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 culture 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. 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.

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.

No abstract

Aberrant lipid accumulation and marked changes in cellular lipid profiles are related to breast cancer metabolism and disease progression. In vitro , these phenomena are primarily studied using cells cultured in monolayers (2D). Here, we employ multicellular spheroids, generated using the MCF10A cell line series of increasing malignancy potential, to better recapitulate the 3D microenvironmental conditions that cells experience in vivo . Breast cancer cell lipid compositions were assessed in 2D and 3D culture models as a function of malignancy using liquid chromatography coupled with mass spectrometry. Further, the spatial distribution of lipids was examined using Raman chemical imaging and lipid staining. We show that with changes in the cellular microenvironment when moving from 2D to 3D cell cultures, total lipid amounts decrease significantly, while the ratio of acylglycerols to membrane lipids increases. This ratio increase could be associated with the formation of large lipid droplets (>10 μm) that are spatially evident throughout the spheroids but absent in 2D cultures. Additionally, we found a significant difference in lipid profiles between the more and less malignant spheroids, including changes that support de novo sphingolipid production and a reduction in ether-linked lipid fractions in the invasive spheroids. These differences in lipid profiles as a function of cell malignancy and microenvironment highlight the importance of coupled spatial and lipidomic studies to better understand the connections between lipid metabolism and cancer.

Breast cancer frequently metastasizes to bone, causing osteolytic lesions. However, how factors secreted by primary tumors affect the bone microenvironment before the osteolytic phase of metastatic tumor growth remains unclear. Understanding these changes is critical as they may regulate metastatic dissemination and progression. To mimic premetastatic bone adaptation, immunocompromised mice were injected with MDA-MB-231–conditioned medium [tumor-conditioned media (TCM)]. Subsequently, the bones of these mice were subjected to multiscale, correlative analysis including RNA sequencing, histology, micro–computed tomography, x-ray scattering analysis, and Raman imaging. In contrast to overt metastasis causing osteolysis, TCM treatment induced new bone formation that was characterized by increased mineral apposition rate relative to control bones, altered bone quality with less matrix and more carbonate substitution, and the deposition of disoriented mineral near the growth plate. Our study suggests that breast cancer–secreted factors may promote perturbed bone growth before metastasis, which could affect initial seeding of tumor cells.

Dynamic mechanical loading is a strong anabolic signal in the skeleton, increasing osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs) and increasing the bone-forming activity of osteoblasts, but its role in bone metastatic cancer is relatively unknown. In this study, we integrated a hydroxyapatite-containing three-dimensional (3D) scaffold platform with controlled mechanical stimulation to investigate the effects of cyclic compression on the interplay between breast cancer cells and BM-MSCs as it pertains to bone metastasis. BM-MSCs cultured within mineral-containing 3D poly(lactide-co-glycolide) (PLG) scaffolds differentiated into mature osteoblasts, and exposure to tumor-derived soluble factors promoted this process. When BM-MSCs undergoing osteogenic differentiation were exposed to conditioned media collected from mechanically loaded breast cancer cells, their gene expression of osteopontin was increased. This was further enhanced when mechanical compression was simultaneously applied to BM-MSCs, leading to more uniformly deposited osteopontin within scaffold pores. These results suggest that mechanical loading of 3D scaffold-based culture models may be utilized to evaluate the role of physiologically relevant physical cues on bone metastatic breast cancer. Furthermore, our data imply that cyclic mechanical stimuli within the bone microenvironment modulate interactions between tumor cells and BM-MSCs that are relevant to bone metastasis.

Microcalcifications serve as diagnostic markers for breast cancer, yet their formation pathway(s) and role in cancer progression are debated due in part to a lack of relevant 3D culture models that allow studying the extent of cellular regulation over mineralization. Previous studies have suggested processes ranging from dystrophic mineralization associated with cell death to bone-like mineral deposition. Here, we evaluated microcalcification formation in 3D multicellular spheroids, generated from non-malignant, pre-cancer, and invasive cell lines from the MCF10A human breast tumor progression series. The spheroids with greater malignancy potential developed necrotic cores, thus recapitulating spatially distinct viable and non-viable areas known to regulate cellular behavior in tumors in vivo. The spatial distribution of the microcalcifications, as well as their compositions, were characterized using nanoCT, electron-microscopy, and X-ray spectroscopy. Apatite microcalcifications were primarily detected within the viable cell regions and their number and size increased with malignancy potential of the spheroids. Levels of alkaline phosphatase decreased with malignancy potential, whereas levels of osteopontin increased. These findings support a mineralization pathway in which cancer cells induce mineralization in a manner that is linked to their malignancy potential, but that is distinct from physiological osteogenic mineralization.

No abstract