Long-term primary culture of a clear cell ovarian carcinoma reveals an epithelial-mesenchymal cooperative interaction.
ABSTRACT: BACKGROUND:We studied a primary culture developed from a biopsy of a clear cell carcinoma of the ovary (O-CCC) by (a) assessing its capacity to retain in vitro pathological features of the tumor of origin; (b) characterizing the main cells released from the complex mass without forced purification of any particular cellular entity; and (c) investigating its long-term proliferative capacity. METHODS:A primary cell culture was developed from a pelvic mass diagnosed as an O-CCC. The morphological analysis of the cell culture was carried out by phase contrast microscopy. Markers of epithelial, mesenchymal, and tumor initiating cells were evaluated by immunocytochemistry. Cell proliferation was studied by detection of bromodeoxyuridine (BrdU) incorporated into newly synthesized DNA. As a biomarker of O-CCC, we assessed the expression of hepatocyte nuclear factor (HNF) 1?. RESULTS:We show that cells with epithelial morphological features express E-cadherin and expand with time in culture, a fact that the incorporation of BrdU confirms. Cells with mesenchymal-like characteristics that express the mesenchymal marker vimentin, however, allocate to the edges of the epithelial compartment. Moreover, we found that some cells with epithelial features also expressed vimentin. At the beginning of incubation, over 60 % of primary cells expressed the O-CCC marker HNF1?; such percentage declined upon passaging. We show that epithelial not mesenchymal cells undergo DNA replication, and that few cells in both epithelial and mesenchymal compartments express the stem-like tumor antigen CD133. CONCLUSIONS:We provide proof-of-principle that cells separated in bulk from a biopsy of an O-CCC can be maintained in culture for several months, and that two consistent cellular compartments-one epithelial that retains the O-CCC marker HNF1?, and another mesenchymal-persist, and seem to have a cooperative interaction leading to the multiplication of epithelial cells within a mesenchymal cellular environment.
Project description:NUCOLL43 is a novel ovarian clear cell carcinoma (O-CCC) cell line that arose from a primary culture of a patient's malignant ascites. The cells grow reliably in cell culture with a doubling time of approx. 45 hours and form colonies at high efficiency. They have a very high degree of loss of heterozygosity (LOH) affecting approximately 85% of the genome, mostly copy neutral and almost identical to the original tumor. The cells express epithelial (pan-cytokeratin) and mesenchymal (vimentin) characteristics, CA125 and p16, like the original tumor. They also express ARID1A but not HNF-1β and, like the original tumor, and are negative for p53 expression, with no evidence of p53 function. NUCOLL43 cells express all other DNA damage response proteins investigated and have functional homologous recombination DNA repair. They are insensitive to cisplatin, the PARP inhibitor rucaparib, and MDM2 inhibitors but are sensitive to camptothecin, paclitaxel, and NVP-BEZ235. The NUCOLL43 cell line represents a distinct subtype of O-CCC that is p53 and HNF-1β null but expresses ARID1A. Its high degree of similarity with the original tumor genomically and proteomically, as well as the high level of LOH, make this an interesting cell line for O-CCC research. It has been deposited with Ximbio.
