A multi-modal exploration of heterogeneous physico-chemical properties of DCIS breast microcalcifications

Abstract

Ductal carcinoma in situ (DCIS) is frequently associated with breast calcification. This study combines multiple analytical techniques to investigate the heterogeneity of these calcifications at the micrometre scale. X-ray diffraction, scanning electron microscopy and Raman and Fourier-transform infrared spectroscopy were used to determine the physicochemical and crystallographic properties of type II breast calcifications located in formalin fixed paraffin embedded DCIS breast tissue samples. Multiple calcium phosphate phases were identified across the calcifications, distributed in different patterns. Hydroxyapatite was the dominant mineral, with magnesium whitlockite found at the calcification edge. Amorphous calcium phosphate and octacalcium phosphate were also identified close to the calcification edge at the apparent mineral/matrix barrier. Crystallographic features of hydroxyapatite also varied across the calcifications, with higher crystallinity centrally, and highest carbonate substitution at the calcification edge. Protein was also differentially distributed across the calcification and the surrounding soft tissue, with collagen and β-pleated protein features present to differing extents. Combination of analytical techniques in this study was essential to understand the heterogeneity of breast calcifications and how this may link crystallographic and physicochemical properties of calcifications to the surrounding tissue microenvironment.

Fig. 1. Multimodal mapping approaches for breast calcification investigation. (a) H&E stains of the calcifications investigated with regions of interest for each technique marked for SEM (white), FTIR (black), Raman (yellow) and XRD (blue). Where FTIR mineral and protein areas differ, FTIR protein ROIs are marked with a dashed black line. FTIR images show the peak intensity for the phosphate peak, Raman images show phosphate/amide ratio and XRD maps show total crystalline material present, calculated from total diffracting area. Scale bars = 150 μm. (b) table showing a summary of key histological features quantified relative to the five calcifications.

Fig. 2. Standard patterns for calcium phosphate phases and PCA loadings. (a) Standard diffractograms of HAP and WH for XRD. (b) Standard spectra of HAP, ACP, OCP and WH for Raman. (c) PCA loadings for FTIR showing WH and OCP peaks (red boxes, WH and OCP cannot be separated using FTIR analysis, therefore are presented as a single image from PCA analysis). (d) PCA loadings for COD in Raman analysis.

Fig. 3. Additional phases maps across calcifications. Maps of calcifications showing distribution of whitlockite (WH), octacalcium phosphate (OCP), amorphous calcium phosphate (ACP) and calcium oxalate dihydrate (COD). Scale bars = 150 μm.

Fig. 4. Hydroxyapatite feature patterns across calcifications. Maps of calcifications for total carbonate content (%) measured using Raman; length of the unit cell ‘a’ axis (nm) and coherence length (nm) measured using XRD; and crystallinity (cm) measured with Raman using 1/FWHM of the phosphate peak at 960 cm⁻¹. Scale bars = 150 μm.

Fig. 5. Carbonate identification. (a) and (b) Mean Raman spectra for each calcification around the phosphate peak at 960 cm⁻¹ (a) and carbonate peak at 1070 cm⁻¹ (b) used to identify mineral phases present and carbonate weight percentage. (c) Key peaks fitted in Raman analysis in the regions presented in (a) and (b). (d) Point spectra of each calcification from FTIR analysis. Single spectrum from each calcification showing the presence of B-type carbonate, evidenced by a peak at 870 cm⁻¹. The spectrum from calcification 2A does not contain this peak.

Fig. 6. Protein distribution across calcifications. (a) Maps of amide I peak intensity and k-means clusters exhibiting protein signals from FTIR and Raman analysis. Scale bars = 150 μm. (b) Mean spectra for the calcification-adjacent centroid (pink/purple) and connective tissue centroid (orange) from Raman analysis. (c) Table showing relative contributions of summed spectra for each centroid in Raman analysis. (d) Mean spectra for the centroids associated with necrosis (blue), calcification (red) and connective tissue (orange) for calcification 2A from FTIR analysis.