Spatial Proteomics of the Normal Breast Collagen Stroma: Links to Density and Body Mass Index

Collagen breast stroma can become a breast cancer risk factor, yet proteomic regulation of normal breast stroma remains poorly defined. This study evaluates the spatial regulation of the collagen proteome from normal breast tissue. Normal breast tissue sections from the Susan G. Komen tissue bank were used (n = 40), with data including genetic ancestry (n = 20 African ancestry; n = 20 European ancestry), body-mass-index (BMI), age, and mammogram density by the Breast Imaging Reporting and Data System (BI-RADS). 10-plex cell marker staining showed CD44 and COL1A1 markers modulated with BMI. Collagen fiber widths by second harmonic generation microscopy contrasted in BMI categories by genetic ancestry. Targeted extracellular matrix proteomics mass spectrometry imaging showed the collagen alpha-1(I) chain proteome was spatially heterogeneous across the normal breast microenvironment with site-specific post-translational modification of proline hydroxylation. Signatures computationally extracted from stroma-rich regions reported that 47 collagen peptides distinguished BI-RADS categories (area under the receiver operating curve >0.7; p-value >0.05). Multivariate modeling of collagen peptides, fiber metrics, and clinical features supported a strong positive association with BMI as a determinant of collagen alterations in the normal breast. This study provides a foundation for larger studies investigating the clinical value of spatial collagen proteome alterations in human breast.