Biomineralogical signatures of breast microcalcifications.

Microcalcifications, primarily biogenic apatite, occur in cancerous and benign breast pathologies and are key mammographic indicators. Outside the clinic, numerous microcalcification compositional metrics (e.g., carbonate and metal content) are linked to malignancy, yet microcalcification formation is dependent on microenvironmental conditions, which are notoriously heterogeneous in breast cancer. We interrogate multiscale heterogeneity in 93 calcifications from 21 breast cancer patients using an omics-inspired approach: For each microcalcification, we define a "biomineralogical signature" combining metrics derived from Raman microscopy and energy-dispersive spectroscopy. We observe that (i) calcifications cluster into physiologically relevant groups reflecting tissue type and local malignancy; (ii) carbonate content exhibits substantial intratumor heterogeneity; (iii) trace metals including zinc, iron, and aluminum are enhanced in malignant-localized calcifications; and (iv) the lipid-to-protein ratio within calcifications is lower in patients with poor composite outcome, suggesting that there is potential clinical value in expanding research on calcification diagnostic metrics to include "mineral-entrapped" organic matrix.

Multiple Pathways for Pathological Calcification in the Human Body.

Biomineralization of skeletal components (e.g., bone and teeth) is generally accepted to occur under strict cellular regulation, leading to mineral-organic composites with hierarchical structures and properties optimized for their designated function. Such cellular regulation includes promoting mineralization at desired sites as well as inhibiting mineralization in soft tissues and other undesirable locations. In contrast, pathological mineralization, with potentially harmful health effects, can occur as a result of tissue or metabolic abnormalities, disease, or implantation of certain biomaterials. This progress report defines mineralization pathway components and identifies the commonalities (and differences) between physiological (e.g., bone remodeling) and pathological calcification formation pathways, based, in part, upon the extent of cellular control within the system. These concepts are discussed in representative examples of calcium phosphate-based pathological mineralization in cancer (breast, thyroid, ovarian, and meningioma) and in cardiovascular disease. In-depth mechanistic understanding of pathological mineralization requires utilizing state-of-the-art materials science imaging and characterization techniques, focusing not only on the final deposits, but also on the earlier stages of crystal nucleation, growth, and aggregation. Such mechanistic understanding will further enable the use of pathological calcifications in diagnosis and prognosis, as well as possibly provide insights into preventative treatments for detrimental mineralization in disease.

Hydroxyapatite mineral enhances malignant potential in a tissue-engineered model of ductal carcinoma in situ (DCIS).

While ductal carcinoma in situ (DCIS) is known as a precursor lesion to most invasive breast carcinomas, the mechanisms underlying this transition remain enigmatic. DCIS is typically diagnosed by the mammographic detection of microcalcifications (MC). MCs consisting of non-stoichiometric hydroxyapatite (HA) mineral are frequently associated with malignant disease, yet it is unclear whether HA can actively promote malignancy. To investigate this outstanding question, we compared phenotypic outcomes of breast cancer cells cultured in control or HA-containing poly(lactide-co-glycolide) (PLG) scaffolds. Exposure to HA mineral in scaffolds increased the expression of pro-tumorigenic interleukin-8 (IL-8) among transformed but not benign cells. Notably, MCF10DCIS.com cells cultured in HA scaffolds adopted morphological changes associated with increased invasiveness and exhibited increased motility that were dependent on IL-8 signaling. Moreover, MCF10DCIS.com xenografts in HA scaffolds displayed evidence of enhanced malignant progression relative to xenografts in control scaffolds. These experimental findings were supported by a pathological analysis of clinical DCIS specimens, which correlated the presence of MCs with increased IL-8 staining and ductal proliferation. Collectively, our work suggests that HA mineral may stimulate malignancy in preinvasive DCIS cells and validate PLG scaffolds as useful tools to study cell-mineral interactions.

Understanding the Stiff-to-Compliant Transition of the Meniscal Attachments by Spatial Correlation of Composition, Structure, and Mechanics.

Recently, the scientific community has shown considerable interest in engineering tissues with organized compositional and structural gradients to mimic hard-to-soft tissue interfaces. This effort is hindered by an incomplete understanding of the construction of native tissue interfaces. In this work, we combined Raman microscopy and confocal elastography to map compositional, structural, and mechanical features across the stiff-to-compliant interface of the attachments of the meniscus in the knee. This study provides new insight into the methods by which biology mediates multiple orders of magnitude changes in stiffness over tens of microns. We identified how the nano- to mesoscale architecture mediates complex microscale transitional regions across the interface: two regions defined by chemical composition, five distinguished by structural features, and three mechanically distinct regions. We identified three major components that lead to a robust interface between a soft tissue and bone: mobile collagen fiber units, a continuous interfacial region, and a local stiffness gradient. This tissue architecture allows for large displacements of collagen fibers in the attachments, enabling meniscal movement without localizing strains to the soft tissue-to-bone interface. The interplay of these regions reveals a method relying on hierarchical structuring across multiple length scales to minimize stress concentrators between highly dissimilar materials. These insights inspire new design strategies for synthetic soft tissue-to-bone attachments and biomimetic material interfaces.

Mapping and Profiling Lipid Distribution in a 3D Model of Breast Cancer Progression.

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.

Crystallinity of hydroxyapatite drives myofibroblastic activation and calcification in aortic valves.

