Multiscale characterization of the mineral phase at skeletal sites of breast cancer metastasis
Significance Hydroxyapatite (HA) nanocrystals are key constituents of the bone extracellular matrix and thus likely to influence the pathogenesis of breast cancer skeletal metastasis. However, there is currently an insufficient understanding of HA nanocrystal properties at sites prone to bone metastasis formation. Here we report a novel application of X-ray scattering and Raman imaging to characterize HA nanostructure in mouse models of breast cancer. Our results suggest that bone regions linked with the initiation of metastasis contain less-mature HA nanocrystals and that mammary tumors enhance HA nanocrystal immaturity in these regions even prior to secondary tumor formation. Insights from this work will significantly advance the development of mineralized culture models to investigate how the bone microenvironment regulates breast cancer metastasis. 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.