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Breast cancer-associated fibroblasts (BCAFs), the most abundant non-cancer stromal cells of the breast tumor microenvironment (TME), dramatically sustain breast cancer (BC) progression by interacting with BC cells. BCAFs, as well as myofibroblasts, display an up regulation of activation and inflammation markers represented by α-smooth muscle actin (α-SMA) and cyclooxygenase 2 (COX-2). BCAF aggregates have been identified in the peripheral blood of metastatic BC patients. We generated an in vitro stromal model consisting of human primary BCAFs grown as monolayers or 3D cell aggregates, namely spheroids and reverted BCAFs, obtained from BCAF spheroids reverted to 2D cell adhesion growth after 216 h of 3D culture. We firstly evaluated the state of activation and inflammation and the mesenchymal status of the BCAF monolayers, BCAF spheroids and reverted BCAFs. Then, we analyzed the MCF-7 cell viability and migration following treatment with conditioned media from the different BCAF cultures. After 216 h of 3D culture, the BCAFs acquired an inactivated phenotype, associated with a significant reduction in α-SMA and COX-2 protein expression. The deactivation of the BCAF spheroids at 216 h was further confirmed by the cytostatic effect exerted by their conditioned medium on MCF-7 cells. Interestingly, the reverted BCAFs also retained a less activated phenotype as indicated by α-SMA protein expression reduction. Furthermore, the reverted BCAFs exhibited a reduced pro-tumor phenotype as indicated by the anti-migratory effect exerted by their conditioned medium on MCF-7 cells. The deactivation of BCAFs without drug treatment is possible and leads to a reduced capability of BCAFs to sustain BC progression in vitro. Consequently, this study could be a starting point to develop new therapeutic strategies targeting BCAFs and their interactions with cancer cells. [ABSTRACT FROM AUTHOR]

The article presents a study on analysis of breast cancer-associated fibroblasts in 2D and 3D cell culture systems. Topics include information on development of an in vitro model to deactivate cancer-associated fibroblasts and study their influence on cancer growth; breast cancer (BC) characterized by an abnormal tumor microenvironment (TME); and cells supporting BC initiation and progression through interactions with BC cells.

Breast cancer associated fibroblasts (BCAFs) represent the most abundant cell type among non-cancer stromal cells of the breast tumor microenvironment (TME). It is known that BCAFs, by interacting with cancer cells, contribute dramatically to breast cancer growth and dissemination. This cell type, as well as myofibroblasts, displays up-regulation of activation and inflammation markers represented by α-smooth muscle actin (a-SMA) and cyclooxygenase 2 (COX-2). Moreover, BCAF aggregates have been detected in the peripheral blood of patients with metastatic breast cancer. Therefore, we have developed and characterized an in vitro stromal model constituted by BCAF monolayers, BCAF aggregates, such as spheroids collected after 72 or 216 h of three dimensional (3 D) culture, and BCAFs reverted to adhesion growth after 216 h of 3 D culture (reverted-BCAFs). BCAF spheroids collected at 216 h display an inactivated phenotype as indicated by a significant reduction of α-SMA and COX-2 proteins, compared with control BCAF monolayers. The deactivated phenotype of BCAF spheroids collected at 216 h is confirmed by the cytostatic effect of their conditioned medium on MCF-7 cells. It is noteworthy that also reverted-BCAFs exhibit an inactivated phenotype linked to significant α-SMA protein level reduction compared with BCAF monolayers. Of note, the conditioned medium of reverted-BCAFs decreases significantly the migratory capability of MCF-7 cells compared with the conditioned medium of BCAF monolayers. The study of this in vitro experimental model suggests that the activated phenotype of BCAFs is reversible and it could be a starting point to develop new therapeutic strategies targeting BCAFs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]