Project description:UNLABELLED: Mesenchymal-epithelial transition events are related to embryonic development, tissue construction, and wound healing. Stem cells are involved in all of these processes, at least in part. However, the direct evidence of mesenchymal-epithelial transition associated with stem cells is unclear. To determine whether mesenchymal-epithelial transition occurs in liver development and/or the differentiation process of hepatic stem cells in vitro, we analyzed a variety of murine liver tissues from embryonic day 11.5 to adults and the colonies derived from hepatic stem/progenitor cells isolated with flow cytometry. The results of gene expression, immunohistochemistry and Western blot showed that as liver develops, the expression of epithelial markers such as Cytokeratin18 and E-cadherin increase, while expression of mesenchymal markers such as vimentin and N-cadherin decreased. On the other hand, in freshly isolated hepatic stem cells, the majority of cells (65.0%) co-express epithelial and mesenchymal markers; this proportion is significantly higher than observed in hematopoietic cells, non-hematopoietic cells and non-stem cell fractions. Likewise, in stem cell-derived colonies cultured over time, upregulation of epithelial genes (Cytokeratin-18 and E-cadherin) occurred simultaneously with downregulation of mesenchymal genes (vimentin and Snail1). Furthermore, in the fetal liver, vimentin-positive cells in the non-hematopoietic fraction had distinct proliferative activity and expressed early the hepatic lineage marker alpha-fetoprotein. CONCLUSION: Hepatic stem cells co-express mesenchymal and epithelial markers; the mesenchymal-epithelial transition occurred in both liver development and differentiation of hepatic stem/progenitor cells in vitro. Besides as a mesenchymal marker, vimentin is a novel indicator for cell proliferative activity and undifferentiated status in liver cells.
Project description:?-cells undergo an epithelial to mesenchymal transition (EMT) when expanded in monolayer culture and give rise to highly proliferative mesenchymal cells that retain the potential to re-differentiate into insulin-producing cells.To investigate whether EMT takes place in the endocrine non-? cells of human islets.Human islets isolated from 12 multiorgan donors were dissociated into single cells, purified by magnetic cell sorting, and cultured in monolayer.Co-expression of insulin and the mesenchymal marker vimentin was identified within the first passage (p1) and increased subsequently (insulin+vimentin+ 7.2±6% at p1; 43±15% at p4). The endocrine non-?-cells did also co-express vimentin (glucagon+vimentin+ 59±1.5% and 93±6%, somatostatin+vimentin+ 16±9.4% and 90±10% at p1 and p4 respectively; PP+vimentin+ 74±14% at p1; 88±12% at p2). The percentage of cells expressing only endocrine markers was progressively reduced (0.6±0.2% insulin+, 0.2±0.1% glucagon+, and 0.3±0.2% somatostatin+ cells at p4, and 0.7±0.3% PP+ cells at p2. Changes in gene expression were also indicated of EMT, with reduced expression of endocrine markers and the epithelial marker CDH-1 (p<0.01), and increased expression of mesenchymal markers (CDH-2, SNAI2, ZEB1, ZEB2, VIM, NT5E and ACTA2; p<0.05). Treatment with the EMT inhibitor A83-01 significantly reduced the percentage of co-expressing cells and preserved the expression of endocrine markers.In adult human islets, all four endocrine islet cell types undergo EMT when islet cells are expanded in monolayer conditions. The presence of EMT in all islet endocrine cells could be relevant to design of strategies aiming to re-differentiate the expanded islet cells towards a ?-cell phenotype.
Project description:It has been reported previously that: (1) normal-breast epithelial cells that are CD24-/44+ express higher levels of stem/progenitor cell-associated genes; (2) cancer cells that have undergone epithelial to mesenchymal transition display CD24-/44+ cell-surface expression, a marker for breast cancer stem cells; (3) loss of E-cadherin is a preliminary step in epithelial to mesenchymal transition; and (4) vimentin is a marker of mesenchymal phenotype. We hypothesized that stem cell subpopulations would be more frequent in metastatic than in primary tumors. Therefore we assessed by immunohistochemical analysis, tissue microarrays containing tissue from primary and associated metastatic breast cancers for expression of CD24, CD44, E-cadherin and vimentin to evaluate candidate cancer-initiating cell populations in breast cancer subtypes and metastatic lesions. The occurrence of CD24-/44+ and CD24+/44- cells did not differ in primary vs matched lymph node or distant and locoregional metastatic lesions; E-cadherin expression was decreased in primary vs lymph node metastases (P=0.018) but not decreased in distant and locoregional metastases relative to primary tumor, whereas vimentin, was more frequently expressed in lymph node and distant and locoregional metastases (P=0.013, P=0.004) than in matched primary cancers. Thus, the frequency of CD24-/44+ cells does not differ in metastases relative to the primary breast cancer but differs by tumor stage and subtype.
