown to influence mammary parenchyma development in a paracrine manner [54, 55]. HGF is a multi-functional cytokine stimulating invasion, motility, and morphogenesis. Its presence was found in conditioned media from human mammary fibroblasts [56, 57]. Fibroblast-derived conditioned media containing HGF were shown to induce tubulogenesis and branching morphogenesis of TAC-2 mouse mammary epithelial cell line [20]. In addition, it is well documented that fibroblastic HGF mediates the proliferation of estrogen receptor positive (ER+) mammary epithelial cells [43]. HGF was identified as one of the major mediators of this effect, because in in vitro experiments the proliferative activity of MECs cultured in fibroblast-derived conditioned medium was completely abolished by a neutralizing antibody against HGF [41].

Another important growth factor—TGF-β1, secreted by the mammary stroma, acts in an auto/ paracrine manner to regulate glandular morphogenesis and remodeling by preventing inappropriate side branching. The presence of TGF-β1 was detected in mature periductal ECM in mice, and it was specifically downregulated at sites where side branches were being initiated [58]. Furthermore, TGF-β1 plays an important role in regulation of growth and activity of fibroblasts. This growth factor functions by signaling to cell surface type II receptors, which recruit type I receptors, resulting in activation of downstream signaling cascades, including canonical Smad pathways that modulate gene transcription [59]. TGF-β signaling in fibroblasts functions to modulate expression of tissue remodeling factors, including ECM proteins, proteases, and angiogenic factors. During lactation, the expression of TGF-β1 is significantly downregulated, which may prevent TGF-β1 from negatively regulating the expression of milk proteins. Upon the onset of involution, when the gland remodels toward its pre-pregnant state, there is an upregulation of TGF-β1 transcripts. TGF-β1 signaling may further contribute to the remodeling of the involuting gland by inducing ECM production, upregulating MMPs expression, and by recruiting immune cells [14, 19]. Recent studies revealed that TGF-β1 promotes mammary fibroblast proliferation and may cause severe side effect in mammary gland structure and function in dairy cows [60]. TGF-β1 not only affects the development of the epithelial compartment by inhibiting formation and differentiation of mammary ducts and induction of apoptosis. Treatment of bovine mammary fibroblasts with TGF-β1 significantly promoted their proliferation and accelerated the cell cycle. Further research using a mouse model showed that TGF-β1 significantly increased the proportion of fibroblasts and accelerated the cell transition from the G1 to G2/M phases. Thus, TGF-β1 is a cytokine which may also cause negative effect in the mammary gland by contributing to the development of mammary gland fibrosis [60].

cells [5]. MMP14 intracellular domain interacts with β1-integrin on the basal surface of cells, and this interaction is required for transducing the extracellular signals needed for epithelial

Stromal-Epithelial Interactions during Mammary Gland Development

http://dx.doi.org/10.5772/intechopen.80405

101

The role of fibroblasts should also be described in the context of the mammary gland remodeling observed extensively during post-lactating involution. Mammary involution is analogous to a wound healing response, involving complex epithelial-stromal cell interactions, degradation of basement membrane driven by protease production originating from fibroblasts. Stromal fibroblasts contain elevated fibronectin, laminins, and higher level of fibrillar collagens to remodel mammary tissue during involution [48]. Fibrillar collagen-epithelial interactions, especially collagen I, III, and V, are crucial during this process [14]. Studies revealed that the epithelial compartment is highly malleable and that cell fate and tissue function are

When discussing the role of stromal fibroblasts in mammary gland biology, one needs to mention about epithelial-stromal interactions in the context of breast cancer development. Fibroblasts arising from tumor stroma, described as cancer-associated fibroblasts (CAFs), compared to normal fibroblasts, have acquired distinct properties mainly leading to the promotion of cancer cell proliferation and invasion. CAFs, which are characterized by their high expression of alpha smooth muscle actin, are detected in large numbers in malignant breast cancers and their presence is correlated with poor clinical outcome [62]. Particularly in breast cancer, the progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) is believed to be actively driven by complex interactions with the surrounding microenvironment including interactions with various activated stromal fibroblasts [63]. It is believed that CAFs contribute to cancer cell survival and progression not only through enhanced secretion of cytokines, growth factors, and proteases such as TGFβ1, HGF, SDF-1, and MMP2, respectively, but also by secreting high levels of nutrient-rich ECM, promoting persistent chronic inflammation within the tumor microenvironment and inducing epithelialto-mesenchymal transition (EMT) of tumor cells [48]. During EMT, downregulation or loss of the epithelial adhesion molecule E-cadherin and upregulation of N-cadherin represent a key step in the acquisition of the phenotype for many tumors. Interestingly, normal fibroblasts induce a strong E-cadherin enhancement even in cancer mammary epithelial cells; thus, these fibroblasts appear to favor the maintenance of the normal tissue architecture [64]. In vitro studies investigating the relationship between mammary carcinoma cells and stromal cells revealed that normal mammary fibroblasts function to suppress tumor progression by negatively regulating expression of oncogenic signaling factors [65]. Furthermore, co-culture of cancerous cells with stromal fibroblasts has been shown to induce significant changes in

