Search

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]

Skeletal metastases, the leading cause of death in advanced breast cancer patients, depend on tumor cell interactions with the mineralized bone extracellular matrix. Bone mineral is largely composed of hydroxyapatite (HA) nanocrystals with physicochemical properties that vary significantly by anatomical location, age, and pathology. However, it remains unclear whether bone regions typically targeted by metastatic breast cancer feature distinct HA materials properties. Here we combined high-resolution X-ray scattering analysis with large-area Raman imaging, backscattered electron microscopy, histopathology, and microcomputed tomography to characterize HA in mouse models of advanced breast cancer in relevant skeletal locations. The proximal tibial metaphysis served as a common metastatic site in our studies; we identified that in disease-free bones this skeletal region contained smaller and less-oriented HA nanocrystals relative to ones that constitute the diaphysis. We further observed that osteolytic bone metastasis led to a decrease in HA nanocrystal size and perfection in remnant metaphyseal trabecular bone. Interestingly, in a model of localized breast cancer, metaphyseal HA nanocrystals were also smaller and less perfect than in corresponding bone in disease-free controls. Collectively, these results suggest that skeletal sites prone to tumor cell dissemination contain less-mature HA (i.e., smaller, less-perfect, and less-oriented crystals) and that primary tumors can further increase HA immaturity even before secondary tumor formation, mimicking alterations present during tibial metastasis. Engineered tumor models recapitulating these spatiotemporal dynamics will permit assessing the functional relevance of the detected changes to the progression and treatment of breast cancer bone metastasis. [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]