Abstract
Microcalcifications are vital mammographic indicators contributing to the early detection of up to 50% of non-palpable tumours and may also be valuable as prognostic markers. However, the precise mechanism by which they form remains incompletely understood. Following development of an in vitro model using human breast cancer cells lines cultured with a combination of mineralisation-promoting reagents, analysis of calcium deposition, alkaline phosphatase (ALP) activity and changes in expression of key genes was used to monitor the calcification process. Two cell lines were identified as successfully mineralising in vitro, MDA-MB-231 and SKBR3. Mineralising cell lines displayed higher levels of ALP activity that was further increased by addition of mineralisation promoting media. qPCR analysis revealed changes in expression of both pro- (RUNX2) and anti- (MGP, ENPP1) mineralisation genes. Mineralisation was suppressed by chelation of intracellular Ca2+ and inhibition of TRPM7, demonstrating a functional role for the channel in formation of microcalcifications. Increased Mg2+ was also found to effectively reduce calcium deposition. These results expand the number of human breast cancer cell lines with a demonstrated in vitro mineralisation capability, provide further evidence for the role of an active, cellular process of microcalcification formation and demonstrate for the first time a role for TRPM7 mediated Ca2+ transport.
Highlights
Breast cancer survival rates have risen significantly in recent decades, due to a combination of improved treatments options and early detection
Media was removed and either replaced with fresh growth media (Control), media supplemented with an osteogenic cocktail (OC; 10 mM β-glycerophosphate and 50 μg/mL ascorbic acid) or media supplemented with OC and 100 nM dexamethasone (Dex)
Previous work from our group demonstrated successful mineralisation in breast cancer cell lines grown in media supplemented with an osteogenic cocktail (OC), consisting of β-glycerophosphate and ascorbic acid
Summary
Breast cancer survival rates have risen significantly in recent decades, due to a combination of improved treatments options and early detection. Mammographic detection of microcalcifications has since come to be regarded as a highly useful marker of breast cancer, with somewhere between 30 and 50% of non-palpable tumours found in screening identified solely due to the presence of microcalcifications[6,7] They are present in the majority of ductal carcinoma in situ (DCIS) cases[8]. Microcalcifications detected by mammography can be categorized based on their size, shape, chemical composition and spatial distribution within the breast, allowing for assessment as a benign or suspicious finding[9] In addition to their utility as a detection marker, the presence of microcalcifications within a breast tumour may be of prognostic significance, with many studies highlighting links between calcifications and www.nature.com/scientificreports/. A thorough examination of the mineralisation potential of a representative selection of human breast cancer cells is merited
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