Fibroblasts are the principal component of the stromal connective tissue. These cells are responsible for ECM remodeling and secrete FGFs and ECM components, such as collagens,

strongly influenced by the stromal compartment of the gland [48].

tumor development and progression [56].

*4.2.5. Summary*

*4.2.4. Different properties of fibroblasts derived from normal and cancerous stroma*

cells to invade [5].

## *4.2.3. Fibroblast-derived extracellular matrix components*

As mentioned earlier, fibroblasts together with other stromal cells synthesize the main amount of ECM components, such as collagens (collagen I, III, and V), proteoglycans, elastin, integrins, and fibronectin; thus, these stromal cells are responsible for mammary tissue architecture and stiffness [5, 48]. ECM can be described as an interconnected meshwork of secreted proteins interacting with cells to form a functional unit [14]. Additionally, mammary gland fibroblasts synthesize many matrix metalloproteinases (MMPs), like MMP2, MMP3, MMP14, that are able to remodel the ECM and release growth factors and cytokines harbored or embedded within the ECM [19]. MMPs consist of a family of over 20 zinc-dependent proteinases synthesized as latent enzymes, in a zymogen form, activated post-translationally and regulated by endogenous inhibitors referred to as tissue inhibitors of metalloproteinases (TIMPs) [5, 56, 57]. MMPs are secreted by stromal cells, but MMP2 and MPP3 exclusively by fibroblasts [61]. MMPs are important for ECM remodeling as well as for the microenvironmental signaling necessary to carry out morphogenic programs within the mammary gland [5]. Increased level of the active MMP3 leads to excessive side branching, and advanced alveolar morphogenesis but as a side effect is responsible for causing production of reactive oxygen species (ROS) leading to genomic instability [5]. MMP3, described also as stromelysin 1 (Str1), is expressed by mammary fibroblasts in vivo at elevated levels in the glands of virgin animals during ductal elongation. The highest level of MMP3 is found around the end buds and rear branch points, where mammary epithelial cells display the highest mitotic activity [57]. Overexpression of another matrix metalloproteinase—MMP14 in the mammary gland was demonstrated to cause excessive side branching and advanced alveolar morphogenesis [56]. The hemopexin domain of MMP14 is important for sorting mammary epithelial cells to points of branching. It has also been shown that only the short intracellular domain of MMP14, which does not contain kinase activity, is needed to resource branching morphogenesis in MMP14-deficient cells [5]. MMP14 intracellular domain interacts with β1-integrin on the basal surface of cells, and this interaction is required for transducing the extracellular signals needed for epithelial cells to invade [5].

The role of fibroblasts should also be described in the context of the mammary gland remodeling observed extensively during post-lactating involution. Mammary involution is analogous to a wound healing response, involving complex epithelial-stromal cell interactions, degradation of basement membrane driven by protease production originating from fibroblasts. Stromal fibroblasts contain elevated fibronectin, laminins, and higher level of fibrillar collagens to remodel mammary tissue during involution [48]. Fibrillar collagen-epithelial interactions, especially collagen I, III, and V, are crucial during this process [14]. Studies revealed that the epithelial compartment is highly malleable and that cell fate and tissue function are strongly influenced by the stromal compartment of the gland [48].

