*1.1.3. Human breast myoepithelial cells*

Myoepithelial cells lie between the epithelial cell layer and the basal lamina, where they establish a network of slender processes covering the overlying epithelial cells. The branching cytoplasmic network of the myoepithelial cell processes can be seen especially in scanning electron micrographs [41]. Spindle-shaped ductal myoepithelial cells lie parallel to the long axis of the duct and form a continuous layer. Contraction of myoepithelial cells in lobules and around ducts contributes to the flow of milk during lactation. The location of the myoepithelial cells between luminal epithelial cells and the basal lamina is an ideal location for them favoring communication with both compartments.

The histologic appearance and immunoreactivity of myoepithelial cells are highly variable, especially in pathologic conditions, and depend on the degree to which the myoid or epithelial phenotype is accentuated in a particular situation. Myoepithelial cells usually display nuclear reactivity for p63, which is the most useful marker for detecting these cells in normal and lesional tissues. Epithelioid myoepithelial cells can have reduced p63 reactivity. Other useful myoepithelial markers are α-smooth muscle actin, calponin, CD10, CKS/6, myosin, p75, and S100 (**Figure 1**, *left*). The presence or absence of myoepithelial cells, at least as demonstrated by routinely used immunostains and is very valuable in discriminating against neoplastic in situ lesions (**Figure 1**, *right*) versus malignant, infiltrative ones.

While the luminal epithelial cell has received plenty of attention as the most functionally active milk-producing cells and as the most probable target cell for tumorigenesis, attention on myoepithelial cells has begun to grow with the acknowledgment that these cells play an active role in branching morphogenesis and tumor suppression.

**Figure 1.** (Left) Immunostain for S100 highlighting the myoepithelial cells layer in a normal terminal ductal lobular unit (TDLU)—green arrow and some adjacent adipocytes—yellow arrow showing strong nuclear and cytoplasmic positivity (immunostain S100 with DAB chromogen, 100× magnification). (Right) Immunostain for S100 highlighting the intact myoepithelial cell layer (blue arrow) at the periphery of some foci of high-grade ductal carcinoma in situ (DCIS) with central comedo-type necrosis (red triangle). Also, note the adjacent normal TDLU (green arrow) and adipocytes (yellow arrow). Immunostain S100 with DAB chromogen, 40× objective.

The function of myoepithelial cells is strongly dependent on regulation of intracellular calcium. These cells contract in response to oxytocin secretion during lactation to generate the contractile force required for milk ejection. It is difficult to understand whether the alteration of calcium metabolism of myoepithelial cells plays a role, if any, in carcinogenesis. Even if it has been demonstrated that store-operated Ca2+ entry was mediated by a functional Orai3 in estrogen receptor-expressing (ER<sup>+</sup> ) breast cancer cells [42], the tumorigenesis impact of these findings on myoepithelial cell remains largely unknown, because it is known that most breast carcinomas originate in the epithelial cells and the spectrum of myoepithelial proliferative breast lesions in scarce.

However, disruption in calcium metabolism may alter the functionality of myoepithelial cells. On this issue, some studies have shown the important role of Orai1 store-operated calcium channels in lactation [43]. Davis et al., using genetically modified mouse models, observed that the store-operated Ca2+ channel Orai1 delivers over 50% of the calcium ions present in the secreted milk. They also demonstrated the role of Orai1 as a principal regulator of oxytocinmediated alveolar unit contractility, milk ejection, general myoepithelial function, and survival.

S100 protein is expressed in myoepithelial cells. The S100 gene family is a Ca2+-binding protein with low molecular weight [44]. The members of the S100 family have a myriad of cell functions such as cell proliferation, apoptosis, differentiation, cancer invasion, and metastasis. S100A2 is involved in breast tumorigenesis being downregulated in some cases, which led to the invasion of breast cancer cells [45]. The S100A4 expression is associated with tumor progression and metastatic potential [46]. In a similar manner, S100A7 is not only overexpressed in high-grade ductal breast carcinoma but also in in situ high-grade lesion (DCIS), and some studies suggested that the concomitant expression of S100A7, S100A8, and S100A9 in a class of breast cancers was associated with poor prognosis [47].

