Abstract P6-02-06: A 3D tri-culture model of normal mammary gland. A tool for breast cancer initiation studies
Authors: Nash CE , Holliday DL , Mavria G , Tomlinson DC , Hanby AM , Speirs V 
Published: August 2014

The mechanisms involved in breast cancer initiation are not well understood. This may in part be due to a lack of an in vitro model that faithfully recapitulates the morphology, phenotype and in vivo architecture of the human mammary gland. Most in vitro models of normal breast have relied on the use of reconstituted basement membrane gels to induce luminal epithelial cell polarity and have neglected the role of myoepithelial cells and fibroblasts in this process. We aimed to develop a 3D in vitro culture system of normal breast which includes three of the major functional cell types embedded in a more physiologically relevant collagen 1 matrix. We used this system to investigate the mechanisms behind breast cancer initiation via genetic manipulation of some well known oncogenes and tumour suppressors involved in breast cancer progression.

Myoepithelial cells (Myo1089) and fibroblasts (generated in house) were isolated and immortalised from breast reduction mammoplasty samples collected with ethical approval. Following characterisation, these were virally transfected with Enhanced Green Fluorescent Protein (EGFP) and dsRed protein respectively to enable tracking. 3D cultures were established in collagen 1 and included the non-tumorigenic luminal epithelial cell line HB2, EGFP Myo1089 cells and dsRed fibroblasts. Cells were cultured for 3 weeks in Transwell™ cell culture inserts. Following fixation these were analysed by H&E, confocal microscopy and immunohistochemistry. Morphology and immunostaining profiles were compared to sections of normal breast tissue.

Immunohistochemical characterisation using the following antibodies: E-cadherin, epithelial membrane antigen, vimentin, laminin 5, collagen 4 plus luminal and basal cytokeratins, demonstrated polarised epithelial structures with lumen formation and basement membrane production with a similar immunostaining profile to normal breast tissue. The importance of including myoepithelial cells and fibroblasts in maintaining these structures was demonstrated. We established this model is amenable to genetic manipulation by overexpressing Her2 in HB2 cells, and knocking out ERβ1 in EGFP Myo1089 cells and Rac1 and Dock4 in dsRed fibroblast cell lines using shRNA techniques. These were included in separate models with morphological and phenotypic effects determined by confocal microscopy.

In summary we have developed an in vitro model of normal breast tissue that includes three of the major functional breast cell types cultured in a physiologically relevant 3D matrix. We have validated the morphology and protein expression profile against human breast tissue specimens and confirm that this is a suitable model of normal breast. The model is suitable for experimental manipulation and cell behaviour can be easily visualised using standard laboratory techniques. We conclude this is a robust in vitro model of normal breast tissue offering an alternative cost-effective method of studying genes involved in breast cancer initiation.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-02-06.