Novel multicellular organotypic models of normal and malignant breast: tools for dissecting the role of the microenvironment in breast cancer progression

Abstract

Introduction: There is increasing recognition of the role of the microenvironment in the control of both normal and tumour cell behaviour. In the breast, myoepithelial cells and fibroblasts can influence tumour cell behaviour, with myoepithelial cells exhibiting a broad tumour-suppressor activity while fibroblasts frequently promote tumour growth and invasion. This study describes the development of physiologically relevant three-dimensional heterotypic culture systems containing mixed normal or tumour-derived breast populations and shows how such models can be used to dissect the interactions that influence cell behaviour.

Methods: Populations of luminal cells, myoepithelial cells and fibroblasts were isolated from normal and malignant breast tissue, characterised and compared with immortalised cell lines. Co-localisation of normal and malignant luminal cells with myoepithelial cells alone or with either normal or tumour-derived fibroblasts was studied. Cultures were grown for seven days, and then gels were fixed and whole gel immunofluorescence carried out to assess co-localisation and polarisation. The potential role of matrix metalloproteinases (MMP) or hepatocyte growth factor(HGF)-c-met signalling in disrupting cellular organisation was investigated by incorporating inhibitors into cultures either alone or in combination.

Results: Over a culture period of seven days, myoepithelial cells organised themselves around luminal cell populations forming dual-cell co-units. Characterisation of co-units showed established basal polarity and differentiation analogous to their in vivo counterparts. Tumour cell co-units revealed subtle differences to normal co-units including disruption of basement membrane and loss of beta4-integrin, as described in ductal carcinoma in situ (DCIS) in vivo. Inclusion of normal fibroblasts had no influence on co-unit formation; however, inclusion of tumour-associated fibroblasts lead to disruption of co-unit organisation, and this was significantly inhibited in the presence of MMP and/or c-met inhibitors.

Conclusions: To the best of the authors' knowledge, this study describes for the first time a co-culture model comprising three major components of normal and malignant breast: luminal cells, myoepithelial cells and stromal fibroblasts. These cells organise into structures recapitulating normal and DCIS breast, with homing of myoepithelial cells around the luminal population. Importantly, differences are exhibited between these systems reflecting those described in tissues, including a central role for tumour-associated fibroblasts and MMPs in mediating disruption of normal structures. These findings support the value of these models in dissecting normal and tumour cell behaviour in an appropriate microenvironment.

Figure 1

Characterisation of cell populations. The purity and phenotypic integrity of isolated primary and immortalised cell populations was confirmed by immunofluorescence. (a) Primary myoepithelial cells expressed of CK-14, CK-17, CK5/6, desmoglein (Dsg) 2, Dsg3, β4-integrin, vimentin, E-cadherin (E-Cad) and weak expression of α-smooth muscle actin (SMA). No expression of the luminal associated epithelial membrane antigen (EMA) and CK-18 was observed. (b) Isolated luminal cells expressed EMA and CK-18 along with expression of Dsg-2 and E-cadherin. (c) Isolated fibroblasts expression vimentin and a proportion of the cells expressed α-SMA but there was no expression of epithelial, macrophage of endothelial associated markers. The immortalised cell lines HB4a (luminal), MYO1089 (myoepithelial), HMFU19 (normal fibroblast) and hfff2 (fetal fibroblast) exhibited identical patterns of marker expression in comparison to their primary counterparts (data not shown).

Figure 2

Generation of epithelial-myoepithelial co-units. Cell populations were cultured in collagen gels for seven days with preaggregation of the luminal cell population. (a) (a)i MCF-7 cells alone formed tight spheres whereas (a)ii luminal cells formed loosely cohesive structures. Co-culture of (a)iii primary luminal/myoepithelial cells and (a)iv MCF-7/myoepithelial cells showed organisation of the myoepithelial cells (cell trackered in red) around the luminal population (nuclear labelling blue with 4',6-diamidino-2-phenylindole (DAPI)) forming a dual-cell co-unit. Co-unit formation was shown to be myoepithelial specific by the lack of organisation of (a)v luminal cells (cell trackered red) around pre-aggregated MCF-7 cells. A time course to show co-unit formation from cells without prior preaggregation was performed. (b) At (b)i,ii day 1 and (b)iii,iv day 3 MCF-7 cells and primary myoepithelial cells were intermingled. At (b)v,vi day 5 cells began to aggregate and by (b)vii,viii day 7 co-units were formed suggesting that organisation of co-units is through an active homing process.

