Investigation of the Functional Roles of Calcium Channels, Inflammatory Cytokines and Tumour Micro-Environmental Factors in a Human In Vitro Model of Breast Cancer Calcification

Microcalcifications are frequently the sole mammographic indicator of breast cancer with several studies suggesting that their presence may also be indicative of increased tumour grade, HER2 overexpression and poor prognosis. Despite their widespread diagnostic usage, the mechanism by which microcalcifications form and their contribution to tumour progression remain poorly understood. Previously, our lab established an in vitro model using a murine cell line. In order to advance our understanding of these vital diagnostic markers, we have expanded these findings and developed a human in vitro model of microcalcification formation.

Following extensive testing of a panel of human breast cancer cell lines, the MDAMB-231 and SKBR3 cell lines were identified as suitable for our studies. Positive mineralisation was observed in one out of three tested HER2+ cell lines, indicating HER2 overexpression by itself is insufficient to induce in vivo mineralisation. Formation of calcifications was accompanied by increased expression of the osteogenic markers RUNX2, BMP2, and ALP.

Inhibition of the cation channel TRPM7 resulted in a dose-dependent decrease in calcium deposition, implicating a functional role for the channel. Calcification was also inhibited by the intracellular Ca2+ chelator BAPTA-AM and by Mg2+ . The role of other calcium handling proteins including PMCA1 and PMCA2 was also investigated.

Finally, we investigated possible links between microcalcification and pro-tumourigenic effects. Culturing breast cancer cells in mineralisation media increased formation of tumourspheres, indicating an increase in cancer stem-cell properties. We also observed an increased IL-1β, IL-6 and COX2 in both breast cancer cells and associated macrophages following stimulation with hydroxyapatite. Inflammatory cytokines were also observed to modulate in vitro mineralisation, suggesting a complex interplay between the formation and release of calcified particles and their subsequent interaction with surrounding tumour cells.

The results of this thesis reaffirms and expands on our labs previous findings, that breast cancer cell lines can be utilised as effective in vitro model for tumour calcification and that formation of these calcifications is the result of an active, cellmediated process, involving interaction between mineralisation associated proteins including BMP2 and ALP, as well as Ca2+ channels and tumour associated cytokines.