Tumour Micro-Environmental Factors And The Development Of Mammary Calcifications: Recent Findings From An In-Vitro Model

The presence of microcalcifications on mammographic images represents a highly valuable tool in the early detection of breast cancer, often allowing radiographers to diagnose mammary tumours at an early, in-situ stage. The presence of microcalcifications has also been linked to a number of unfavourable prognostic factors including decreased survival and an increased probability of relapse. Despite their long history of use in the clinic, the precise mechanisms by which microcalcifications are formed remains poorly understood. Pathological soft-tissue calcification is often considered the result of an imbalance between pro- and anti-calcifying factors. However, the possible impact of such an imbalance has not been studied in the context of breast calcification. We have established an in-vitro model of microcalcification formation using the triple-negative adenocarcinoma cell line MDA-MB-231. When cultured in the presence of the osteogenesis-promoting reagents β-glycerophosphate, ascorbic acid and dexamethasone, calcified deposits begin to form by Day 14, as verified by Alizarin Red staining and the quantitative o-cresolphthalein assay. Following the establishment of our model, we set out to identify the underlying molecular triggers initiating the calcification process, in particular the role of micro-environmental factors. Altered magnesium homeostasis has been suggested as an important mediator of tissue calcification. We found that a slight increase in magnesium concentration almost completely blocked calcium deposition in our model. Previous studies in other tissues have shown this protective effect to be dependent on the cation channel TRPM7 which is increased in breast cancer patients with calcifications compared to those without. Unexpectedly, inhibition of the TRPM7 channel by two separate compounds (2-APB and NS-8593) not only failed to reverse the inhibition of microcalcification formation by exogenous magnesium, but actually further decreased calcium deposition by Day 28, suggesting that calcium influx via the TRPM7 channel may be promoting development of mammary calcifications. Finally, we investigated the effect of several tumour associated cytokines on the rate of calcium deposition and found that IL-1β and TNF-α blocked mineralisation whilst IL-6 and BMP2 lead to an increase. Interestingly, co-administration of IL-6 alongside a soluble form of its receptor (sIL-6R) was observed to promote mineralisation even in the absence of dexamethasone, which had previously been essential to the formation of calcifications in our model. To date, our model has yielded a number of important insights into the formation of calcifications in breast cell lines, many of which recapitulate observations from patient studies. It is hoped that this work will contribute to our understanding of the origin of these pre-invasive diagnostic clues. Citation Format: Shane O9Grady, Maria P. Morgan. Tumour micro-environmental factors and the development of mammary calcifications: Recent findings from an in-vitro model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 799. doi:10.1158/1538-7445.AM2017-799