A time-course Raman spectroscopic analysis of spontaneous in vitro microcalcifications in a breast cancer cell line

Laboratory Investigation (2021) 101:1267–1280 https://doi.org/10.1038/s41374-021-00619-0 ARTICLE A time-course Raman spectroscopic analysis of spontaneous in vitro microcalcifications in a breast cancer cell line Pascaline Bouzy 1 Shane O’Grady2 Honey Madupalli3 Mary Tecklenburg3 Keith Rogers4 Francesca Palombo1 Maria P. Morgan 2 Nicholas Stone 1 ● ● ● ● ● ● ● 1234567890();,: 1234567890();,: Received: 18 February 2021 / Revised: 17 May 2021 / Accepted: 17 May 2021 / Published online: 11 June 2021 © The Author(s) 2021. This article is published with open access Abstract Microcalcifications are early markers of breast cancer and can provide valuable prognostic information to support clinical decision-making. Current detection of calcifications in breast tissue is based on X-ray mammography, which involves the use of ionizing radiation with potentially detrimental effects, or MRI scans, which have limited spatial resolution. Additionally, these techniques are not capable of discriminating between microcalcifications from benign and malignant lesions. Several studies show that vibrational spectroscopic techniques are capable of discriminating and classifying breast lesions, with a pathology grade based on the chemical composition of the microcalcifications. However, the occurrence of microcalcifications in the breast and the underlying mineralization process are still not fully understood. Using a previously established model of in vitro mineralization, the MDA-MB-231 human breast cancer cell line was induced using two osteogenic agents, inorganic phosphate (Pi) and β-glycerophosphate (βG), and direct monitoring of the mineralization process was conducted using Raman microspectroscopy. MDA-MB-231 cells cultured in a medium supplemented with Pi presented more rapid mineralization (by day 3) than cells exposed to βG (by day 11). A redshift of the phosphate stretching peak for cells supplemented with βG revealed the presence of different precursor phases (octacalcium phosphate) during apatite crystal formation. These results demonstrate that Raman micro-spectroscopy is a powerful tool for nondestructive analysis of mineral species and can provide valuable information for evaluating mineralization dynamics and any associated breast cancer progression, if utilized in pathological samples. Introduction In 2018, breast cancer was one of the most commonly diagnosed types of cancer and the second cause of death from Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41374021-00619-0. * Nicholas Stone 1 School of Physics and Astronomy, University of Exeter, Exeter, UK 2 School of Pharmacy and Biomolecular Science, Royal College of Surgeons in Ireland, Dublin, 2, Ireland 3 Department of Chemistry and Biochemistry and Science of Advanced Materials Program, Central Michigan University, Mt. Pleasant, MI, USA 4 Cranfield Forensic Institute, Cranfield University, Shrivenham, UK cancer worldwide (after lung cancer) [1]. Around 2.1 million women across the world were diagnosed with breast cancer in 2018 [1] with survival rates which are predicted to increase over the next decades, mainly due to changes in detection practice by screening individuals more often and earlier [2]. Early-stage diagnosis is important to improve the survival rate and treatment response, and in this context microcalcifications appear to be the most valuable marker of breast cancer [3]. Xray and vibrational spectroscopy techniques are widely used to investigate the composition of these crystals [4–9]. For instance, it has already been shown that type I microcalcifications are composed of calcium oxalate dihydrate and are observed only in benign lesions, whilst type II microcalcifications are mainly composed of calcium hydroxyapatite (Hap) and are associated with both proliferative benign lesions and malignant lesions [10]. Moreover, in biological tissue, the Hap crystal is not found in its stoichiometric form as the lattice contains carbonates (CO32− ions) which reduce the stability of the crystal and increase its solubility [11–13] with detrimental effects. Two types of substitution are found in the Hap lattice: type A, in which carbonate replaces a hydroxyl ion (OH−), and 1268 P. Bouzy et al. Fig. 1 Experimental design and specific Raman features of mineralization. a Protocol used for 14-days mineralization of MDA-MB-231 cells (left panel). Acronyms denote OC osteogenic cocktail, Dex dexamethasone, Pi inorganic phosphate, DMEM Dulbecco’s modified Eagle medium, βG βglycerophosphate, AA ascorbic acid. Representative images of MDA-MB-231 cells growing at different time point of mineralization (Day 3, 7, 11, and 14) (right panel). Scale bar: 20 µm and ×100 magnification. b Raman spectra acquired from mineral deposits in cell culture after 3 days and c 11 days of mineralization. Labels denote Phe Phenylalanine, Cys Cysteine, Tyr Tyrosine. Cells were treated with Pi + Dex (blue line) and OC + Dex (or βG) (green line). Non-treated cells were considered as control (red line). Each spectrum is an average of 40 spectra for each condition. type B, where carbonate replaces a phosphate ion (PO43−) [14]. Baker et al. have previously demonstrated using FTIR microspectroscopy that the extent of carbonate substitution within breast microcalcifications directly correlates with the degree of pathology [5], a finding that is in line with results of other Raman studies [4, 5, 15]. However, further analysis shows that the calcification’s chemical composition is more complex and suggests the presence of a close interplay between microcalcifications and their microenvironments, potentially affecting both formation and maturation of the crystals. It is known that the regulation of tumor pH plays an important role in cell proliferation and cancer progression. Several studies have shown that cells within a tumor experience hypoxia and upregulation of metabolic A time-course Raman spectroscopic analysis of spontaneous in vitro microcalcifications in a breast. . . processes. Changes in metabolic activity lead to an increase of intracellular concentrations of H+ hydrogen ions and carbon dioxide, which are then released (via different transporters) into the microenvironment causing a decrease of pH [16] and promoting breast cancer progression [17]. The acidic tumor microenvironment can give rise to different precursors in microcalcifications [18]. In addition, recent studies suggest that microcalcifications may contain other mineral phases such as magnesium-substituted β-tricalcium phosphate (β-TCP or whitlockite) [15, 19]. In this study, we hypothesized that, in addition to the calcium Hap, different phosphate species as mineral precursors (e.g. octacalcium phosphate (OCP) or amorphous calcium phosphate (ACP)) could be found in the calcium deposits [20]. To gain an insight into the crystal formation process, we evaluated two different pathways of cell mineralization usi (...truncated)