#### *1.1.4. Human breast adipocytes*

in a medium with low calcium levels and antioxidants will be of real use in the future studies of mammary development, breast carcinogenesis, chemoprevention, and cancer therapy.

Myoepithelial cells lie between the epithelial cell layer and the basal lamina, where they establish a network of slender processes covering the overlying epithelial cells. The branching cytoplasmic network of the myoepithelial cell processes can be seen especially in scanning electron micrographs [41]. Spindle-shaped ductal myoepithelial cells lie parallel to the long axis of the duct and form a continuous layer. Contraction of myoepithelial cells in lobules and around ducts contributes to the flow of milk during lactation. The location of the myoepithelial cells between luminal epithelial cells and the basal lamina is an ideal location for them

The histologic appearance and immunoreactivity of myoepithelial cells are highly variable, especially in pathologic conditions, and depend on the degree to which the myoid or epithelial phenotype is accentuated in a particular situation. Myoepithelial cells usually display nuclear reactivity for p63, which is the most useful marker for detecting these cells in normal and lesional tissues. Epithelioid myoepithelial cells can have reduced p63 reactivity. Other useful myoepithelial markers are α-smooth muscle actin, calponin, CD10, CKS/6, myosin, p75, and S100 (**Figure 1**, *left*). The presence or absence of myoepithelial cells, at least as demonstrated by routinely used immunostains and is very valuable in discriminating against neoplastic in

While the luminal epithelial cell has received plenty of attention as the most functionally active milk-producing cells and as the most probable target cell for tumorigenesis, attention on myoepithelial cells has begun to grow with the acknowledgment that these cells play an

**Figure 1.** (Left) Immunostain for S100 highlighting the myoepithelial cells layer in a normal terminal ductal lobular unit (TDLU)—green arrow and some adjacent adipocytes—yellow arrow showing strong nuclear and cytoplasmic positivity (immunostain S100 with DAB chromogen, 100× magnification). (Right) Immunostain for S100 highlighting the intact myoepithelial cell layer (blue arrow) at the periphery of some foci of high-grade ductal carcinoma in situ (DCIS) with central comedo-type necrosis (red triangle). Also, note the adjacent normal TDLU (green arrow) and adipocytes (yellow

*1.1.3. Human breast myoepithelial cells*

170 Calcium and Signal Transduction

favoring communication with both compartments.

situ lesions (**Figure 1**, *right*) versus malignant, infiltrative ones.

active role in branching morphogenesis and tumor suppression.

arrow). Immunostain S100 with DAB chromogen, 40× objective.

Apart from the epigenetic and genetic changes that occur within epithelial cells leading to breast proliferative lesions, it has shown that tumor initiation and progression also depend on the intricate intercellular dialog between tumor epithelial cells and the surrounding stromal cells [48]. Among the different cell types comprised in the breast stroma, the most abundant are those of adipose origin, mainly mature adipocytes, preadipocytes, and adipose-derived stromal/stem cells (ASCs). Besides the structural role that breast tissue has, it also has an important bioactive function [49, 50]. In pathologic condition, it is of great importance the interaction that is established between tumor cells and stromal adipocytes within the invasive front characteristic of breast cancers [51, 52]. Similar to breast myoepithelial cells, breast adipocytes also express s100 protein—**Figure 1** [53].

The role of calcium metabolism in such events is poorly understood, but some studies have found that some proteins such as calpains [54] and calpastatin [55] may be altered in adiposederived stromal cells being responsible for their enhanced invasion potential.

Vitamin D3 triggers apoptosis in breast cancer cells and adipocytes by inducing an apoptotic signal by increasing the concentration of intracellular Ca2+. This signal acts as an apoptotic initiator that bluntly recruits calcium-dependent apoptotic effectors such as calpain and caspase 12, in both breast cancer cells and adipocytes. Some studies suggested that inducing apoptosis with vitamin D3, particularly in the tumor-surrounding adipose tissue involved in tumor progression, can contribute to the antitumoral effects of this hormone and may be of real therapeutic interest to include calcium-dependent apoptotic proteases as molecular targets for new therapeutic and preventive agents in breast cancer and obese patients [56].
