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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.

SUBMITTER: Holliday DL

PROVIDER: S-EPMC2687706 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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

Holliday Deborah L DL Brouilette Kellie T KT Markert Anja A Gordon Linda A LA Jones J Louise JL

Breast cancer research : BCR 20090119 1

<h4>Introduction</h4>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 no ...[more]

PMID: 19152687

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Project description:During epithelial ovarian cancer (EOC) progression, intraperitoneally disseminating tumor cells and multicellular aggregates (MCAs) present in ascites fluid adhere to the peritoneum and induce retraction of the peritoneal mesothelial monolayer prior to invasion of the collagen-rich submesothelial matrix and proliferation into macro-metastases. Clinical studies have shown heterogeneity among EOC metastatic units with respect to cadherin expression profiles and invasive behavior; however, the impact of distinct cadherin profiles on peritoneal anchoring of metastatic lesions remains poorly understood. In the current study, we demonstrate that metastasis-associated behaviors of ovarian cancer cells and MCAs are influenced by cellular cadherin composition. Our results show that mesenchymal N-cadherin-expressing (Ncad+) cells and MCAs invade much more efficiently than E-cadherin-expressing (Ecad+) cells. Ncad+ MCAs exhibit rapid lateral dispersal prior to penetration of three-dimensional collagen matrices. When seeded as individual cells, lateral migration and cell-cell junction formation precede matrix invasion. Neutralizing the Ncad extracellular domain with the monoclonal antibody GC-4 suppresses lateral dispersal and cell penetration of collagen gels. In contrast, use of a broad-spectrum matrix metalloproteinase (MMP) inhibitor (GM6001) to block endogenous membrane type 1 matrix metalloproteinase (MT1-MMP) activity does not fully inhibit cell invasion. Using intact tissue explants, Ncad+ MCAs were also shown to efficiently rupture peritoneal mesothelial cells, exposing the submesothelial collagen matrix. Acquisition of Ncad by Ecad+ cells increased mesothelial clearance activity but was not sufficient to induce matrix invasion. Furthermore, co-culture of Ncad+ with Ecad+ cells did not promote a 'leader-follower' mode of collective cell invasion, demonstrating that matrix remodeling and creation of invasive micro-tracks are not sufficient for cell penetration of collagen matrices in the absence of Ncad. Collectively, our data emphasize the role of Ncad in intraperitoneal seeding of EOC and provide the rationale for future studies targeting Ncad in preclinical models of EOC metastasis.

Dataset Information

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

Publications

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

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