epithelial transition states
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TGFβ has roles in inflammation, wound healing, epithelial to mesenchymal transition (EMT), and cancer stem cell states, and acts as a tumor suppressor gene for squamous cell carcinoma (SCC). SCCs are also characterized by high levels of ΔNp63, which induces epithelial cell phenotypes and maintains squamous stem cells. Previous studies indicate a complex interplay between ΔNp63 and TGFβ signaling, with contradictory effects reported. We investigated the effects of TGFβ on p63 isoform proteins and mRNAs in non-malignant squamous and SCC cells, and the role of either canonical or non-canonical TGFβ signaling pathways. TGFβ selectively increased ΔNp63 protein levels in non-malignant keratinocytes in association with SMAD3 activation and was prevented by TGFβ receptor inhibition, indicating activation of canonical TGFβ pathway signaling. TP63 isoform mRNAs showed discordance from protein levels, with an initial increase in both TAP63 and ΔNP63 mRNAs followed by a decrease at later times. These data demonstrate complex and heterogeneous effects of TGFβ in squamous cells that depend on the extent of canonical TGFβ pathway aberrations. The interplay between TGFβ and p63 is likely to influence the magnitude of EMT states in SCC, with clinical implications for tumor progression and response to therapy.
SIGNIFICANCE: Machine learning is increasingly being applied to the classification of microscopic data. In order to detect some complex and dynamic cellular processes, time-resolved live-cell imaging might be necessary. Incorporating the temporal information into the classification process may allow for a better and more specific classification. AIM: We propose a methodology for cell classification based on the time-lapse quantitative phase images (QPIs) gained by digital holographic microscopy (DHM) with the goal of increasing performance of classification of dynamic cellular processes. APPROACH: The methodology was demonstrated by studying epithelial-mesenchymal transition (EMT) which entails major and distinct time-dependent morphological changes. The time-lapse QPIs of EMT were obtained over a 48-h period and specific novel features representing the dynamic cell behavior were extracted. The two distinct end-state phenotypes were classified by several supervised machine learning algorithms and the results were compared with the classification performed on single-time-point images. RESULTS: In comparison to the single-time-point approach, our data suggest the incorporation of temporal information into the classification of cell phenotypes during EMT improves performance by nearly 9% in terms of accuracy, and further indicate the potential of DHM to monitor cellular morphological changes. CONCLUSIONS: Proposed approach based on the time-lapse images gained by DHM could improve the monitoring of live cell behavior in an automated fashion and could be further developed into a tool for high-throughput automated analysis of unique cell behavior.
BACKGROUND: During cancer progression, epithelial cancer cells can be reprogrammed into mesenchymal-like cells with increased migratory potential through the process of epithelial-mesenchymal transition (EMT), representing an essential step of tumor progression towards metastatic state. AGR2 protein was shown to regulate several cancer-associated processes including cellular proliferation, survival and drug resistance. METHODS: The expression of AGR2 was analyzed in cancer cell lines exposed to TGF-β alone or to combined treatment with TGF-β and the Erk1/2 inhibitor PD98059 or the TGF-β receptor specific inhibitor SB431542. The impact of AGR2 silencing by specific siRNAs or CRISPR/Cas9 technology on EMT was investigated by western blot analysis, quantitative PCR, immunofluorescence analysis, real-time invasion assay and adhesion assay. RESULTS: Induction of EMT was associated with decreased AGR2 along with changes in cellular morphology, actin reorganization, inhibition of E-cadherin and induction of the mesenchymal markers vimentin and N-cadherin in various cancer cell lines. Conversely, induction of AGR2 caused reversion of the mesenchymal phenotype back to the epithelial phenotype and re-acquisition of epithelial markers. Activated Smad and Erk signaling cascades were identified as mutually complementary pathways responsible for TGF-β-mediated inhibition of AGR2. CONCLUSION: Taken together our results highlight a crucial role for AGR2 in maintaining the epithelial phenotype by preventing the activation of key factors involved in the process of EMT.