Project description:The early phases of carcinogenesis resemble embryonic development, often involving the reexpression of embryonic mesenchymal genes. The NCI60 panel of human tumor cell lines can genetically be subdivided into two superclusters (SCs) that correspond to CD95 Type I and II cells. SC1 cells are characterized by a mesenchymal and SC2 cells by an epithelial gene signature, suggesting that SC1 cells represent less differentiated, advanced stages of cancer. miRNAs are small 20- to 22-nucleotide-long noncoding RNAs that inhibit gene expression at the posttranscriptional level. By performing miRNA expression analysis on 10 Type I and 10 Type II cells, we have determined that SC1 cells express low and SC2 cells high levels of the miRNA let-7, respectively, suggesting that let-7 is a marker for less advanced cancers. Expression of the let-7 target high-mobility group A2 (HMGA2), an early embryonic gene, but not of classical epithelial or mesenchymal markers such as E-cadherin or vimentin, inversely correlated with let-7 expression in SC1 and SC2 cells. Using ovarian cancer as a model, we demonstrate that expression of let-7 and HMGA2 is a better predictor of prognosis than classical markers such as E-cadherin, vimentin, and Snail. These data identify loss of let-7 expression as a marker for less differentiated cancer.
Project description:The collagen gel contraction assay measures gel size to assess the contraction of cells embedded in collagen gel matrices. Using the assay with lung fibroblasts is useful in studying the lung tissue remodeling process in wound healing and disease development. However, the involvement of bronchial epithelial cells in this process should also be investigated.We applied a layer of mucociliary differentiated bronchial epithelial cells onto collagen gel matrices with lung fibroblasts. This co-culture model enables direct contact between epithelial and mesenchymal cells. We stimulated the culture with transforming growth factor (TGF) ?1 as an inducer of tissue remodeling for 21 days, and measured gel size, histological changes, and expression of factors related to extracellular matrix homeostasis.TGF-?1 exerted a concentration-dependent effect on collagen gel contraction and on contractile myofibroblasts in the mesenchymal collagen layer. TGF-?1 also induced expression of the mesenchymal marker vimentin in the basal layer of the epithelium, suggesting the induction of epithelial-mesenchymal transition. In addition, the expression of various genes encoding extracellular matrix proteins was upregulated. Fibrotic tenascin-C accumulated in the sub-epithelial region of the co-culture model.Our findings indicate that TGF-?1 can affect both epithelial and mesenchymal cells, and induce gel contraction and structural changes. Our novel in vitro co-culture model will be a useful tool for investigating the roles of epithelial cells, fibroblasts, and their interactions in the airway remodeling process.
Project description:<h4>Background</h4>Vimentin is a ubiquitous mesenchymal intermediate filament supporting mechano-structural integrity of quiescent cells while participating in adhesion, migration, survival, and cell signaling processes via dynamic assembly/disassembly in activated cells. Soft tissue sarcomas and some epithelial cancers exhibiting "epithelial to mesenchymal transition" phenotypes express vimentin. Withaferin-A, a naturally derived bioactive compound, may molecularly target vimentin, so we sought to evaluate its effects on tumor growth in vitro and in vivo thereby elucidating the role of vimentin in drug-induced responses.<h4>Methods and findings</h4>Withaferin-A elicited marked apoptosis and vimentin cleavage in vimentin-expressing tumor cells but significantly less in normal mesenchymal cells. This proapoptotic response was abrogated after vimentin knockdown or by blockade of caspase-induced vimentin degradation via caspase inhibitors or overexpression of mutated caspase-resistant vimentin. Pronounced anti-angiogenic effects of Withaferin-A were demonstrated, with only minimal effects seen in non-proliferating endothelial cells. Moreover, Withaferin-A significantly blocked soft tissue sarcoma growth, local recurrence, and metastasis in a panel of soft tissue sarcoma xenograft experiments. Apoptosis, decreased angiogenesis, and vimentin degradation were all seen in Withaferin-A treated specimens.<h4>Conclusions</h4>In light of these findings, evaluation of Withaferin-A, its analogs, or other anti-vimentin therapeutic approaches in soft tissue sarcoma and "epithelial to mesenchymal transition" clinical contexts is warranted.
