3D culture reveals a signaling network

The behavior of a cell is significantly influenced by its context. Epithelial cells derived from glandular organs such as the breast recreate their glandular organization when grown under 3D culture conditions. While traditional monolayer cultures are powerful tools to understand how cells proliferate, grow and respond to stress, they do not recreate the 3D property observed in vivo. Multiple studies demonstrate that 3D organization can reveal novel and unexpected insights into the mechanisms by which normal and tumorderived epithelial cells function. In the present article we comment on a study that reports identification of a RasV12-induced IL-6 signaling network in mammary epithelial cells in 3D cultures

Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

<p>Abstract</p> <p>Background</p> <p>Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D) co-culture assay.</p> <p>Methods</p> <p>Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy.</p> <p>Results</p> <p>In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors.</p> <p>Conclusion</p> <p>Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.</p

Finite proliferative lifespan in vitro of a human breast cancer cell strain isolated from a metastatic lymph node

We recently described culture conditions that allow proliferation of metastatic human breast cancer cells from biopsy specimens of certain patient samples. These conditions resulted in the development of an immortalized cell strain designated SUM-44PE. These same culture conditions were used to isolate a human breast cancer cell strain from a metastatic lymph node of a separate breast cancer patient. The SUM-16LN human breast cancer cells isolated from this specimen were cultured either in serum-free medium or serum-containing medium supplemented with insulin and hydrocortisone. Unlike the SUM-44PE cells that have proliferated in culture continuously for over two years, SUM-16LN cells proliferated in culture for approximately 200 days and underwent 15 to 20 population doublings before undergoing cell senescence. No cells of this strain proliferated beyond passage 8. SUM-16LN cells were keratin-19 positive and had an aneuploid karyotype. These cells overexpressed p53 protein and had an amplified epidermal growth factor (EGF) receptor gene that resulted in high level expression of tyrosine phosphorylated EGF receptor protein. Despite the presence of high levels of tyrosine phosphorylated EGF receptor in these cells, they proliferated in serum-free, EGF-free medium and did not secrete detectable levels of EGF-like mitogenic growth factor. In addition, these cells were potently growth inhibited by all concentrations of exogenous EGF tested and by the neutralizing EGF receptor antibody Mab 425. These results suggest that the high level of tyrosine phosphorylated EGF receptor present in these cells is the direct result of receptor overexpression and not the result of the presence of a simulatory ligand. Thus, SUM-16LN represents a human breast cancer cell strain that exhibited genetic and cellular characteristics of advanced human breast cancer cells. Nevertheless, these cells exhibited a finite proliferative lifespan in culture, suggesting that cellular immortalization is not a phenotype expressed by all human breast cancer cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44199/1/10549_2004_Article_BF00666588.pd

Adenomyoepithelial tumours and myoepithelial carcinomas of the breast – a spectrum of monophasic and biphasic tumours dominated by immature myoepithelial cells

BACKGROUND: Adenomyoepithelial tumours and myoepithelial carcinomas of the breast are primarily defined by the presence of neoplastic cells with a myoepithelial immunophenotype. Current classification schemes are based on purely descriptive features and an assessment of individual prognosis is still problematic. METHODS: A series of 27 adenomyoepithelial tumours of the breast was analysed immunohistochemically with antibodies directed against various cytokeratins, p63, smooth muscle alpha-actin (SMA) and vimentin. Additionally, double immunofluorescence and comparative genomic hybridisation (CGH) was performed. RESULTS: Immunohistochemically, all the tumours showed a constant expression of high molecular weight cytokeratins (Ck) Ck5 and Ck14, p63, SMA and vimentin. With exception of one case diagnosed as myoepithelial carcinoma, all tested tumours expressed low molecular weight cytokeratin Ck18 in variable proportions of cells. Even in monophasic tumours lacking obvious glandular differentiation in conventional staining, a number of neoplastic cells still expressed those cytokeratins. Double immunofluorescence revealed tumour cells exclusively staining for Ck5/Ck14 in the presence of other cell populations that co-expressed high molecular weight Ck5/Ck14 as well as either low molecular weight Ck8/18 or SMA. Based on morphology, we assigned the series to three categories, benign, borderline and malignant. This classification was supported by a stepwise increase in cytogenetic alterations on CGH. CONCLUSION: Adenomyoepithelial tumours comprise a spectrum of neoplasms consisting of an admixture of glandular and myoepithelial differentiation patterns. As a key component SMA-positive cells co-expressing cytokeratins could be identified. Although categorisation of adenomyoepithelial tumours in benign, borderline and malignant was supported by results of CGH, any assessment of prognosis requires to be firmly based on morphological grounds. At present it is not yet clear, if and to what extent proposed Ck5-positive progenitor cells contribute to the immunohistochemical and morphological heterogeneity of these neoplasms of the breast

Regulation of mammary gland branching morphogenesis by the extracellular matrix and its remodeling enzymes.

