Abstract P6-01-03: Exploring the relationship between an in vitro model of breast cancer cell mineralisation and the cancer grade specific composition of ex vivo microcalcifications
Authors: Bouzy P , O'Grady S , Palombo F , Morgan MP , Stone N 
Published: May 2019

Abstract
Background: Microcalcifications resulting from calcium deposition in the mammary gland play a central role in the early detection of breast cancer [1]. However, the relationship between their occurrence in the breast and cancer progression remains poorly understood. Our approach is to use vibrational spectroscopy and imaging, which is non-invasive, non-destructive, label-free and chemically specific, to assess the composition and distribution of the deposits in an in vitro cancer cell model of mineralisation [2]. In parallel we will utilise the same methods to measure the biochemical composition of microcalcifications found in breast biopsies from different grades of cancer. The ultimate aim of the study is to link the changes identified during the in vitro mineralisation process with the different stages of breast cancer. Vibrational spectroscopic methods can provide incredibly detailed biomolecular fingerprints enabling us to elucidate both the compositional changes with advancing pathology and the spatial distribution of those changes within the calcification and the surrounding tissue.
Methods: The breast cancer cell line MDA-MB-231 has the ability to produce mineralisation. This mineralisation was assessed over a 14-day period in the presence of different osteogenic cocktails: one composed of ascorbic acid, β-glycerophosphate (βG) and dexamethasone (Dex), and another one composed of inorganic phosphate (Pi). Fixed cells were analysed using Raman spectroscopy and micro-FTIR imaging at different time points (3, 7, 11 and 14 days). Tissue sections from patients with microcalcifications identified in histopathology will be sectioned to 3 mm and imaged with infrared (Agilent 670 FTIRinterferometer and Focal Plane Array imaging microscope) and Raman (Renishaw inVia) microspectrometers.
Results: We observed distinct and specific phosphate peak (PO43-) at 960 and 1020 cm-1 in Raman and FTIR spectra, respectively, corresponding to hydroxyapatite crystal and indicating the presence of microcalcification formation. Treatment with Pi induced a faster mineralisation (day 3) compared to cells treated with βG (day 11) and different spectral profiles during this development phase. In addition, there are changes in both the relative DNA and protein concentrations in the cells following 11 days exposure to the osteogenic agents.
It has been shown that the level of carbonate substitution in the calcium hydroxyapatite crystal correlates directly with the pathology of the tissue surrounding the microcalcification. Here we compare the mineral composition found ex vivo versus the in vitro model.
Conclusion: It could be possible to link the progressive biophysical changes associated with mineralisation to distinct stages of breast cancer pathology based on protein, lipid and carbonated apatite contents of the mineralised cells.
Support:This work was conducted as part of the Marie Curie Innovative Training Network Mid-TECH [H2020-MSCA-ITN-2014-642661]
References:
[1] R. Baker, KD. Rogers, N. Shepherd and N. Stone. British Journal of Cancer. 103, 1034-1039 (2010)
[2] RF. Cox, A. Hernandez-Santana, S. Ramdass, G. McMahon, JH. Harmey and MP. Morgan. British Journal of Cancer. 106, 525–537 (2012)
Citation Format: Bouzy P, O'Grady S, Palombo F, Morgan MP, Stone N. Exploring the relationship between an in vitro model of breast cancer cell mineralisation and the cancer grade specific composition of ex vivo microcalcifications [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-01-03.