- MeSH
- buněčná adheze genetika MeSH
- epitelo-mezenchymální tranzice účinky léků genetika MeSH
- genový knockdown MeSH
- kadheriny metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- pohyb buněk genetika MeSH
- proteiny Smad metabolismus MeSH
- proteiny genetika MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- signální transdukce účinky léků MeSH
- transformující růstový faktor beta farmakologie MeSH
- vimentin metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Expression of acute kidney injury-associated (AKI-associated) transcripts in kidney transplants may reflect recent injury and accumulation of epithelial cells in "failed repair" states. We hypothesized that the phenomenon of failed repair could be associated with deterioration and failure in kidney transplants. METHODS: We defined injury-induced transcriptome states in 4,502 kidney transplant biopsies injury-induced gene sets and classifiers previously developed in transplants. RESULTS: In principal component analysis (PCA), PC1 correlated with both acute and chronic kidney injury and related inflammation and PC2 with time posttransplant. Positive PC3 was a dimension that correlated with epithelial remodeling pathways and anticorrelated with inflammation. Both PC1 and PC3 correlated with reduced survival, with PC1 effects strongly increasing over time whereas PC3 effects were independent of time. In this model, we studied the expression of 12 "new" gene sets annotated in single-nucleus RNA-sequencing studies of epithelial cells with failed repair in native kidneys. The new gene sets reflecting epithelial-mesenchymal transition correlated with injury PC1 and PC3, lower estimated glomerular filtration rate, higher donor age, and future failure as strongly as any gene sets previously derived in transplants and were independent of nephron segment of origin and graft rejection. CONCLUSION: These results suggest 2 dimensions in the kidney transplant response to injury: PC1, AKI-induced changes, failed repair, and inflammation; and PC3, a response involving epithelial remodeling without inflammation. Increasing kidney age amplifies PC1 and PC3. TRIAL REGISTRATION: INTERCOMEX (ClinicalTrials.gov NCT01299168); Trifecta-Kidney (ClinicalTrials.gov NCT04239703). FUNDING: Genome Canada; Natera, Inc.; and Thermo Fisher Scientific.
- MeSH
- akutní poškození ledvin * patologie genetika MeSH
- analýza hlavních komponent MeSH
- biopsie MeSH
- dospělí MeSH
- epitelo-mezenchymální tranzice genetika MeSH
- epitelové buňky * patologie metabolismus MeSH
- ledviny patologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- průřezové studie MeSH
- rejekce štěpu * patologie genetika MeSH
- senioři MeSH
- transkriptom MeSH
- transplantace ledvin * škodlivé účinky MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- pozorovací studie MeSH
The diagnosis of solid tumors of epithelial origin (carcinomas) represents a major part of the workload in clinical histopathology. Carcinomas consist of malignant epithelial cells arranged in more or less cohesive clusters of variable size and shape, together with stromal cells, extracellular matrix, and blood vessels. Distinguishing stroma from epithelium is a critical component of artificial intelligence (AI) methods developed to detect and analyze carcinomas. In this paper, we propose a novel automated workflow that enables large-scale guidance of AI methods to identify the epithelial component. The workflow is based on re-staining existing hematoxylin and eosin (H&E) formalin-fixed paraffin-embedded sections by immunohistochemistry for cytokeratins, cytoskeletal components specific to epithelial cells. Compared to existing methods, clinically available H&E sections are reused and no additional material, such as consecutive slides, is needed. We developed a simple and reliable method for automatic alignment to generate masks denoting cytokeratin-rich regions, using cell nuclei positions that are visible in both the original and the re-stained slide. The registration method has been compared to state-of-the-art methods for alignment of consecutive slides and shows that, despite being simpler, it provides similar accuracy and is more robust. We also demonstrate how the automatically generated masks can be used to train modern AI image segmentation based on U-Net, resulting in reliable detection of epithelial regions in previously unseen H&E slides. Through training on real-world material available in clinical laboratories, this approach therefore has widespread applications toward achieving AI-assisted tumor assessment directly from scanned H&E sections. In addition, the re-staining method will facilitate additional automated quantitative studies of tumor cell and stromal cell phenotypes.
- MeSH
- barvení a značení MeSH
- deep learning * MeSH
- eosin MeSH
- epitelové buňky MeSH
- hematoxylin MeSH
- keratiny * MeSH
- lidé MeSH
- umělá inteligence MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Occupational exposure to diesel exhaust may cause lung cancer in humans. Mechanisms include DNA-damage and inflammatory responses. Here, the potential of NIST SRM2975 diesel exhaust particles (DEP) to transform human bronchial epithelial cells (HBEC3) in vitro was investigated. Long-term exposure of HBEC3 to DEP led to increased colony growth in soft agar. Several DEP-transformed cell lines were established and based on the expression of epithelial-to-mesenchymal-transition (EMT) marker genes, one of them (T2-HBEC3) was further characterized. T2-HBEC3 showed a mesenchymal/fibroblast-like morphology, reduced expression of CDH1, and induction of CDH2 and VIM. T2-HBEC3 had reduced migration potential compared with HBEC3 and little invasion capacity. Gene expression profiling showed baseline differences between HBEC3 and T2-HBEC3 linked to lung carcinogenesis. Next, to assess differences in sensitivity to DEP between parental HBEC3 and T2-HBEC3, gene expression profiling was carried out after DEP short-term exposure. Results revealed changes in genes involved in metabolism of xenobiotics and lipids, as well as inflammation. HBEC3 displayed a higher steady state of IL1B gene expression and release of IL-1β compared with T2-HBEC3. HBEC3 and T2-HBEC3 showed similar susceptibility towards DEP-induced genotoxic effects. Liquid-chromatography-tandem-mass-spectrometry was used to measure secretion of eicosanoids. Generally, major prostaglandin species were released in higher concentrations from T2-HBEC3 than from HBEC3 and several analytes were altered after DEP-exposure. In conclusion, long-term exposure to DEP-transformed human bronchial epithelial cells in vitro. Differences between HBEC3 and T2-HBEC3 regarding baseline levels and DEP-induced changes of particularly CYP1A1, IL-1β, PGE2, and PGF2α may have implications for acute inflammation and carcinogenesis.
