Development and characterisation of a 3D multi-cellular in vitro model of normal human breast: a tool for cancer initiation studies

Abstract

Multicellular 3-dimensional (3D) in vitro models of normal human breast tissue to study cancer initiation are required. We present a model incorporating three of the major functional cell types of breast, detail the phenotype and document our breast cancer initiation studies. Myoepithelial cells and fibroblasts were isolated and immortalised from breast reduction mammoplasty samples. Tri-cultures containing non-tumorigenic luminal epithelial cells HB2, or HB2 overexpressing different HER proteins, together with myoepithelial cells and fibroblasts were established in collagen I. Phenotype was assessed morphologically and immunohistochemically and compared to normal breast tissue. When all three cell types were present, polarised epithelial structures with lumens and basement membrane production were observed, akin to normal human breast tissue. Overexpression of HER2 or HER2/3 caused a significant increase in size, while HER2 overexpression resulted in development of a DCIS-like phenotype. In summary, we have developed a 3D tri-cellular model of normal human breast, amenable to comparative analysis after genetic manipulation and with potential to dissect the mechanisms behind the early stages of breast cancer initiation.

Keywords: 3D cell culture; HER2; breast.

Figure 1. Comparison of morphology of 3D tri-culture model with normal human breast tissue

When cultured alone (a, b), HB2 cells formed tight spherical units of varying size with occasional scattered cells. A similar phenotype was observed when HB2 were co-cultured with Myo1089 cells (c, d). In tri-cultures containing HB2, Myo1089 and fibroblasts, ductal structures with hollow lumens (dotted arrow) were observed surrounded by a border of clear myoepithelial cells (arrow; e) akin to normal human breast acini (f). Immunohistochemical characterisation using tGFP and dsRed antibodies confirmed the distribution of myoepithelial cells at the outer edges of HB2 structures with hollow lumens (dotted arrow) (arrows; g) and fibroblasts loosely distributed throughout collagen gels (arrows; h), respectively. Scale bars = 100 μm.

Figure 2. Comparison of immunohistochemical profiles of 3D tri-cultures with those of normal breast tissue

The phenotype of 3D in vitro tri-cultures (a-d; i-l) was assessed by immunohistochemistry and compared to cross sections of breast reduction mammoplasty tissue (e-h; m-p). Negative controls (a, e). E-cadherin was expressed by HB2 epithelial cells at cell-cell junctions (b, arrow) similar to luminal epithelium of breast acini (f, arrow). EMA expression was concentrated at the HB2 cell-lumen interface in co-units (c) and was specific to apical membranes of luminal epithelium of breast acini (g, arrow). The majority of cells within HB2 co-units expressed Ki67 (d) with scattered positivity within breast acini (h). M30 staining demonstrated apoptosis within lumens of HB2 co-units (i, arrow) and also in breast acini (m, arrow). α-SMA was restricted to myoepithelial cells on the periphery of HB2 co-units (j, arrow) while a continuous layer of α-SMA positive myoepithelial cells surround luminal epithelium of breast acini (n, arrow). Collagen IV was concentrated around outside edges of HB2 co-units (k, arrow) and similarly is demonstrated encapsulating breast acini (o, arrow). Vimentin was expressed in both Myo1089 cells (M) and fibroblasts (F) in the tri-culture model (l) analogous to Vimentin positivity in myoepithelial cells (M) and fibroblasts (F) in normal breast tissue (p). Original magnification for tri-culture model = 20x; for normal tissue = 40x.

Figure 3. Overexpression of HER proteins in HB2 cells affects 3D architecture

Stable overexpression of HER2 and HER3 in HB2 cells was shown by western blot (a). WT cells in 3D tri-culture (b) formed rounded units (RU) with lumens (L). When HER2 was overexpressed (c), formation of much larger elongated units (EU) with distorted lumens (DL) and protrusions (PRO) from edges was observed. HER3 overexpression (d) resulted in rounded units with distorted lumens (DL) while overexpression of HER2 and HER3 cells (e) resulted in a combination of rounded units (RU) and elongated units (EU) with distorted lumens (DL). Quantitative analysis confirmed HER2 and HER2/3 overexpression induced significantly larger HB2 unit formation (f). Only HER3 overexpression alone significantly altered the number of HB2 units formed (g) while HER2, HER3 and HER2/3 overexpression significantly reduced lumen formation (h). Bars denote mean from three gel replicates and error bars standard error of the mean (SEM). * = p < 0.05.

Figure 4. Comparison of HER2 overexpressing tri-cultures to human breast tissue containing DCIS

A cross section of human breast tissue (a) shows normal acini which are small and rounded with cleared central lumens (L, inset). Areas of DCIS consist of elongated discohesive cells (DC) with distorted lumens containing necrotic cells (DL). Co-units of the tri-culture model are also small and rounded with central lumens (L), strongly resembling normal breast acini. Overexpression of HER2 in luminal epithelium (c) resulted in an elongated, branched shape, the appearance of distorted lumen with possible necrosis (DL) and also discohesive cells (DC), reminiscent of a DCIS-like phenotype. Original magnification x20 (tri-culture model); 40x (breast tissue).