### *4.2.4. Different properties of fibroblasts derived from normal and cancerous stroma*

When discussing the role of stromal fibroblasts in mammary gland biology, one needs to mention about epithelial-stromal interactions in the context of breast cancer development. Fibroblasts arising from tumor stroma, described as cancer-associated fibroblasts (CAFs), compared to normal fibroblasts, have acquired distinct properties mainly leading to the promotion of cancer cell proliferation and invasion. CAFs, which are characterized by their high expression of alpha smooth muscle actin, are detected in large numbers in malignant breast cancers and their presence is correlated with poor clinical outcome [62]. Particularly in breast cancer, the progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) is believed to be actively driven by complex interactions with the surrounding microenvironment including interactions with various activated stromal fibroblasts [63]. It is believed that CAFs contribute to cancer cell survival and progression not only through enhanced secretion of cytokines, growth factors, and proteases such as TGFβ1, HGF, SDF-1, and MMP2, respectively, but also by secreting high levels of nutrient-rich ECM, promoting persistent chronic inflammation within the tumor microenvironment and inducing epithelialto-mesenchymal transition (EMT) of tumor cells [48]. During EMT, downregulation or loss of the epithelial adhesion molecule E-cadherin and upregulation of N-cadherin represent a key step in the acquisition of the phenotype for many tumors. Interestingly, normal fibroblasts induce a strong E-cadherin enhancement even in cancer mammary epithelial cells; thus, these fibroblasts appear to favor the maintenance of the normal tissue architecture [64]. In vitro studies investigating the relationship between mammary carcinoma cells and stromal cells revealed that normal mammary fibroblasts function to suppress tumor progression by negatively regulating expression of oncogenic signaling factors [65]. Furthermore, co-culture of cancerous cells with stromal fibroblasts has been shown to induce significant changes in tumor development and progression [56].

#### *4.2.5. Summary*

type I receptors, resulting in activation of downstream signaling cascades, including canonical Smad pathways that modulate gene transcription [59]. TGF-β signaling in fibroblasts functions to modulate expression of tissue remodeling factors, including ECM proteins, proteases, and angiogenic factors. During lactation, the expression of TGF-β1 is significantly downregulated, which may prevent TGF-β1 from negatively regulating the expression of milk proteins. Upon the onset of involution, when the gland remodels toward its pre-pregnant state, there is an upregulation of TGF-β1 transcripts. TGF-β1 signaling may further contribute to the remodeling of the involuting gland by inducing ECM production, upregulating MMPs expression, and by recruiting immune cells [14, 19]. Recent studies revealed that TGF-β1 promotes mammary fibroblast proliferation and may cause severe side effect in mammary gland structure and function in dairy cows [60]. TGF-β1 not only affects the development of the epithelial compartment by inhibiting formation and differentiation of mammary ducts and induction of apoptosis. Treatment of bovine mammary fibroblasts with TGF-β1 significantly promoted their proliferation and accelerated the cell cycle. Further research using a mouse model showed that TGF-β1 significantly increased the proportion of fibroblasts and accelerated the cell transition from the G1 to G2/M phases. Thus, TGF-β1 is a cytokine which may also cause negative effect in the mammary gland by contributing to the development of mammary gland fibrosis [60].

As mentioned earlier, fibroblasts together with other stromal cells synthesize the main amount of ECM components, such as collagens (collagen I, III, and V), proteoglycans, elastin, integrins, and fibronectin; thus, these stromal cells are responsible for mammary tissue architecture and stiffness [5, 48]. ECM can be described as an interconnected meshwork of secreted proteins interacting with cells to form a functional unit [14]. Additionally, mammary gland fibroblasts synthesize many matrix metalloproteinases (MMPs), like MMP2, MMP3, MMP14, that are able to remodel the ECM and release growth factors and cytokines harbored or embedded within the ECM [19]. MMPs consist of a family of over 20 zinc-dependent proteinases synthesized as latent enzymes, in a zymogen form, activated post-translationally and regulated by endogenous inhibitors referred to as tissue inhibitors of metalloproteinases (TIMPs) [5, 56, 57]. MMPs are secreted by stromal cells, but MMP2 and MPP3 exclusively by fibroblasts [61]. MMPs are important for ECM remodeling as well as for the microenvironmental signaling necessary to carry out morphogenic programs within the mammary gland [5]. Increased level of the active MMP3 leads to excessive side branching, and advanced alveolar morphogenesis but as a side effect is responsible for causing production of reactive oxygen species (ROS) leading to genomic instability [5]. MMP3, described also as stromelysin 1 (Str1), is expressed by mammary fibroblasts in vivo at elevated levels in the glands of virgin animals during ductal elongation. The highest level of MMP3 is found around the end buds and rear branch points, where mammary epithelial cells display the highest mitotic activity [57]. Overexpression of another matrix metalloproteinase—MMP14 in the mammary gland was demonstrated to cause excessive side branching and advanced alveolar morphogenesis [56]. The hemopexin domain of MMP14 is important for sorting mammary epithelial cells to points of branching. It has also been shown that only the short intracellular domain of MMP14, which does not contain kinase activity, is needed to resource branching morphogenesis in MMP14-deficient

