IMAGING OF PRECANCER TUMOR MODEL TO STUDY THE RELATIONSHIP BETWEEN MICROCALCIFICATIONS AND MALIGNANCY

A long-standing and central challenge in cancer research is preventing the progression of early precancer lesions to invasive and metastatic cancer. The tumor microenvironment (TME) affects tumor progression and can drive cancer invasion in certain conditions. Microcalcification (MCs) are calcium-containing minerals that form in the breast and are a component of the TME. MCs appear in more than 90% of ductal carcinoma in situ (DCIS) precancer cases but are often scientifically overlooked. The genetic events that lead to the progression from precancer to invasive cancer are unknown. However, accumulating evidence suggests a connection between MCs to cancer progression. The prognosis of breast cancers with MCs is typically worse than those without them. There is a correlation between the MC crystal properties and malignancy. Calcium oxalate dehydrate (COD) is associated exclusively with benign lesions and hydroxyapatite (HA) is associated with benign and malignant lesions. In addition, higher carbonate content is associated with lower malignancy. We study the relationship between MC crystal properties and precancer cell malignancy in a 3D breast tumor in vitro model. This system comprises hydrated 3D multicellular spheroids that grow in suspension, cultured with various cancer-related minerals. To obtain a physiologically relevant model, the 3D precancer tumor models have a diameter of up to 500 µm, making it challenging to image the mineral-cell interactions in situ. Hence, our methodology includes advanced 3D imaging techniques such as micro computerized tomography to characterize the sizes, morphology and distribution of mineral particles embedded within the multicellular spheroids. In addition, we use confocal microscopy to characterize the morphology of spheroids. The surface texture and morphology of the mineral particles were characterized using an SEM. The cells appear to be rounder and less spread in our 3D model than 2D cultures, suggesting that 3D spheroids mimic in vivo TME better than 2D precancer cell cultures. The micro-CT shows that the mineral particles are embedded within the spheroid and are distributed throughout it, most likely allowing a high percentage of cell-mineral contact. Based on the characterization of cancer-related markers, the presence of minerals in the spheroid microenvironment most likely promotes precancer malignancy compared to spheroids without minerals. The gained insights from this study may suggest a prognostic value for precancer cases and offer ways of affecting disease progression.