P2-10-01: Extracellular Matrix Stiffness and Mammographic Density in the Human Breast.

Abstract

Abstract Introduction: Mammographic density (MD) is associated with greater risk to malignancy. MD is also correlated to high collagen content in the extra cellular matrix (ECM). Data from our group and others have highlighted the importance of mechanical cues from the ECM in breast tissue homeostasis and tumor progression to invasion [1; 2; reviewed in 3]. Whether the stiffness of the ECM could also initiate breast cancer and if so how remains unknown. Because elevated collagen levels increases ECM stiffness, we hypothesize that MD increases breast cancer risk because the ECM is stiffer. Materials and Methods: We studied breast tissues obtained through prophylactic mastectomy from women with low (BIRADS 1) versus high MD (BIRADS 4). From each surgically excised breast, samples of 0.5cm x 0.5cm x 1cm dimension were removed from the retroareolar region and from 4 peripheral quadrants. Sample sections were subjected to biophysical, morphological and biochemical analysis. Biophysical analysis included the application of Atomic Force Microscopy to obtain an extensive force map of distinct anatomical regions of the ECM associated with the intra-lobular and inter-lobular ECM. Topological analysis of ECM architecture was performed using two photons and SIM-POL imaging coupled with picrosirius staining, polarized light imaging and image quantification. Biochemical and morphological analysis consisted of immunohistochemistry for markers that detect mechano-signaling in the epithelium and stromal fibroblasts, and H&E to visualize cellular and ECM organization. Results and Discussion: We found that the intra-lobular ECM associated with the terminal end-buds in the breast contained anisotropic relaxed collagen fibrils and was very compliant. By contrast, the inter-lobular ECM of the breast contained oriented collagen fibrils and was relatively stiffer. Notably, the ECM associated with the retroareolar region, which is typically detected as very dense using mammographic imaging, contained oriented collagen fibrils, and was significantly stiffer than the ECM associated with the peripheral quadrants. Intriguingly, preliminary data suggested that the ECM associated with the terminal end-buds in the upper outer quadrant showed a trend towards greater stiffness in women with high MD (BIRADS 4) than low MD (BIRADS 1). Although, it is tempting to speculate that ECM stiffness could enhance risk to malignancy, further sample analysis is now necessary. Conclusions: • In the human breast there is anatomical heterogeneity with respect to ECM organization and mechanical properties. High MD appears to reflect elevated ECM stiffness. The intra-lobular ECM is considerably stiffer in the upper outer quadrant than in the other peripheral regions of the breast. Atomic Force Microscopy is a tractable method to monitor ECM stiffness and mechanical heterogeneity in the human breast. Acknowledgements: supported by W81XWH-05-1-0330 and R01 CA138818-01A1 to VMW, 1U01 ES019458-01 to VMW and ZW, and P50 CA 58207 to JG, VW, SH and LC, U54CA143836-01 to JL and VW.