- MeSH
- bronchy účinky léků metabolismus ultrastruktura MeSH
- buněčné kultury MeSH
- epitelo-mezenchymální tranzice účinky léků genetika MeSH
- epitelové buňky účinky léků metabolismus ultrastruktura MeSH
- interleukin-1beta genetika MeSH
- látky znečišťující vzduch toxicita MeSH
- lidé MeSH
- pevné částice toxicita MeSH
- poškození DNA MeSH
- stanovení celkové genové exprese MeSH
- transformované buněčné linie MeSH
- transkriptom účinky léků MeSH
- výfukové emise vozidel toxicita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The role of the local microenvironment in influencing cell behavior is central to both normal development and cancer formation. Here, we show that sprouty 1 (SPRY1) modulates the microenvironment to enable proper mammary branching morphogenesis. This process occurs through negative regulation of epidermal growth factor receptor (EGFR) signaling in mammary stroma. Loss of SPRY1 resulted in up-regulation of EGFR-extracellular signal-regulated kinase (ERK) signaling in response to amphiregulin and transforming growth factor alpha stimulation. Consequently, stromal paracrine signaling and ECM remodeling is augmented, leading to increased epithelial branching in the mutant gland. By contrast, down-regulation of EGFR-ERK signaling due to gain of Sprouty function in the stroma led to stunted epithelial branching. Taken together, our results show that modulation of stromal paracrine signaling and ECM remodeling by SPRY1 regulates mammary epithelial morphogenesis during postnatal development.
- MeSH
- adaptorové proteiny signální transdukční nedostatek metabolismus MeSH
- amfiregulin farmakologie MeSH
- buňky stromatu účinky léků metabolismus MeSH
- časosběrné zobrazování MeSH
- epitel růst a vývoj metabolismus MeSH
- epitelové buňky cytologie účinky léků MeSH
- erbB receptory metabolismus MeSH
- extracelulární matrix metabolismus MeSH
- extracelulárním signálem regulované MAP kinasy metabolismus MeSH
- fibroblasty účinky léků metabolismus MeSH
- fosfoproteiny nedostatek metabolismus MeSH
- fosforylace účinky léků MeSH
- kolagen metabolismus MeSH
- ligandy MeSH
- membránové proteiny nedostatek metabolismus MeSH
- mléčné žlázy zvířat účinky léků metabolismus MeSH
- morfogeneze * účinky léků MeSH
- mutace genetika MeSH
- myši knockoutované MeSH
- myši nahé MeSH
- parakrinní signalizace * účinky léků MeSH
- pohyb buněk účinky léků MeSH
- protoonkogenní proteiny c-akt metabolismus MeSH
- signální transdukce * účinky léků MeSH
- transformující růstový faktor alfa farmakologie MeSH
- vývojová regulace genové exprese účinky léků MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
For the treatment of bilateral limbal stem cell deficiency (LSCD), cell therapy with transplantation of cultivated oral mucosa epithelial cells (COMET) is a promising alternative. Although not yet established, current protocols on the cultivation of oral mucosal epithelial cell (OMECs) sheets are based mainly on substrates and xenobiotic additives that may lead to variable outcomes and undesirable immune responses by the patient. The aim of this study was to characterize OMECs cultivated in xenobiotic-free media (XF) seeded on fibrin gel, in comparison to conventional complex (COM) medium. Oral mucosal biopsies were retrieved from 31 donors. After cultivation in COM or XF medium, OMECs were compared based on growth kinetics, morphology, cell size and viability. Using immunofluorescence and gene expression analyses, the degree of stemness, proliferation and differentiation was evaluated in OMEC cultures. Our findings showed that although OMECs showed a similar morphology and viability, and comparable growth kinetics, immunofluorescence revealed the preservation of stemness (p63 + p40 positivity in cells ≤11 μm) and proliferation in both COM and XF. Gene expression analyses showed that keratin (K)13 and K15 expression levels were significantly higher in XF (adj. p < 0.001), but otherwise COM and XF-treated OMECs had comparable transcriptional profiles in a panel of stemness, proliferation and differentiation genes. These results demonstrate the feasibility of culturing OMECs on fibrin gel without xenogeneic additives, while maintaining their undifferentiated state and preserving stemness. In conclusion, both in terms of results and methodology, the procedures presented here are suitable for implementation in clinical practice.