Calcific aortic valve disease (CAVD) is an inexorably degenerative pathology characterized by progressive calcific lesion formation on the valve leaflets. The interaction of valvular cells in advanced lesion environments is not well understood yet highly relevant as clinically detectable CAVD exhibits calcifications composed of non-stoichiometric hydroxyapatite (HA). In this study, Fourier transform infrared spectroscopic imaging was used to spatially analyze mineral properties as a function of disease progression. Crystallinity (size and perfection) increased with increased valve calcification. To study the relationship between crystallinity and cellular behavior in CAVD, valve cells were seeded into 3D mineral-rich collagen gels containing synthetic HA particles, which had varying crystallinities. Lower crystallinity HA drove myofibroblastic activation in both valve interstitial and endothelial cells, as well as osteoblastic differentiation in interstitial cells. Additionally, calcium accumulation within gels depended on crystallinity, and apoptosis was insufficient to explain differences in HA-driven cellular activity. The protective nature of endothelial cells against interstitial cell activation and calcium accumulation was completely inhibited in the presence of less crystalline HA particles. Elucidating valve cellular behavior post-calcification is of vital importance to better predict and treat clinical pathogenesis, and mineral-containing hydrogel models provide a unique 3D platform to evaluate valve cell responses to a later stage of valve disease. STATEMENT OF SIGNIFICANCE: We implement a 3D in vitro platform with embedded hydroxyapatite (HA) nanoparticles to investigate the interaction between valve interstitial cells, valve endothelial cells, and a mineral-rich extracellular environment. HA nanoparticles were synthesized based on analysis of the mineral properties of calcific regions of diseased human aortic valves. Our findings indicate that crystallinity of HA drives activation and differentiation in interstitial and endothelial cells. We also show that a mineralized environment blocks endothelial protection against interstitial cell calcification. Our HA-containing hydrogel model provides a unique 3D platform to evaluate valve cell responses to a mineralized ECM. This study additionally lays the groundwork to capture the diversity of mineral properties in calcified valves, and link these properties to progression of the disease.

Correlative imaging reveals physiochemical heterogeneity of microcalcifications in human breast carcinomas.

Microcalcifications (MCs) are routinely used to detect breast cancer in mammography. Little is known, however, about their materials properties and associated organic matrix, or their correlation to breast cancer prognosis. We combine histopathology, Raman microscopy, and electron microscopy to image MCs within snap-frozen human breast tissue and generate micron-scale resolution correlative maps of crystalline phase, trace metals, particle morphology, and organic matrix chemical signatures within high grade ductal carcinoma in situ (DCIS) and invasive cancer. We reveal the heterogeneity of mineral-matrix pairings, including punctate apatitic particles (<2 µm) with associated trace elements (e.g., F, Na, and unexpectedly Al) distributed within the necrotic cores of DCIS, and both apatite and spheroidal whitlockite particles in invasive cancer within a matrix containing spectroscopic signatures of collagen, non-collagen proteins, cholesterol, carotenoids, and DNA. Among the three DCIS samples, we identify key similarities in MC morphology and distribution, supporting a dystrophic mineralization pathway. This multimodal methodology lays the groundwork for establishing MC heterogeneity in the context of breast cancer biology, and could dramatically improve current prognostic models.

Synergistic Biomineralization Phenomena Created by a Combinatorial Nacre Protein Model System.

In the nacre or aragonite layer of the mollusk shell, proteomes that regulate both the early stages of nucleation and nano-to-mesoscale assembly of nacre tablets from mineral nanoparticle precursors exist. Several approaches have been developed to understand protein-associated mechanisms of nacre formation, yet we still lack insight into how protein ensembles or proteomes manage nucleation and crystal growth. To provide additional insights, we have created a proportionally defined combinatorial model consisting of two nacre-associated proteins, C-RING AP7 (shell nacre, Haliotis rufescens) and pseudo-EF hand PFMG1 (oyster pearl nacre, Pinctada fucata), whose individual in vitro mineralization functionalities are well-documented and distinct from one another. Using scanning electron microscopy, flow cell scanning transmission electron microscopy, atomic force microscopy, Ca(II) potentiometric titrations, and quartz crystal microbalance with dissipation monitoring quantitative analyses, we find that both nacre proteins are functionally active within the same mineralization environments and, at 1:1 molar ratios, synergistically create calcium carbonate mesoscale structures with ordered intracrystalline nanoporosities, extensively prolong nucleation times, and introduce an additional nucleation event. Further, these two proteins jointly create nanoscale protein aggregates or phases that under mineralization conditions further assemble into protein-mineral polymer-induced liquid precursor-like phases with enhanced ACC stabilization capabilities, and there is evidence of intermolecular interactions between AP7 and PFMG1 under these conditions. Thus, a combinatorial model system consisting of more than one defined biomineralization protein dramatically changes the outcome of the in vitro biomineralization process.

Microscopy techniques for investigating the control of organic constituents on biomineralization.

This article addresses recent advances in the application of microscopy techniques to characterize crystallization processes as they relate to biomineralization and bio-inspired materials synthesis. In particular, we focus on studies aimed at revealing the role organic macromolecules and functionalized surfaces play in modulating the mechanisms of nucleation and growth. In nucleation studies, we explore the use of methods such as in situ transmission electron microscopy, atomic force microscopy, and cryogenic electron microscopy to delineate formation pathways, phase stabilization, and the competing effects of free energy and kinetic barriers. In growth studies, emphasis is placed on understanding the interactions of macromolecular constituents with growing crystals and characterization of the internal structures of the resulting composite crystals using techniques such as electron tomography, atom probe tomography, and vibrational spectromicroscopy. Examples are drawn from both biological and bio-inspired synthetic systems.