A considerable body of research indicates that mammary gland branching morphogenesis is dependent, in part, on the extracellular matrix (ECM), ECM-receptors, such as integrins and other ECM receptors, and ECM-degrading enzymes, including matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). There is some evidence that these ECM cues affect one or more of the following processes: cell survival, polarity, proliferation, differentiation, adhesion, and migration. Both three-dimensional culture models and genetic manipulations of the mouse mammary gland have been used to study the signaling pathways that affect these processes. However, the precise mechanisms of ECM-directed mammary morphogenesis are not well understood. Mammary morphogenesis involves epithelial 'invasion' of adipose tissue, a process akin to invasion by breast cancer cells, although the former is a highly regulated developmental process. How these morphogenic pathways are integrated in the normal gland and how they become dysregulated and subverted in the progression of breast cancer also remain largely unanswered questions

Prognostic Breast Cancer Signature Identified from 3D Culture Model Accurately Predicts Clinical Outcome across Independent Datasets

One of the major tenets in breast cancer research is that early detection is vital for patient survival by increasing treatment options. To that end, we have previously used a novel unsupervised approach to identify a set of genes whose expression predicts prognosis of breast cancer patients. The predictive genes were selected in a well-defined three dimensional (3D) cell culture model of non-malignant human mammary epithelial cell morphogenesis as down-regulated during breast epithelial cell acinar formation and cell cycle arrest. Here we examine the ability of this gene signature (3D-signature) to predict prognosis in three independent breast cancer microarray datasets having 295, 286, and 118 samples, respectively. Our results show that the 3D-signature accurately predicts prognosis in three unrelated patient datasets. At 10 years, the probability of positive outcome was 52, 51, and 47 percent in the group with a poor-prognosis signature and 91, 75, and 71 percent in the group with a good-prognosis signature for the three datasets, respectively (Kaplan-Meier survival analysis, p&lt;0.05). Hazard ratios for poor outcome were 5.5 (95% CI 3.0 to 12.2, p&lt;0.0001), 2.4 (95% CI 1.6 to 3.6, p&lt;0.0001) and 1.9 (95% CI 1.1 to 3.2, p = 0.016) and remained significant for the two larger datasets when corrected for estrogen receptor (ER) status. Hence the 3D-signature accurately predicts breast cancer outcome in both ER-positive and ER-negative tumors, though individual genes differed in their prognostic ability in the two subtypes. Genes that were prognostic in ER+ patients are AURKA, CEP55, RRM2, EPHA2, FGFBP1, and VRK1, while genes prognostic in ER patients include ACTB, FOXM1 and SERPINE2 (Kaplan-Meier p&lt;0.05). Multivariable Cox regression analysis in the largest dataset showed that the 3D-signature was a strong independent factor in predicting breast cancer outcome. The 3D-signature accurately predicts breast cancer outcome across multiple datasets and holds prognostic value for both ER-positive and ER-negative breast cancer. The signature was selected using a novel biological approach and hence holds promise to represent the key biological processes of breast cancer

Derivation of Myoepithelial Progenitor Cells from Bipotent Mammary Stem/Progenitor Cells

There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Recent molecular profiling has identified six major subtypes of breast cancer: basal-like, ErbB2-overexpressing, normal breast epithelial-like, luminal A and B, and claudin-low subtypes. To help understand the relationship among mammary stem/progenitor cells and breast cancer subtypes, we have recently derived distinct hTERT-immortalized human mammary stem/progenitor cell lines: a K5+/K19− type, and a K5+/K19+ type. Under specific culture conditions, bipotent K5+/K19− stem/progenitor cells differentiated into stable clonal populations that were K5−/K19− and exhibit self-renewal and unipotent myoepithelial differentiation potential in contrast to the parental K5+/K19− cells which are bipotent. These K5−/K19− cells function as myoepithelial progenitor cells and constitutively express markers of an epithelial to mesenchymal transition (EMT) and show high invasive and migratory abilities. In addition, these cells express a microarray signature of claudin-low breast cancers. The EMT characteristics of an un-transformed unipotent mammary myoepithelial progenitor cells together with claudin-low signature suggests that the claudin-low breast cancer subtype may arise from myoepithelial lineage committed progenitors. Availability of immortal MPCs should allow a more definitive analysis of their potential to give rise to claudin-low breast cancer subtype and facilitate biological and molecular/biochemical studies of this disease