*4.2.3. Fibroblast-derived extracellular matrix components*

100 Stromal Cells - Structure, Function, and Therapeutic Implications

Fibroblasts are the principal component of the stromal connective tissue. These cells are responsible for ECM remodeling and secrete FGFs and ECM components, such as collagens, fibronectin, laminins, elastin, proteoglycans, and MMPs. Due to their properties, fibroblasts support the luminal epithelial growth and branching morphogenesis as well as participate in the mammary gland tissue remodeling during involution (**Figure 2**).

*4.3.2. Role of eosinophils in mammary gland morphogenesis*

*4.3.3. Role of macrophages in mammary gland development and remodeling*

The role of macrophages at different stages of glandular morphogenesis as well as remodeling are better recognized. In the pubertal mammary gland, macrophages are recruited to the highly mitotic terminal end buds from which ducts elongate and branch to give rise to a mature ductal tree [48]. Macrophage colony stimulating factor-1 (CSF1) secreted by myoepithelial cells is a key cytokine that regulates the recruitment, proliferation, and survival of macrophages [48, 72]. Estrogen-regulated CSF1synthesis is essential for expanding of epithelial ducts and buds and alters structural alignment of collagen fibers around the expanding TEBs [70]. Macrophage abundance changes over the estrous cycle, peaking at metestrus and diestrus phases, and being the lowest at proestrus and estrus [66]. Studies on Csf1op/op mice, which are homozygous for a null mutation in *Csf1* gene, revealed that these animals exhibited multiple defects and had reduced macrophage numbers in most tissues including the mammary gland [72]. Depletion of mammary gland macrophages observed in Csf1op/op mice altered the mammary stem/progenitor cell activity, which was reflected in a substantially reduced outgrowth potential of the mammary epithelium. The mammary glands of Csf1op/op mice displayed lower number of TEBs as well as reduced ductal branching and elongation. During pregnancy, Csf1op/op glands developed precocious alveolar units but failed to switch to the lactational state resulting in impaired lactation [72]. These observations prove a continued requirement for normal macrophages during ductal morphogenesis and their stimulatory role on the putative basal progenitor cells. Macrophages also mediate the switch from

Eosinophils belong to immune cells found around the growing TEBs. These cells are attracted by eotaxin, another chemokine produced by mammary gland [43]. Eosinophil knockdown mice show altered elongation and branching during mammary gland development as well as insufficient milk productions at the time of lactation [48]. Similar abnormalities can be noted in knockout mice with deficiency of interleukin 5 (IL-5), a cytokine to which eosinophils are particularly responsive [71]. Mammary tissues from IL-5-deficient females had fewer TEBs, less well-branched mammary ducts, and lower overall density of the mammary gland structures. Furthermore, IL-5-deficient pups nursed by IL-5-deficient mothers were notably underweight, with a high percentage of pre-weaning mortality, in contrast to well-developed IL-5-deficient mice which were nursed by IL-5-sufficient foster mothers [71]. Interestingly, overabundance of eosinophils during puberty results in retarded morphogenesis of the mammary epithelium, suggesting the existence of mechanisms controlling the number of these cells that reside in the gland and are involved in MECs expansion during morphogenesis [66]. In addition to eosinophils, mast cells were also shown to be important for normal mammary gland development. Mice deficient in mast cells have defective mammary branching during puberty. It may be associated with the lack of vascular endothelial growth factor (VEGF) released by these cells that assist in mast cell degranulation [48]. Through activation of their serine proteases and degranulation, mast cells are involved in normal branching during puberty, and they accumulate and possibly activate plasma kallikrein, thus activating the plasminogen [5]. Furthermore, it was demonstrated that inhibition of this mast cell-associated protease during involution caused an accumulation of fibrillar collagen and delayed repopulation of adipocytes, thus preventing the gland from regaining the pre-pregnant state [14].

Stromal-Epithelial Interactions during Mammary Gland Development

http://dx.doi.org/10.5772/intechopen.80405

103
