Visualizing intracellular and extracellular cell-mineral interactions in a three-dimensional breast tumor model

Microcalcifications (MCs) in breast tissue, with particularly high prevalence in precancerous lesions, such as ductal carcinoma in situ (DCIS), represent important diagnostic markers for cancer screening. Two primary crystal types, hydroxyapatite (HA) and calcium oxalate dihydrate (COD), occur within breast tumor microenvironments. Evidence from clinical samples has demonstrated that COD is usually found as individual crystals, whereas HA tends to aggregate. Notably, MC crystal types and properties are linked to malignancy, with COD crystals being associated with benign lesions. In-vitro studies have shown that COD crystals can suppress cancerous behavior, whereas HA often promotes it. However, crystal-cell interactions in tumor microenvironments that may explain differential responses to COD and HA remain poorly investigated. Thus, to examine mineral-cell interactions based on cell malignancy potential and crystal type, a three-dimensional multicellular model consisting of precancerous and cancerous human cell lines was employed. Through cryo-scanning electron microscopy (cryo-SEM), we visualized mineral-biological interfaces and showed that cellular types and processes govern crystal aggregation patterns and distribution. Both precancerous and invasive models exhibited enhanced COD crystal packing, while HA crystals formed predominantly loose structures with notable malignancy-dependent variations. In all cases, crystals were found both extra- and intracellularly, the latter being located inside vesicles in some cases, especially for COD crystals. The predominance of intracellular vesicles containing COD crystals rather than HA crystals across both cell types may reflect enhanced cellular processes that promote reduced malignant behavior. These findings may explain why seemingly similar calcium-containing minerals associate differently with malignancy and induce distinct cellular responses.