Role of Microcalcifications in Breast Cancer Bone Mimicry

Breast microcalcifications (MCs) are insoluble calcium deposits, that are used as a diagnostic tool to detect breast cancer where up to 93% of cases of ductal carcinoma in situ (DCIS) are determined based on the presence of MCs. Breast MCs are categorized into type I (benign – calcium oxalate (OX)) and type II (malignant – calcium phosphate, hydroxyapatite (HA)) MCs. Breast lesions with the presence of malignant MCs show an increase in the expression of mesenchymal and bone markers, also termed as “bone mimicry”. Breast cancer cells with bone mimicry are known to exhibit propensity to metastasize to bone. Despite correlative evidence linking MCs to bone mimicry and bone mimicry to bone metastasis, the biological role of MCs in promoting invasive phenotype, bone mimicry and subsequent metastasis remains unknown. To address this, our laboratory has developed collagen-inspired extracellular matrices (“ECM-Mimics”) recapitulating benign or malignant MC composition observed in MC-positive DCIS patients. We hypothesize that the presence of malignant microcalcifications in the primary breast tumor can promote invasive and bone mimicry phenotypes. We also hypothesize that breast cancer cells pre-conditioned to malignant MCs create a premetastatic niche in the primary breast tumor where breast cancer cells secrete cytokines and chemokines which are important for remodeling the bone microenvironment. In this MS thesis project, we show that the ECM-mimics are able to deposit and recapitulate the composition of benign (OX) and malignant (HA) microcalcifications found in breast biopsies of cancer patients. When seeded onto the ECM-mimics containing different microcalcifications, non-metastatic T47D breast cancer cells exhibit higher mRNA expression of mesenchymal and bone markers by qPCR analysis. The conditioned media collected from T47D cells exposed to malignant MCs show higher alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) enzyme activity, which are markers for osteoblast and osteoclast activity, respectively. Furthermore, ex vivo human bone explants exposed to conditioned media from the T47D cells seeded on malignant MCs also show significant increase in the bone metabolic activity, ALP enzyme activity, and TRAP enzyme activity of the bones compared to bones exposed to conditioned media from the T47D cells seeded on non-mineralized or benign MC containing ECM-mimics. These results suggest that malignant MCs may play a critical role in metastatic breast cancer progression