Influence of Fibroblasts on Mammary Gland Development, Breast Cancer Microenvironment Remodeling, and Cancer Cell Dissemination

Abstract

The stromal microenvironment regulates mammary gland development and tumorigenesis. In normal mammary glands, the stromal microenvironment encompasses the ducts and contains fibroblasts, the main regulators of branching morphogenesis. Understanding the way fibroblast signaling pathways regulate mammary gland development may offer insights into the mechanisms of breast cancer (BC) biology. In fact, the unregulated mammary fibroblast signaling pathways, associated with alterations in extracellular matrix (ECM) remodeling and branching morphogenesis, drive breast cancer microenvironment (BCM) remodeling and cancer growth. The BCM comprises a very heterogeneous tissue containing non-cancer stromal cells, namely, breast cancer-associated fibroblasts (BCAFs), which represent most of the tumor mass. Moreover, the different components of the BCM highly interact with cancer cells, thereby generating a tightly intertwined network. In particular, BC cells activate recruited normal fibroblasts in BCAFs, which, in turn, promote BCM remodeling and metastasis. Thus, comparing the roles of normal fibroblasts and BCAFs in the physiological and metastatic processes, could provide a deeper understanding of the signaling pathways regulating BC dissemination. Here, we review the latest literature describing the structure of the mammary gland and the BCM and summarize the influence of epithelial-mesenchymal transition (EpMT) and autophagy in BC dissemination. Finally, we discuss the roles of fibroblasts and BCAFs in mammary gland development and BCM remodeling, respectively.

Keywords: ECM remodeling; breast cancer associated fibroblasts (BCAFs); breast cancer microenvironment; fibroblasts; mammary gland; metastasis.

Figure 1

(A,B) Haematoxylin and eosin staining. (A) Normal structure of human terminal duct lobular units (TDLUs), composed of an inner layer of luminal epithelial cells (red arrow), and an outer layer of myoepithelial cell (yellow arrow), separated from the stroma by a basement membrane (blue arrow). (B) Early cancerization of a human mammary duct with initial stromal invasion. Magnification X 20. Whole Slide Imaging were digitized with an Aperio AT2 scanner with 40x optics. (C,D) Schematic representations of (C) terminal end bud (TEB) during mammary branching morphogenesis and (D) ductal and alveolar structure during breast cancer (BC). (C) The figure shows the structure of TEB which consists of inner layers of epithelial precursors, also known as body cells, surrounded by an outer layer of myoepithelial precursors, also known as cap cells. A basement membrane, lining the neck of the TEB and the subtending duct, separates the epithelium and the stroma. In front of the expanding TEB, the basement membrane is transiently disrupted, to allow epithelial growth and ductal expansion. Fibroblasts, together with adipocytes (not shown), represent the major cellular component of the mammary stroma, and play a fundamental role in mammary gland development. Indeed, they sustain mammary gland formation by promoting epithelial cell expansion, normal duct elongation, and invasion into the fat pad. (D) BC cells break the normal architecture of myoepithelial and epithelial cells in the ducts and alveoli of mammary gland. Epithelial cells can undergo malignant transformation. Myoepithelial cells surrounding the neoplastic lesion disappear. Fibroblasts become constitutively activated in breast cancer-associated fibroblasts (BCAFs), and the basement membrane gradually thins out. BCAFs sustain BC growth and progression by enhancing epithelial tumor cell proliferation, migration, and invasion by paracrine interactions. These events lead to BC expansion inside and outside the mammary gland.

Figure 2

Role of fibroblasts in mammary gland branching and BC growth. Mammary branching morphogenesis and BC growth share many cellular processes, including epithelial cell proliferation, migration, invasion, and extracellular matrix (ECM) remodeling. These similarities explain why many morphogenic processes induced by fibroblasts are exploited intensively also in BC. (A) In normal conditions, fibroblasts guide mammary epithelial branching through paracrine signals that are mediated by growth factors, such as insulin-like growth factor (IGF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF)2, and FGF9, low concentrations of transforming growth factor (TGF)-β and tightly regulated activation of the epithelial growth factor receptor (EGFR) signaling pathway. The expression of Sprouty/Spred family members in stromal cells plays a very important role during mammary branching. These morphogenic processes activated by fibroblasts are tightly controlled to ensure normal mammary gland development. (B) However, they become dysregulated and subverted during BC. In fact, BCAFs support BC growth by increasing extensively the same morphogenic pathways required for normal gland development. BCAFs massively upregulate the platelet derived growth factor receptor (PDGFR)α and EGFR signaling pathways, and produce higher levels of FGFs, HGF, and TGF-β than normal fibroblasts. The loss of Spry genes promotes, in fibroblasts, a cancer-associated fibroblast (CAF)-like phenotype and thus leads to BC growth.

Figure 3

Role of BCAFs in BC metastasis. BCAFs can create favorable physical conditions for BC metastasis. Through ECM remodeling, enhanced and aberrant angiogenesis, soluble factor release and establishment of local and distant chemoattractant gradients, BCAFs sustain and enhance BC cell migration, intravasation in blood circulation (as circulating tumor cells (CTCs), CTC aggregate and/or CTC/BCAF aggregate), extravasation, and, ultimately, the homing and colonization of secondary sites. Additionally, through paracrine interactions, BCAFs promote epithelial-mesenchymal transition (EpMT), and the activation of several signaling pathways in BC cells. All of these processes enhance the capability of BC cells to migrate, produce proteases, enter the blood circulation and finally colonize a secondary tumor site. Notably, BCAFs can disseminate through the blood circulation as cBCAFs and/or aggregates without cancer cells, colonize and modify the “soil” in distant organs, thereby creating a receptive microenvironment (the pre-metastatic niche) for distant tumor growth. The color gradient (from lighter to darker violet), starting in the primary tumor, proceeding in the blood stream, and ending in the secondary tumor, indicates the gradual increase of BCAF-derived chemoattractive factors. Lighter colors depict lower concentrations. Darker colors depict higher concentrations.