Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures
TL;DR: A collection of protocols to culture MCF-10A cells, to establish stable pools expressing a gene of interest via retroviral infection, as well as to grow and analyzeMCF- 10A cells in three-dimensional basement membrane culture are provided.
About: This article is published in Methods.The article was published on 2003-07-01. It has received 1957 citations till now. The article focuses on the topics: Cell culture & Basement membrane.
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TL;DR: It is found that tumors are rigid because they have a stiff stroma and elevated Rho-dependent cytoskeletal tension that drives focal adhesions, disrupts adherens junctions, perturbs tissue polarity, enhances growth, and hinders lumen formation.
3,553 citations
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...To address this, we used BM-crosslinked polyacrylamide (PA) gels (BM gel) with calibrated elastic moduli of 150, 400, 675, 1050, and R5000 Pa. Similar to MECs inside a 3D BM (3D BM; Weaver et al., 2002) or on top of a thick BM overlaid with BM (3D over BM; Debnath et al., 2003), by 16–20 days MECs on a compliant PA BM gel overlaid with BM (3D BM gel) formed growtharrested acini with cell-cell localized β-catenin, basally polarized β4 integrin, and apical-lateral cortical actin and assembled an endogenous LN-5 BM (Figure 2C)....
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...C: Phase contrast microscopy and confocal IF images of MEC colonies on 3D BM gels of increasing stiffness (150–5000 Pa), showing colony morphology after 20 days (top); β-catenin before (large image) and after triton extraction (inset; see also Supplemental Data) (green), costained with β4 integrin (large image) or E-cadherin (inset) (red; middle); and actin (green), costained with LN-5 (BM; red; bottom) and nuclei (blue)....
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...The HMT3522 and MCF10A MECs and NIH 3T3 cells were maintained as described (Debnath et al., 2003; Wang et al., 1998; Yeung et al., 2005)....
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...D: Confocal IF images of β1 integrin adhesions (green) in MECs in BM/COL I gels of 175 and 1200 Pa for 16 days (3D BM; soft versus stiff), or 70% confluent monolayers on BM-coated polystyrene (2D stiff), costained for FAKpY397, FAKpY861, vinculin (red), or actin (red) and nuclei (blue)....
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...…150, 400, 675, 1050, and R5000 Pa. Similar to MECs inside a 3D BM (3D BM; Weaver et al., 2002) or on top of a thick BM overlaid with BM (3D over BM; Debnath et al., 2003), by 16–20 days MECs on a compliant PA BM gel overlaid with BM (3D BM gel) formed growtharrested acini with cell-cell localized…...
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TL;DR: It is believed that 3D cultures will have a strong impact on drug screening and will also decrease the use of laboratory animals, for example, in the context of toxicity assays.
Abstract: Cell monolayers have serious limitations for cell biological investigations and for cell-based assays in drug screening and toxicity studies. However, the establishment of three-dimensional cultures as a mainstream approach requires the development of reliable protocols, new cell lines and suitable imaging techniques.
2,413 citations
Cites background from "Morphogenesis and oncogenesis of MC..."
...) is still lacking, despite the recent publication of detailed protocol...
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TL;DR: TEAD is revealed as a new component in the Hippo pathway playing essential roles in mediating biological functions of YAP, and is required for YAP-induced cell growth, oncogenic transformation, and epithelial-mesenchymal transition.
Abstract: The YAP transcription coactivator has been implicated as an oncogene and is amplified in human cancers. Recent studies have established that YAP is phosphorylated and inhibited by the Hippo tumor suppressor pathway. Here we demonstrate that the TEAD family transcription factors are essential in mediating YAP-dependent gene expression. TEAD is also required for YAP-induced cell growth, oncogenic transformation, and epithelial-mesenchymal transition. CTGF is identified as a direct YAP target gene important for cell growth. Moreover, the functional relationship between YAP and TEAD is conserved in Drosophila Yki (the YAP homolog) and Scalloped (the TEAD homolog). Our study reveals TEAD as a new component in the Hippo pathway playing essential roles in mediating biological functions of YAP.
2,003 citations
Cites methods from "Morphogenesis and oncogenesis of MC..."
...Three-dimensional culture of MCF10A cells The 3D culture of MCF10A cells was done as described (Debnath et al. 2003)....
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TL;DR: It is shown that a single cell, marked with a LacZ transgene, can reconstitute a complete mammary gland in vivo and establish that single cells within the Lin-CD29hiCD24+ population are multipotent and self-renewing, properties that define them as MaSCs.
