title: Increased peri-ductal collagen micro-organization may contribute to raised mammographic density.
abstract: BACKGROUND
  High 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.
  
  
  METHODS
  Mammographic 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.
  
  
  RESULTS
  Mammographic 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.
  
  
  CONCLUSIONS
  These 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.
authors.affiliation: Centre for Tissue Injury & Repair, Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK. james.mcconnell@manchester.ac.uk.
authors.forename: James C
authors.initials: JC
authors.lastname: McConnell
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research and Manchester Breast Centre, Faculty of Life Sciences, University of Manchester, Manchester, UK. oliver.oconnell@postgrad.manchester.ac.uk.
authors.forename: Oliver V
authors.initials: OV
authors.lastname: O'Connell
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research and Manchester Breast Centre, Faculty of Life Sciences, University of Manchester, Manchester, UK. keith.brennan@manchester.ac.uk.
authors.forename: Keith
authors.initials: K
authors.lastname: Brennan
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research and Manchester Breast Centre, Faculty of Life Sciences, University of Manchester, Manchester, UK. weiping.li-2@manchester.ac.uk.
authors.forename: Lisa
authors.initials: L
authors.lastname: Weiping
authors.affiliation: University Hospital of South Manchester, Manchester, UK. miles.howe@uhsm.nhs.uk.
authors.forename: Miles
authors.initials: M
authors.lastname: Howe
authors.affiliation: University Hospital of South Manchester, Manchester, UK. leena.joseph@uhsm.nhs.uk.
authors.forename: Leena
authors.initials: L
authors.lastname: Joseph
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research and Manchester Breast Centre, Faculty of Life Sciences, University of Manchester, Manchester, UK. david.knight@manchester.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Knight
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research and Manchester Breast Centre, Faculty of Life Sciences, University of Manchester, Manchester, UK. ronan.o'cualain@manchester.ac.uk.
authors.forename: Ronan
authors.initials: R
authors.lastname: O'Cualain
authors.affiliation: University Hospital of South Manchester, Manchester, UK. yit.lim@uhsm.nhs.uk.
authors.forename: Yit
authors.initials: Y
authors.lastname: Lim
authors.affiliation: Manchester Cancer Research Centre Tissue Biobank, University of Manchester, Manchester, UK. angela.leek@uhsm.nhs.uk.
authors.forename: Angela
authors.initials: A
authors.lastname: Leek
authors.affiliation: Manchester Cancer Research Centre Tissue Biobank, University of Manchester, Manchester, UK. rachael.waddington@uhsm.nhs.uk.
authors.forename: Rachael
authors.initials: R
authors.lastname: Waddington
authors.affiliation: Manchester Cancer Research Centre Tissue Biobank, University of Manchester, Manchester, UK. jane.rogan@christie.nhs.uk.
authors.forename: Jane
authors.initials: J
authors.lastname: Rogan
authors.affiliation: Centre for Imaging Sciences, Institute of Population Health, Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK. sue.astley@manchester.ac.uk.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Astley
authors.affiliation: University Hospital of South Manchester, Manchester, UK. ashu.gandhi@uhsm.nhs.uk.
authors.forename: Ashu
authors.initials: A
authors.lastname: Gandhi
authors.affiliation: Institute of Cancer Sciences, Manchester Academic Health Sciences Centre, University Hospital of South Manchester, University of Manchester, Manchester, UK. cliona.kirwan@manchester.ac.uk.
authors.forename: Cliona C
authors.initials: CC
authors.lastname: Kirwan
authors.affiliation: Centre for Tissue Injury & Repair, Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK. michael.sherratt@manchester.ac.uk.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sherratt
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research and Manchester Breast Centre, Faculty of Life Sciences, University of Manchester, Manchester, UK. cstreuli@manchester.ac.uk.
authors.forename: Charles H
authors.initials: CH
authors.lastname: Streuli
mesh_terms: D000368:Aged; D000818:Animals; D000071060:Breast Density; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D015815:Cell Adhesion Molecules / Q000378:metabolism; D003094:Collagen / Q000378:metabolism* / Q000648:ultrastructure; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D042361:Mammary Glands, Human / Q000002:abnormalities*; D008327:Mammography / Q000379:methods*; D018625:Microscopy, Atomic Force; D008875:Middle Aged; D040901:Proteomics; D051381:Rats; D012307:Risk Factors
chemical_list: C481130:COL16A1 protein, human; D015815:Cell Adhesion Molecules; D016326:Extracellular Matrix Proteins; C512835:POSTN protein, human; D003094:Collagen