Microcalcifications In Breast Cancer: Novel Insights Into The Molecular Characteristics And Mechanism Based On Raman Spectroscopy

Microcalcifications in the breast are an important indicator for cancer, and often the only early mammographic sign of breast cancer. Mammography is currently the single most effective routine screening method for early detection of breast cancer. Therefore, there is a clinical need for a tool that can detect microcalcifications in the breast tissues. Raman spectroscopy is capable of distinguishing between the two types of calcifications in deparaffinized fixed breast tissue sections. But the fluorescence occurring as a background on the Raman spectrum could not be reliably analyzed, as a result of the fixation process. Despite the importance of mammary microcalcifications for the early detection of breast cancer and the potential prognostic and biological relevance, little research has been carried out to investigate the molecular mechanisms involved in their formation. To date, no in vitro models of mammary cell mineralisation exist to study the molecular mechanisms involved in this process. Thus, the aim of this study was to explore an approach to concomitantly diagnose microcalcification status and local pathological state featuring SHINERS and multivariate data analysis of spectral markers reflecting molecular expression. In this study, we seek to exploit the heightened sensitivity and intrinsic specificity offered by the Au@Si O2 shell-isolated nanoparticles in elucidating the biochemical content of fresh frozen specimen and, thus, in differentiating between normal breast tissue, fibroadenoma, atypical ductal hyperplasia, ductal carcinoma in situ, and invasive ductal carcinoma, espeacially for the microcalcifications in the diseased breast tissues. Next was to establish and characterise a reproducible in vitro model of mammary cell mineralisation, from which the molecular mechanisms underlying mammary mineralisation can begin to be elucidated, and then to explore the realationship between the expression of osteopontin in breast cancer cell lines and the formation of breast microcalcifications in these cell lines.(1) The molecular characteristics of microcalcifications in breast cancer tissues based on Raman spectroscopy.Although tissue staining followed by morphologic identification remains the gold standard for diagnosis of most cancers, such determinations relying solely on morphology are often hampered by inter- and intra-observer variability. Vibrational spectroscopic techniques, in contrast, offer objective markers for diagnoses and can afford disease detection prior to alterations in cellular and extracellular architecture by furnishing a rapid “omics”-like view of the biochemical status of the probed specimen. Here, we report a classification approach to concomitantly detect microcalcification status and local pathological state in breast tissue, featuring a combination of vibrational spectroscopy that focuses on the tumor and its microenvironment, and multivariate data analysis of spectral markers reflecting molecular expression. We employ the unprecedented sensitivity and exquisite molecular specificity offered by Au@Si O2 shell-isolated nanoparticle-enhanced Raman spectroscopy(SHINERS) to probe the presence of calcified deposits and distinguish between normal breast tissues, fibroadenoma, atypical ductal hyperplasia, ductal carcinoma in situ(DCIS), and invasive ductal carcinoma(IDC).By correlating the spectra with the corresponding histologic assessment, we developed partial least squares-discriminant analysis derived decision algorithm that provides excellent diagnostic power in the fresh frozen sections(overall accuracy of 99.4% and 93.6% using SHINs for breast lesions with and without microcalcifications, respectively). The performance of this decision algorithm is competitive with or supersedes that of analogous algorithms employing spontaneous Raman spectroscopy while enabling facile detection due to the considerably higher intensity of SHINERS. Our results pave the way for rapid tissue spectral pathology measurements using SHINERS that can offer a novel stain-free route to accurate and economical diagnoses without human interpretation.(2) The mechanism of microcalcifications in breast cancer cell lines based on Raman spectroscopy.Breast microcalcifications are the sole early stage diagnostic markers of breast cancer. The association of mineralization(especially type II microcalcifications) with both benign and malignant lesions often leads to unnecessary biopsies. The processes by which these ectopic microcalcifications form are unknown. In the current work, we attempted to explore the possibility of obtaining genes responsible for the formation of microcalcifications in breast cancer cell lines at cellular level and understand their potential involvement in disease progression and distant metastases. First, we selected three genes(Osteopontin, Osteonectin, and Collagen I) from the online databases and analyzed the expression of these three genes(8 cell lines, MDA-MB-231, SUM149, SUM159, MDA-MB-468, MCF-7, BT-474, T-47 D, SKBR-3) with and without osteogenic cocktail(OC) by western bolt. The results showed that the OPN expression increased significantly after we added OC to the medium, whereas the expression of the other two genes did not show significant difference.We next analyzed the expression of the OPN genes with and without osteogenic cocktail(OC) in 8 breast cancer cell lines by performing western bolt and q PCR. And we also quantified the presence of calcifications in these 8 cell lines by staining them with Alizarin after culturing them in OC for 1 week. The results show that the higher metastatic cell line, MDA-MB-231 shows the highest OPN expression and the highest calcifications after adding OC to the medium.Next, we used the sh RNA to silence the OPN gene of MDA-MB-231 cells. Then we quantified the levels of calcifications in these OPN knockdown cell lines after staining them with Alizarin. The levels of calcification across these lines are found to be correlated with the OPN expression levels of these cell lines to a significant degree. Also, there is a significant reduction of calcification in the knockdown lines when compared to the control MDA-MB-231 line.We have successfully shown that OPN gene is directly associated with the formation and regulation of hydroxyapatite formation in MDA-MB-231 breast cancer cells through sh RNA knockdown study. The knockdown of this gene not only reduced the formation of microcalcifications in the cells in response to osteogenic cocktail but also affected their migration and invasion characteristics. The observed dual roles of the OPN gene encourage us to probe further into the possible existence of a direct relationship between microcalcifications and ability to metastasize to distant organs mediated by common genetic factors in the future.