Abstract 2345: Establishment and characterization of a novel in vitro model of mammary cell mineralization
Authors: Cox Rachel F. , Hernandez-Santana Aaron , McMahon Gillian , Morgan Maria 
Published: February 2011

Abstract
Mammary microcalcifications represent one of the most reliable mammographic features of non-palpable breast cancer and are often the sole indicator of the disease. There are two types of calcifications associated with breast disease; calcium oxalate and hydroxyapatite. Calcium oxalate is associated with benign lesions of the breast, whereas hydroxyapatite is associated with both benign and malignant tumors. The aims of this study were to (1) determine whether mammary cell lines are capable of calcifying in vitro, (2) characterize the mineral species deposited using Raman spectroscopy and (3) determine whether the characteristics are associated with cell phenotype.
Human and mouse mammary cell lines (Hs578T, Hs578Ts(i)8, MCF10a, 4T1, 4T1.2) were grown in either regular growth media or an osteogenic cocktail (ascorbic acid, β-glycerophosphate, +/-dexamethasone) for up to 4 weeks in 2D culture. Alizarin red S staining, von Kossa staining and a colorimetric calcium assay were used to monitor mineralization. Raman spectroscopy was used to identify and characterize the calcium species of any mineral deposited by the cells.
The metastatic mouse mammary cells 4T1 and 4T1.2, as well as the highly invasive human mammary cell line Hs578Ts(i)8 were capable of generating mineral in vitro when treated with the osteogenic cocktail. This was confirmed by alizarin red S, von Kossa stains and calcium assay. In contrast the less invasive human mammary cell line Hs578T and the normal human mammary cell line MCF10a did not mineralize under any conditions. The calcium species deposited by all mineralizing cell lines were examined by Raman spectroscopy and found to have a symmetric phosphate stretch at ∼960cm−1, which is typical for a carbonated form of hydroxyapatite. In addition, it was found that the Raman peak of the Hs578Ts(i)8 cell line deposits has a narrower full-width at half height (FWHH; 15.85 +/- 1.7 cm−1) than that found in the 4T1 (23.89 +/- 3 cm−1) and 4T1.2 (22.72 +/- 2.5 cm−1) cell lines. This is associated with higher crystallinity and lower carbonate content. There is evidence in the literature to suggest that these characteristics are associated with malignant tumors, which is reflective of the highly invasive nature of the Hs578Ts(i)8 cells.
This study is the first to demonstrate that certain mammary cells lines are capable of mineralizing in vitro. Mineralization potential of mammary cell lines appears to be associated with cell phenotype. This is evident by the finding that more invasive cell lines mineralize in vitro, whereas normal immortalized mammary cells do not. The crystallinity of the mineral species deposited also correlates with tumorigenic potential as shown by the FWHH of Raman spectroscopic analysis. Further studies are currently underway to elucidate the molecular mechanism involved in formation of mammary calcifications using our unique mineralization model.
Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2345.