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Breast microcalcifications are routinely explored for mammographic detection of breast cancer and are primarily composed of non-stoichiometric hydroxyapatite (Ca 10− x (PO 4 ) 6− x (CO 3 ) x (OH) 2− x ) (HA). Interestingly, HA morphology and carbonate substitution vary in malignant vs. benign lesions. However, whether or not these changes (i) are functionally linked and (ii) impact malignancy remains unclear due in part to lack of model systems that permit evaluating these possibilities. Here, we have adapted a 96 well-based mineralized culture platform to investigate breast cancer cell behavior in response to systematic changes in the chemical and physical properties of HA. By adjusting the carbonate content of the simulated body fluid (SBF) solutions used during growth, we can control the morphology and carbonate substitution of the deposited HA. Our results suggest that both the combined and individual effects of these differences alter breast cancer cell growth and secretion of tumorigenic interleukin-8 (IL-8). Consequently, changes in both HA carbonate incorporation and morphology impact the behavior of breast cancer cells. Collectively, our data underline the importance of biomineralized culture platforms to evaluate the functional contribution of HA material properties to the pathogenesis of breast cancer. Statement of Significance Breast microcalcifications are small mineral deposits primarily composed of hydroxyapatite (HA). HA physicochemical properties have been of considerable interest, as these are often altered during breast cancer progression and linked to malignancy. However, the functional relationship between these changes and malignancy remains unclear due in part to lack of model systems. Here, we have adapted a previously developed a 96 well-based culture platform to evaluate breast cancer cell behavior in response to systematic changes in HA properties. Our results demonstrate that changes in HA morphology and carbonate content influence breast cancer cell growth and interleukin-8 secretion, and suggest that characterizing the effect of HA properties on breast cancer cells may improve our understanding of breast cancer development and progression. [ABSTRACT FROM AUTHOR]

Bone metastasis represents the leading cause of breast cancer related-deaths. However, the effect of skeleton-associated biomechanical signals on the initiation, progression, and therapy response of breast cancer bone metastasis is largely unknown. This review seeks to highlight possible functional connections between skeletal mechanical signals and breast cancer bone metastasis and their contribution to clinical outcome. It provides an introduction to the physical and biological signals underlying bone functional adaptation and discusses the modulatory roles of mechanical loading and breast cancer metastasis in this process. Following a definition of biophysical design criteria, in vitro and in vivo approaches from the fields of bone biomechanics and tissue engineering that may be suitable to investigate breast cancer bone metastasis as a function of varied mechano-signaling will be reviewed. Finally, an outlook of future opportunities and challenges associated with this newly emerging field will be provided. [ABSTRACT FROM AUTHOR]

Abstract Bone metastasis is a leading cause of death in patients with breast cancer, but the underlying mechanisms are poorly understood. While much work focuses on the molecular and cellular events that drive breast cancer bone metastasis, it is mostly unclear what role bone extracellular matrix (ECM) properties play in this process. Bone ECM primarily consists of mineralized collagen fibrils, which are composed of non-stoichiometric carbonated apatite (HA) and collagen type I. Reduced bone mineral content is epidemiologically linked with increased risk of bone metastasis. Yet elucidating the potential functional impact of collagen mineralization on breast cancer cells has remained challenging because of a lack of model systems that allow studying tumor cell behavior as a function of physiological, intrafibrillar collagen mineralization. Here, we have developed cell culture substrates composed of mineralized collagen type I fibrils using a polymer-induced liquid-precursor (PILP) process. Intrafibrillar HA decreased breast cancer cell adhesion forces and accordingly reduced collagen fiber alignment relative to cells cultured on control collagen. The resulting mineral-mediated changes in collagen network characteristics and mechanosignaling correlated with increased cell motility, but inhibited directed migration of breast cancer cells. These results suggest that physiological mineralization of collagen fibrils reduces tumor cell adhesion with potential functional consequences on skeletal homing of disseminated tumor cells in early stages of breast cancer metastasis. [ABSTRACT FROM AUTHOR]

Adipose-derived stem cells (ASCs) are abundantly present in the mammary microenvironment and can promote breast cancer malignancy by differentiating into myofibroblasts. However, it remains largely unclear which role tumor-derived extracellular vesicles (TEVs) play in this process. Here, we used microfabricated, type I collagen-based 3-D tissue culture platforms to investigate the effect of breast cancer cell-derived TEVs on ASCs myofibroblast differentiation and consequential changes in extracellular matrix remodeling and vascular sprouting. TEVs collected from MDA MB-231 human metastatic breast cancer cells (MDAs) promoted ASC myofibroblast differentiation in both 2-D and 3-D cultures as indicated by increased alpha smooth muscle actin (α-SMA) and fibronectin (Fn) levels. Correspondingly, TEV-treated ASCs were more contractile, secreted more vascular endothelial growth factor (VEGF), and promoted angiogenic sprouting of human umbilical vein endothelial cells (HUVECs). These changes were dependent on transforming growth factor beta (TGF-β)-related signaling and tumor cell glutaminase activity as their inhibition decreased TEV-related myofibroblastic differentiation of ASCs and related functional consequences. In summary, our data suggest that TEVs are important signaling factors that contribute to ASC desmoplastic reprogramming in the tumor microenvironment, and suggest that tumor cell glutamine metabolism may be used as a therapeutic target to interfere with this process. [ABSTRACT FROM AUTHOR]

