Calcifications Can Trigger or Suppress Breast Precancer Malignancy Potential as a Function of Mineral Type in a 3D Tumor Model

The breast precancer stage will not necessarily develop into invasive breast cancer and theoretically does not always require treatment. However, it is impossible to predict whether precancers will become invasive cancer. More than 90% of breast precancer cases contain pathological minerals called microcalcifications. Most microcalcifications are calcium phosphate in the form of apatite, and their composition and crystal properties correlate with malignancy in clinical samples and may have a prognostic value. In clinical samples, apatite crystals with high and low carbonate content are associated with benign and malignant lesions, respectively, and calcium oxalate dihydrate (COD) is always found in benign lesions. Recent <i>in vitro</i> and <i>in vivo</i> studies show that specific changes in the properties of apatite crystals can trigger precancer progression.<br/>Here, we developed a breast tumor <i>in vitro</i> model consisting of synthetic microcalcification analogs embedded in precancer multicellular spheroids that mimic the 3D microenvironment of mammary ducts. As microcalcification analogs, we use various synthetic mineral types that are physiologically relevant: apatite with high and low carbonate content and COD. Our methodology includes micro-CT for 3D imaging of mineral particles within soft tissues and scanning electron microscopy and vibrational spectroscopy for mineral characterization.<br/>We show that cell proliferation and the expression of proteins associated with cancer change with the properties of the embedded mineral in our <i>in vitro </i>model. Low carbonate apatite promotes precancer malignancy compared to other types of minerals or conditions of no added mineral. Surprisingly, COD suppresses precancer malignancy compared to apatite or conditions of no added mineral. This finding suggests that microcalcifications can affect precancer cells by either triggering them to proliferate or inhibiting their proliferation, even compared to conditions of no minerals. Possibly, the presence of COD crystals only in benign clinical tissues is not random but is a result of its tumor-suppressing qualities. Insights from this study may suggest in the future ways of affecting disease progression by manipulating the crystal properties of breast microcalcifications <i>in situ</i>.