Abstract: The existence of mammary stem cells (MaSCs) has been postulated from evidence that the mammary gland can be regenerated by transplantation of epithelial fragments in mice. Interest in MaSCs has been further stimulated by their potential role in breast tumorigenesis. However, the identity and purification of MaSCs has proved elusive owing to the lack of defined markers. We isolated discrete populations of mouse mammary cells on the basis of cell-surface markers and identified a subpopulation (Lin-CD29hiCD24+) that is highly enriched for MaSCs by transplantation. Here we show that a single cell, marked with a LacZ transgene, can reconstitute a complete mammary gland in vivo. The transplanted cell contributed to both the luminal and myoepithelial lineages and generated functional lobuloalveolar units during pregnancy. The self-renewing capacity of these cells was demonstrated by serial transplantation of clonal outgrowths. In support of a potential role for MaSCs in breast cancer, the stem-cell-enriched subpopulation was expanded in premalignant mammary tissue from MMTV-wnt-1 mice and contained a higher number of MaSCs. Our data establish that single cells within the Lin-CD29hiCD24+ population are multipotent and self-renewing, properties that define them as MaSCs.
1,919 citations
Journal Article•
TL;DR: In this article, the mammary gland can be functionally regenerated in mice by serial transplantation of epithelial fragments, providing evidence for the existence of self-renewing, multipotential mammary stem cells (MaSCs).
Abstract: 4839 The mammary gland can be functionally regenerated in mice by serial transplantation of epithelial fragments, providing evidence for the existence of self-renewing, multipotential mammary stem cells (MaSCs). Recently the concept has emerged that MaSCs play a central role in breast tumorigenesis. However, the identity and purification of MaSC has proved elusive due to the lack of defined markers. Using specific cell surface markers and flow cytometry, we have identified a distinct subpopulation that is enriched for MaSCs, demonstrated by transplantation into cleared mammary fat pads at limiting dilution. Remarkably, a single mammary epithelial cell from this population, carrying the lacZ transgene, was found to generate a complete mammary gland in vivo. These cells contributed to both the luminal and myoepithelial lineages in transplanted virgin mammary glands, and extensive lobuloalveolar units were generated during pregnancy. Serial transplantation of the clonal outgrowths also yielded complete mammary glands, confirming that the cells were capable of self-renewal. These data establish that single cells from the enriched population have multipotential and self-renewing capacity, a hallmark of stem cells. It will be of interest to determine whether the stem cell is a target of transformation mammary tumorigenesis models.
1,810 citations
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TL;DR: Variation in gene expression patterns in a set of 65 surgical specimens of human breast tumours from 42 different individuals were characterized using complementary DNA microarrays representing 8,102 human genes, providing a distinctive molecular portrait of each tumour.
Abstract: Human breast tumours are diverse in their natural history and in their responsiveness to treatments. Variation in transcriptional programs accounts for much of the biological diversity of human cells and tumours. In each cell, signal transduction and regulatory systems transduce information from the cell's identity to its environmental status, thereby controlling the level of expression of every gene in the genome. Here we have characterized variation in gene expression patterns in a set of 65 surgical specimens of human breast tumours from 42 different individuals, using complementary DNA microarrays representing 8,102 human genes. These patterns provided a distinctive molecular portrait of each tumour. Twenty of the tumours were sampled twice, before and after a 16-week course of doxorubicin chemotherapy, and two tumours were paired with a lymph node metastasis from the same patient. Gene expression patterns in two tumour samples from the same individual were almost always more similar to each other than either was to any other sample. Sets of co-expressed genes were identified for which variation in messenger RNA levels could be related to specific features of physiological variation. The tumours could be classified into subtypes distinguished by pervasive differences in their gene expression patterns.
14,768 citations
TL;DR: Survival analyses on a subcohort of patients with locally advanced breast cancer uniformly treated in a prospective study showed significantly different outcomes for the patients belonging to the various groups, including a poor prognosis for the basal-like subtype and a significant difference in outcome for the two estrogen receptor-positive groups.
