Correlative imaging reveals physiochemical heterogeneity of microcalcifications in human breast carcinomas

Abstract

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.

Studying biomineralization pathways in a 3D culture model of breast cancer microcalcifications

“…The mineralized particle sizes reported here ranged from sub-micron to 20 μm, below the resolution limit of mammography. Similar sizes of MCs were recently reported in human breast tumor tissue samples and could represent early stages of mineralization that then grow to the larger sizes detected by mammography[15]. 3D localization of sub-micron particles within the spheroids, however, will require the use of higher resolution volume techniques, such as Focused Ion Beam-Scanning Electron Microscopy imaging[64,65].…”

“…Our SEM data (Fig. 2a–c) and previous results[15], however, showed that mineralization also occurs in core areas, both in vitro and in tissues. MCs in the core most likely form by unregulated mineralization[56–59], but possibly via a dysregulated process followed by cell death.…”

“…Using high resolution imaging, synchrotron XRF mapping, and XANES, we identified apatite particles formed in both types of the malignant spheroids. As it is known that human MCs are either calcium oxalate[55,60] or calcium phosphate minerals[1,2,15,59,61–63], the fact that the spheroids reported here can produce non-stoichiometric apatite, the most common calcium phosphate mineral found in breast tissues, shows the relevance of this model to MC mechanistic studies. Future studies comparing Ca-edge and P-edge XANES of the mineral particles found in the spheroids to microcalcifications from human tissue samples may further inform the relevance of the model.…”

“…Pathological mineral formation can follow cellularly ‘unregulated’, ‘regulated’ and ‘dysregulated’ pathways. Often MCs are observed in necrotic areas of human breast tumors and are most likely the result of unregulated mineralization[14,15]. Unregulated mineralization occurs in areas of cell death and most likely results from some combination of abnormal homeostasis in injured or necrotic cells, local increases in calcium and phosphate concentrations, and apatite nucleation on cellular debris[16–18].…”

Pathological Mineralization: The Potential of Mineralomics

“…This association of breast mineralization to cancer has, therefore, led to a number of research initiatives aiming to identify specific differences between benign and malignant mineralization [125,126,127]. Through electron and light microscopy, X-ray diffraction, and microprobe analysis, two chemically different types of minerals were associated with breast carcinomas: calcium oxalate and apatite [13,14]. Calcium oxalate has been identified mostly in the context of benign diseases [128], although some recent studies failed to identify its presence [129].…”