Breast cancer cells recruit surrounding stromal cells, such as cancer-associated fibroblasts (CAFs), to remodel their extracellular matrix (ECM) and promote invasive tumor growth. Two major ECM components, fibronectin (Fn) and collagen I (Col I), are known to interact with each other to regulate cellular behavior. In this study, we seek to understand how Fn and Col I interplay and promote a dysregulated signaling pathway to facilitate tumor progression. Specifically, we investigated the evolution of tumor-conditioned stromal ECM composition, structure, and relaxation. Furthermore, we assessed how evolving Fn-Col I interactions gradually affected pro-angiogenic signaling. Our data first indicate that CAFs initially assembled a strained, viscous, and unfolded Fn matrix. This early altered Fn matrix was later remodeled into a thick Col I-rich matrix that was characteristic of a dense tumor mass. Next, our results suggest that this ECM remodeling was primarily mediated by matrix metalloproteinases (MMPs). This MMP activity caused profound structural and mechanical changes in the developing ECM, which then modified vascular endothelial growth factor (VEGF) secretion by CAFs and matrix sequestration. Collectively, these findings enhance our understanding of the mechanisms by which Fn and Col I synergistically interplay in promoting a sustained altered signaling cascade to remodel the breast tumor stroma for invasive breast tumor growth. [ABSTRACT FROM AUTHOR]

Fibronectin (Fn) forms a fibrillar network that controls cell behavior in both physiological and diseased conditions including cancer. Indeed, breast cancer-associated stromal cells not only increase the quantity of deposited Fn but also modify its conformation. However, (i) the interplay between mechanical and conformational properties of early tumor-associated Fn networks and (ii) its effect on tumor vascularization remain unclear. Here, we first used the Surface Forces Apparatus to reveal that 3T3-L1 preadipocytes exposed to tumor-secreted factors generate a stiffer Fn matrix relative to control cells. We then show that this early matrix stiffening correlates with increased molecular unfolding in Fn fibers, as determined by Förster Resonance Energy Transfer. Finally, we assessed the resulting changes in adhesion and proangiogenic factor (VEGF) secretion of newly seeded 3T3-L1s, and we examined altered integrin specificity as a potential mechanism of modified cell–matrix interactions through integrin blockers. Our data indicate that tumor-conditioned Fn decreases adhesion while enhancing VEGF secretion by preadipocytes, and that an integrin switch is responsible for such changes. Collectively, our findings suggest that simultaneous stiffening and unfolding of initially deposited tumor-conditioned Fn alters both adhesion and proangiogenic behavior of surrounding stromal cells, likely promoting vascularization and growth of the breast tumor. This work enhances our knowledge of cell – Fn matrix interactions that may be exploited for other biomaterials-based applications, including advanced tissue engineering approaches. [ABSTRACT FROM AUTHOR]

The physical microenvironment of tumor cells plays an important role in cancer initiation and progression. Here, we present evidence that confinement – a new physical parameter that is apart from matrix stiffness - can also induce malignant transformation in mammary epithelial cells. We discovered that MCF10A cells, a benign mammary cell line that forms growth-arrested polarized acini in Matrigel, transforms into cancer-like cells within the same Matrigel material following confinement in alginate shell hydrogel microcapsules. The confined cells exhibited a range of tumor-like behaviors, including uncontrolled cellular proliferation and invasion. Additionally, 4–6 weeks after transplantation into the mammary fad pads of immunocompromised mice, the confined cells formed large palpable masses that exhibited histological features similar to that of carcinomas. Taken together, our findings suggest that physical confinement represents a previously unrecognized mechanism for malignancy induction in mammary epithelial cells and also provide a new, microcapsule-based, high throughput model system for testing new breast cancer therapeutics. [ABSTRACT FROM AUTHOR]

The NAD+-dependent deacetylase Sirtuin 1 (SIRT1) is down-regulated in triple-negative breast cancer. To determine the mechanistic basis by which reduced SIRT1 expression influences processes related to certain aggressive cancers, we examined the consequences of depleting breast cancer cells of SIRT1. We discovered that reducing SIRT1 levels decreased the expression of one particular subunit of the vacuolar-type H+ ATPase (V-ATPase), which is responsible for proper lysosomal acidification and protein degradation. This impairment in lysosomal function caused a reduction in the number of multi-vesicular bodies (MVBs) targeted for lysosomal degradation and resulted in larger MVBs prior to their fusing with the plasma membrane to release their contents. Collectively, these findings help explain how reduced SIRT1 expression, by disrupting lysosomal function and generating a secretome comprising exosomes with unique cargo and soluble hydrolases that degrade the extracellular matrix, can promote processes that increase breast-cancer-cell survival and invasion. • SIRT1 expression is frequently decreased in aggressive breast cancers • Reducing SIRT1 levels in breast cancer cells impairs lysosomal acidification • Loss of SIRT1 gives rise to a secretome that promotes cell invasion and survival • SIRT1 mediates these effects by regulating V-ATPase expression Sirtuin 1 (SIRT1) expression is down-regulated in triple-negative breast cancer. Latifkar et al. show how reducing SIRT1 levels inhibit proper lysosomal function and, in doing so, results in the generation of a secretome with unique components, i.e., exosomes and resident lysosomal hydrolases, that promote the aggressiveness of breast cancer cells. [ABSTRACT FROM AUTHOR]