Abstract: The purpose of this study was to classify breast carcinomas based on variations in gene expression patterns derived from cDNA microarrays and to correlate tumor characteristics to clinical outcome. A total of 85 cDNA microarray experiments representing 78 cancers, three fibroadenomas, and four normal breast tissues were analyzed by hierarchical clustering. As reported previously, the cancers could be classified into a basal epithelial-like group, an ERBB2-overexpressing group and a normal breast-like group based on variations in gene expression. A novel finding was that the previously characterized luminal epithelial/estrogen receptor-positive group could be divided into at least two subgroups, each with a distinctive expression profile. These subtypes proved to be reasonably robust by clustering using two different gene sets: first, a set of 456 cDNA clones previously selected to reflect intrinsic properties of the tumors and, second, a gene set that highly correlated with patient outcome. Survival analyses on a subcohort of patients with locally advanced breast cancer uniformly treated in a prospective study showed significantly different outcomes for the patients belonging to the various groups, including a poor prognosis for the basal-like subtype and a significant difference in outcome for the two estrogen receptor-positive groups.
10,791 citations
TL;DR: The basic components of the death machinery are reviewed, how they interact to regulate apoptosis in a coordinated manner is described, and the main pathways that are used to activate cell death are discussed.
Abstract: Apoptosis - the regulated destruction of a cell - is a complicated process. The decision to die cannot be taken lightly, and the activity of many genes influence a cell's likelihood of activating its self-destruction programme. Once the decision is taken, proper execution of the apoptotic programme requires the coordinated activation and execution of multiple subprogrammes. Here I review the basic components of the death machinery, describe how they interact to regulate apoptosis in a coordinated manner, and discuss the main pathways that are used to activate cell death.
7,255 citations
TL;DR: Although the Ki‐67 protein is well characterized on the molecular level and extensively used as a proliferation marker, the functional significance still remains unclear; there are indications, however, that Ki‐ 67 protein expression is an absolute requirement for progression through the cell‐division cycle.
Abstract: The expression of the human Ki-67 protein is strictly associated with cell proliferation. During interphase, the antigen can be exclusively detected within the nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. The fact that the Ki-67 protein is present during all active phases of the cell cycle (G(1), S, G(2), and mitosis), but is absent from resting cells (G(0)), makes it an excellent marker for determining the so-called growth fraction of a given cell population. In the first part of this study, the term proliferation marker is discussed and examples of the applications of anti-Ki-67 protein antibodies in diagnostics of human tumors are given. The fraction of Ki-67-positive tumor cells (the Ki-67 labeling index) is often correlated with the clinical course of the disease. The best-studied examples in this context are carcinomas of the prostate and the breast. For these types of tumors, the prognostic value for survival and tumor recurrence has repeatedly been proven in uni- and multivariate analysis. The preparation of new monoclonal antibodies that react with the Ki-67 equivalent protein from rodents now extends the use of the Ki-67 protein as a proliferation marker to laboratory animals that are routinely used in basic research. The second part of this review focuses on the biology of the Ki-67 protein. Our current knowledge of the Ki-67 gene and protein structure, mRNA splicing, expression, and cellular localization during the cell-division cycle is summarized and discussed. Although the Ki-67 protein is well characterized on the molecular level and extensively used as a proliferation marker, the functional significance still remains unclear. There are indications, however, that Ki-67 protein expression is an absolute requirement for progression through the cell-division cycle.
4,359 citations
Journal Article•
TL;DR: The data suggest that the early stages of mitogen stimulation represent initial sequences of proliferation and not parts of the cell cycle, and immunostaining with monoclonal antibody Ki-67 provides a reliable means of rapidly evaluating the growth fraction of normal and neoplastic human cell populations.
Abstract: The monoclonal antibody Ki-67 detects a nuclear antigen that is present only in proliferating cells. The aim of the present investigation was to clarify whether the Ki-67 nuclear antigen is restricted in its expression to certain phases of the cell cycle. All experiments consistently showed that the Ki-67 nuclear antigen is present in S, G2, and M phase, but is absent in G0. However, the results concerning Ki-67 antigen expression in G1 phase varied: cells passing the early events of mitogen triggered transition from G0 to G1, i.e., G1T and first G1A, lacked the Ki-67 nuclear antigen, whereas G1 cells after mitosis were constantly Ki-67-positive. This result suggests that after mitosis cells might not follow the same metabolic pathways as G0 cells do when entering G1 for the first time. Therefore, we suggest that the early stages of mitogen stimulation represent initial sequences of proliferation and not parts of the cell cycle. Because our data show that the Ki-67 nuclear antigen is present throughout the cell cycle, immunostaining with monoclonal antibody Ki-67 provides a reliable means of rapidly evaluating the growth fraction of normal and neoplastic human cell populations.
4,093 citations