Project description:Embryonic epiboly has become an important developmental model for studying the mechanisms underlying collective movements of epithelial cells. In the last couple of decades, most studies of epiboly have utilized Xenopus or zebrafish as genetically tractable model organisms, while the avian epiboly model has received virtually no attention. Here, we re-visit epiboly in quail embryos and characterize several molecular markers of epithelial-to-mesenchymal transition (EMT) in the inner zone of the extraembryonic Area Opaca and at the blastoderm edge. Our results show that the intermediate filament vimentin, a widely-used marker for the mesenchymal phenotype, is strongly expressed in the edge cells compared to the cells in the inner zone. Laminin, an extracellular matrix protein that is a major structural and adhesive component of the epiblast basement membrane and the inner zone of the Area Opaca, is notably absent from the blastoderm edge. While these expression profiles are consistent with a mesenchymal phenotype, several other epithelial markers, including cytokeratin, ?-catenin, and E-cadherin, are present in the blastoderm edge cells. Moreover, the results of a BrDU proliferation assay strongly suggest that expansion of the edge cell population is primarily due to recruitment of cells from the inner zone, as opposed to proliferation. Taken together, our data show that the edge cells of the avian blastoderm have characteristics of both epithelial and mesenchymal cells, and that the avian epiboly model, which has been dormant for so many years, may yet again prove to be helpful as a unique developmental model for studying partial EMT in the context of collective epithelial cell migration.
Project description:During embryonic development bipotential hepatoblasts differentiate into hepatocytes and cholangiocytes- the two main cell types within the liver. Cell fate decision depends on elaborate interactions between distinct signalling pathways, namely Notch, WNT, TGF?, and Hedgehog. Several in vitro protocols have been established to differentiate human pluripotent stem cells into either hepatocyte or cholangiocyte like cells (HLC/CLC) to enable disease modelling or drug screening. During HLC differentiation we observed the occurrence of epithelial cells with a phenotype divergent from the typical hepatic polygonal shape- we refer to these as endoderm derived epithelial cells (EDECs). These cells do not express the mature hepatocyte marker ALB or the progenitor marker AFP. However they express the cholangiocyte markers SOX9, OPN, CFTR as well as HNF4?, CK18 and CK19. Interestingly, they express both E Cadherin and Vimentin, two markers that are mutually exclusive, except for cancer cells. EDECs grow spontaneously under low density cell culture conditions and their occurrence was unaffected by interfering with the above mentioned signalling pathways.
Project description:Vimentin is used widely as a marker of the epithelial to mesenchymal transitions (EMTs) that take place during embryogenesis and metastasis, yet the functional implications of the expression of this type III intermediate filament (IF) protein are poorly understood. Using form factor analysis and quantitative Western blotting of normal, metastatic, and vimentin-null cell lines, we show that the level of expression of vimentin IFs (VIFs) correlates with mesenchymal cell shape and motile behavior. The reorganization of VIFs caused by expressing a dominant-negative mutant or by silencing vimentin with shRNA (neither of which alter microtubule or microfilament assembly) causes mesenchymal cells to adopt epithelial shapes. Following the microinjection of vimentin or transfection with vimentin cDNA, epithelial cells rapidly adopt mesenchymal shapes coincident with VIF assembly. These shape transitions are accompanied by a loss of desmosomal contacts, an increase in cell motility, and a significant increase in focal adhesion dynamics. Our results demonstrate that VIFs play a predominant role in the changes in shape, adhesion, and motility that occur during the EMT.