- MeSH
- buněčná diferenciace MeSH
- buněčné kultury * MeSH
- deficit limbálních kmenových buněk MeSH
- dospělí MeSH
- epitelové buňky * metabolismus účinky léků MeSH
- fibrin * MeSH
- gely MeSH
- kmenové buňky * metabolismus cytologie MeSH
- kultivační média MeSH
- kultivované buňky MeSH
- lidé středního věku MeSH
- lidé MeSH
- limbus corneae * cytologie patologie metabolismus MeSH
- nemoci rohovky patologie farmakoterapie metabolismus MeSH
- proliferace buněk účinky léků MeSH
- rohovkový epitel metabolismus cytologie účinky léků patologie MeSH
- senioři MeSH
- transplantace kmenových buněk metody MeSH
- ústní sliznice * cytologie MeSH
- viabilita buněk MeSH
- xenobiotika farmakologie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
When defined in terms of markers for normal cell lineages, most invasive breast cancer cells correspond to the phenotype of the common luminal epithelial cell found in the terminal ductal lobular units. Luminal epithelial cells cultured from milk, which have limited proliferative potential, have now been immortalized by introducing the gene encoding simian virus 40 large tumor (T) antigen. Infection with a recombinant retrovirus proved to be 50-100 times more efficient than calcium phosphate transfection, and of the 17 cell lines isolated, only 5 passed through a crisis period as characterized by cessation of growth. When characterized by immunohistochemical staining with monoclonal antibodies, 14 lines showed features of luminal epithelial cells and of these, 7 resembled the common luminal epithelial cell type in the profile of keratins expressed. These cells express keratins 7, 8, 18, and 19 homogeneously and do not express keratin 14 or vimentin; a polymorphic epithelial mucin produced in vivo by luminal cells is expressed heterogeneously and the pattern of fibronectin staining is punctate. Although the cell lines have a reduced requirement for added growth factors, they do not grow in agar or produce tumors in the nude mouse. When the v-Ha-ras oncogene was introduced into two of the cell lines by using a recombinant retrovirus, most of the selected clones senesced, but one entered crisis and emerged after 3 months as a tumorigenic cell line.
- MeSH
- antigeny transformující polyomavirové * genetika MeSH
- buněčné dělení MeSH
- buněčné linie MeSH
- DNA virů genetika MeSH
- epitelové buňky MeSH
- fluorescenční protilátková technika MeSH
- genetické vektory MeSH
- keratiny metabolismus MeSH
- lidé MeSH
- mateřské mléko * cytologie MeSH
- myši nahé MeSH
- myši MeSH
- onkogenní protein p21(ras) genetika MeSH
- opičí virus SV40 genetika MeSH
- prsy * cytologie MeSH
- Retroviridae genetika MeSH
- Southernův blotting MeSH
- techniky in vitro MeSH
- virová transformace buněk * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
Macrophages located in airways and the alveolar space are continually exposed to different signals from the respiratory mucosa. In this respect, epithelial cells represent an important source of cytokines and mediators modulating the state of activation and/or differentiation of mononuclear phagocytes. Many of the proinflammatory genes induced in macrophages during immune and immunopathological reactions are regulated by transcription factor NF kappa B. The aim of our study was to characterize changes in the expression of genes associated with NF kappa B activation and signalling in THP-1 human macrophages co-cultured with A549 respiratory epithelial cells. At least 4-fold upregulation of mRNA level was found in 29 of 84 tested genes including genes for multiple cytokines and chemokines, membrane antigens and receptors, and molecules associated with NF kappa B signalling. The mRNA induction was confirmed at the level of protein expression by evaluating the release of IL-6 and IL-8 and by ICAM-1 expression. Blocking of one NFκB subunit by p65 siRNA inhibited the production of IL-6 in both cell types while IL-8 release from THP-1 cells did not seem to be affected. We conclude from our data that unstimulated respiratory epithelial cells regulate genes associated with NF kappa B dependent immune responses in human macrophages and that these interactions may play a key role in immediate responses in the respiratory mucosa.
- MeSH
- buněčné linie MeSH
- cytokiny sekrece MeSH
- epitelové buňky metabolismus MeSH
- kokultivační techniky MeSH
- lidé MeSH
- makrofágy imunologie metabolismus MeSH
- messenger RNA genetika MeSH
- mezibuněčná adhezivní molekula-1 genetika metabolismus MeSH
- NF-kappa B antagonisté a inhibitory metabolismus MeSH
- regulace genové exprese MeSH
- respirační sliznice imunologie metabolismus MeSH
- signální transdukce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
