BackgroundHigh mammographic density is a therapeutically modifiable risk factor for breast cancer. Although mammographic density is correlated with the relative abundance of collagen-rich fibroglandular tissue, the causative mechanisms, associated structural remodelling and mechanical consequences remain poorly defined. In this study we have developed a new collaborative bedside-to-bench workflow to determine the relationship between mammographic density, collagen abundance and alignment, tissue stiffness and the expression of extracellular matrix organising proteins.MethodsMammographic density was assessed in 22 post-menopausal women (aged 54–66 y). A radiologist and a pathologist identified and excised regions of elevated non-cancerous X-ray density prior to laboratory characterization. Collagen abundance was determined by both Masson’s trichrome and Picrosirius red staining (which enhances collagen birefringence when viewed under polarised light). The structural specificity of these collagen visualisation methods was determined by comparing the relative birefringence and ultrastructure (visualised by atomic force microscopy) of unaligned collagen I fibrils in reconstituted gels with the highly aligned collagen fibrils in rat tail tendon. Localised collagen fibril organisation and stiffness was also evaluated in tissue sections by atomic force microscopy/spectroscopy and the abundance of key extracellular proteins was assessed using mass spectrometry.ResultsMammographic density was positively correlated with the abundance of aligned periductal fibrils rather than with the abundance of amorphous collagen. Compared with matched tissue resected from the breasts of low mammographic density patients, the highly birefringent tissue in mammographically dense breasts was both significantly stiffer and characterised by large (>80 μm long) fibrillar collagen bundles. Subsequent proteomic analyses not only confirmed the absence of collagen fibrosis in high mammographic density tissue, but additionally identified the up-regulation of periostin and collagen XVI (regulators of collagen fibril structure and architecture) as potential mediators of localised mechanical stiffness.ConclusionsThese preliminary data suggest that remodelling, and hence stiffening, of the existing stromal collagen microarchitecture promotes high mammographic density within the breast. In turn, this aberrant mechanical environment may trigger neoplasia-associated mechanotransduction pathways within the epithelial cell population.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0664-2) contains supplementary material, which is available to authorized users.

Introduction High mammographic density (MD) in women is strongly associated with breast cancer risk. However the structural and compositional differences between dense and non-dense breast tissues are not well defined. We determined the relationship between MD, collagen deposition and fibril alignment, and tissue micro-stiffness, in similarly aged individuals without adjacent cancer. Methods Fresh tissue samples were collected from post-menopausal women undergoing breast screening. Collagen deposition and fibril organisation were analysed using light microscopy of wax-sections stained with H&E, Trichrome or Picrosirius Red (with polarising light), and quantified using ImageJ. Local tissue stiffness was measured using atomic force microscopy (AFM) of hydrated tissue. For AFM, 3 x 25 µm2 areas of each sample were indented at 400 equally spaced points with a 1 µm diameter spherical probe at a loading rate of 1 Hz (Reduced Modulus; YM). Results Volumetric MD (VolparaTM) was determined in 22 women (54-66y) undergoing risk-reducing surgery or mastectomy. Localised regions of elevated density, determined from digital mammograms, were isolated from patients of low and high overall MD, using a new collaborative workflow linking radiologist, surgeon, pathologist, and tissue biobank. All elevated-density regions contained considerable amounts of stromal connective tissue. However, there were significant differences in these regions from women with low vs high overall MD. Picrosirius Red staining of the localised areas of density revealed that the percentage organised fibrillar collagen content, particularly in the periductal breast stroma, strongly correlated with overall MD. AFM showed that the localised micro-stiffness of dense areas increased significantly in the breast stroma of patients with high overall MD (Volpara score > 15) compared with those of low overall MD (Volpara score < 5). Conclusions High MD is a significant risk factor for breast cancer, yet its molecular determinants in the normal, non-cancerous breast are poorly defined. We have shown that high MD is associated with more organised fibrillar collagen, leading to increased stiffness of the periductal breast stroma. Women with low and high MD all have regions with localised density, which contain both stromal connective tissue and epithelial ducts/lobules. However, our results show that these localised areas have differences in collagen organisation and tissue micro-mechanics. We now hypothesise that in the connective tissue of women with high MD, altered synthesis, deposition and turnover of stromal proteins alters the local biomechanical properties within the breast, providing a stiffer microenvironment, and contributing to cancer onset. Citation Format: Ashu Gandhi, Cliona C Kirwan, James C McConnell, Oliver V O'Connell, Michael J Sherratt, Charles H Streuli. High mammographic density is associated with deposition of organised fibrillar collagen and increased stiffness in periductal breast stroma [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-14.