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.

微钙化可作为乳腺癌的诊断标志物，但其形成途径和在癌症进展中的作用受到争议，部分原因是缺乏相关的3D培养模型，该模型无法研究细胞对矿化作用的调控程度。先前的研究表明，其过程从与细胞死亡相关的营养不良矿化到骨样矿物质沉积。在这里，我们评估了3D多细胞球体中的微钙化形成，该球体是由MCF10A人乳腺肿瘤进展系列的非恶性，癌前期和浸润性细胞系产生的。具有更大恶性潜能的球体会形成坏死核心，从而概括了已知在体内调节肿瘤细胞行为的空间上不同的可行和非可行区域。使用nanoCT，电子显微镜和X射线光谱对微钙化的空间分布及其组成进行了表征。磷灰石微钙化主要在活细胞区域内检测到，其数量和大小随球体的恶性程度增加而增加。碱性磷酸酶的水平随着恶性潜能的降低而降低，而骨桥蛋白的水平升高。这些发现支持矿化途径，其中癌细胞以与其恶性潜能相关的方式诱导矿化，但不同于生理成骨性矿化。