An Evaluation of Matrix-Containing and Humanised Matrix-Free 3-Dimensional Cell Culture Systems for Studying Breast Cancer.

BACKGROUND: 3D cell cultures are emerging as more physiologically meaningful alternatives to monolayer cultures for many biological applications. They are attractive because they more closely mimic in vivo morphology, especially when co-cultured with stromal fibroblasts. METHODOLOGY/PRINCIPAL FINDINGS: We compared the efficacy of 3 different 3D cell culture systems; collagen I, low attachment culture vessels and a modification of Fibrolife®, a specialised humanised cell culture medium devoid of animal-derived components, using breast cancer cell lines representative of the different molecular subtypes of breast cancer, cultured alone or with human mammary fibroblasts with a view to developing matrix-free humanised systems. 3D collagen I culture supported the growth of a range of breast cancer cell lines. By modifying the composition of Fibrolife® to epiFL, matrix-free cell culture was possible. During sequential transfer to epiFL breast cancer cells gradually detached from the flask, growing progressively as spheroids. Phenotype was stable and reversible with cells remaining actively proliferating and easily accessible throughout culture. They could also be revived from frozen stocks. To achieve co-culture with fibroblasts in epiFL required use of low attachment culture vessels instead of standard plastic as fibroblasts remained adherent in epiFL. Here, cancer cell spheroids were allowed to form before adding fibroblasts. Immunohistochemical examination showed fibroblasts scattered throughout the epithelial spheroid, not dissimilar to the relationship of tumour stroma in human breast cancer. CONCLUSIONS: Because of its ease of handling, matrix-free 3D cell culture may be a useful model to study the influence of fibroblasts on breast cancer epithelial cells with use of epiFL culture medium taking this a step further towards a fully humanised 3D model. This methodology could be applied to other types of cancer cell lines, making this a versatile technique for cancer researchers wishing to use in vitro systems that better reflect cancer in vivo.

Down-Regulation of miR-92 in Breast Epithelial Cells and in Normal but Not Tumour Fibroblasts Contributes to Breast Carcinogenesis.

BACKGROUND: MicroRNA (miR) expression is commonly dysregulated in many cancers, including breast. MiR-92 is one of six miRs encoded by the miR-17-92 cluster, one of the best-characterised oncogenic miR clusters. We examined expression of miR-92 in the breast epithelium and stroma during breast cancer progression. We also investigated the role of miR-92 in fibroblasts in vitro and showed that down-regulation in normal fibroblasts enhances the invasion of breast cancer epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: We used laser microdissection (LMD) to isolate epithelial cells from matched normal, DCIS and invasive tissue from 9 breast cancer patients and analysed miR-92 expression by qRT-PCR. Expression of ERß1, a direct miR-92 target, was concurrently analysed for each case by immunohistochemistry. LMD was also used to isolate matched normal (NFs) and cancer-associated fibroblasts (CAFs) from 14 further cases. Effects of miR-92 inhibition in fibroblasts on epithelial cell invasion in vitro was examined using a Matrigel™ assay. miR-92 levels decreased in microdissected epithelial cells during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p<0.01) and being lowest in invasive breast tissue (p<0.01). This was accompanied by a shift in cell localisation of ERß1 from nuclear expression in normal breast epithelium to increased cytoplasmic expression during progression to DCIS (p = 0.0078) and invasive breast cancer (p = 0.031). ERß1 immunoreactivity was also seen in stromal fibroblasts in tissues. Where miR-92 expression was low in microdissected NFs this increased in matched CAFs; a trend also seen in cultured primary fibroblasts. Down-regulation of miR-92 levels in NFs but not CAFs enhanced invasion of both MCF-7 and MDA-MB-231 breast cancer epithelial cells. CONCLUSIONS: miR-92 is gradually lost in breast epithelial cells during cancer progression correlating with a shift in ERß1 immunoreactivity from nuclei to the cytoplasm. Our data support a functional role in fibroblasts where modification of miR-92 expression can influence the invasive capacity of breast cancer epithelial cells. However in silico analysis suggests that ERß1 may not be the most important miR-92 target in breast cancer.

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

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.

3-D tissue modelling and virtual pathology as new approaches to study ductal carcinoma in situ.

Widespread screening mammography programmes mean that ductal carcinoma in situ (DCIS), a pre-invasive breast lesion, is now more frequently diagnosed. However, not all diagnosed DCIS lesions progress to invasive breast cancer, which presents a dilemma for clinicians. As such, there is much interest in studying DCIS in the laboratory, in order to help understand more about its biology and determine the characteristics of those that progress to invasion. Greater knowledge would lead to targeted and better DCIS treatment. Here, we outline some of the models available to study DCIS, with a particular focus on animal-free systems.