Figure 3

Phenotypic analysis of co-units. (a) Co-units formed by primary myoepithelial cells (red) surrounding the normal luminal epithelial cells exhibited polarity detected by luminal expression of epithelial membrane antigen (EMA) localised to (a)i, green stain: the epithelial population. (a)ii, green stain: E-cadherin was expressed at cell-cell junctions. (a)iii, green stain: Myoepithelial cells laid down the matrix protein tenascin-C (TN-C) and (a)iv, green stain: expressed β4-integrin in hemidesmosome like structures at the myoepithelial-gel interface. (b) In the myoepithelial/MCF-7 co-units, (b)i, green stain: MCF-7 tumour cells expressed EMA and (b)ii, green stain: E-cadherin. A less organised basement membrane was present, (b)iii, green stain: shown by TN-C staining and (b)iv, green stain: marked down-regulation or loss of β4-integrin expression was observed. Quantitation of co-unit formation was carried by counting the number of co-units in 10 microscopic fields per culture to give an average number of co-units/field. A co-unit was defined as aggregated luminal or MCF-7 cells which were at least 70% enclosed by myoepithelial cells. (c) No significant difference in the two types of co-units was observed. To quantitate the presence of β4-integrin and TN-C the percentage of co-units expressing the proteins were counted and expressed as a percentage of total co-units. (d) A significant decrease in the levels of β4 integrin and TN-C were observed in the MCF-7-myoepithelial cell cultures compared with the luminal-myoepithelial cell cultures. (p = 0.001).

Figure 4

Effect of fibroblasts on co-unit structure. Incorporation of normal breast fibroblasts into myoepithelial/MCF-7 cultures. Non pre-aggregated cells were mixed and cultured in a collagen gel for up to one week to establish if fibroblasts influence epithelial co-unit formation. (a) (a)i,ii: At day 1, MCF-7 cells, myoepithelial cells (cell trackered red) and fibroblasts (cell trackered green) were intermingled. (a)iii,iv: By day 3, MCF-7 clusters were beginning to form and (a)v,vi these were more prominent by day 5. (a)vii,viii: Co-units were formed by day 7 with normal fibroblasts located around the epithelial structures (low power image). Incorporation of tumour-associated fibroblast (TAF) into myoepithelial/MCF-7 into 7 day co-cultures. (b) (b)i,ii: The homing of myoepithelial cells to MCF-7 cells is disrupted on addition of TAFs, with the cells reverting to an intermingled mixed population. (b)iii: The effect on the culture is particularly evident at low-power magnification. (c) Quantitation of co-units revealed a decrease from 7.5 to 1.2 in the presence of TAFs compared with normal fibroblasts (p = 0.001). (d) These effects could be replicated in cultures grown for seven days in the presence of conditioned medium from the fibroblast populations with disruption of co-unit architecture (d)ii in the presence of TAF conditioning media but not (d)i normal fibroblast conditioning media.

Figure 5

Effect of inhibitors on fibroblast-induced co-unit disruption. (a) Cultures were generated with MCF-7 cells, myoepithelial cells and tumour-associated fibroblasts (TAFs) without prior aggregation, and grown in the (a)i,ii presence of dimethyl sulfoxide (DMSO) (10 μM), a specific inhibitor for c-met, (a)iii,iv the receptor for HGF, (PHA 665752, 100 nM), (a)iv,vii a broad spectrum matrix metalloproteinase (MMP) inhibitor (10 μM) individually, or (a)vii,viii in combination. Inhibitors were replenished at two-day intervals and the cultures harvested at seven days. (b) In the vehicle control cultures exhibited an average of two co-units per microscopic field. This was significantly increased in the presence of the c-met inhibitor (average co-units 4.6, p = 0.016), the MMP inhibitor (average co-units 7, p ≤ 0.001) and the inhibitors in combination (average co-units 7.5, p = 0.001).