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.

• Klíčová slova
• digital holographic microscopy, epithelial–mesenchymal transition, quantitative phase imaging, supervised machine learning,
• MeSH
• algoritmy MeSH
• časosběrné zobrazování MeSH
• epitelo-mezenchymální tranzice * MeSH
• holografie * MeSH
• strojové učení MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• Klíčová slova
• EMT, Squamous cell carcinoma, TAp63, TGFβ, ΔNp63,
• MeSH
• epitelo-mezenchymální tranzice * genetika   MeSH
• epitelové buňky metabolismus   MeSH
• lidé MeSH
• protein - isoformy genetika   metabolismus   MeSH
• spinocelulární karcinom * genetika   metabolismus   MeSH
• transformující růstový faktor beta MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protein - isoformy MeSH
• transformující růstový faktor beta MeSH

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.

• Klíčová slova
• AGR2, E-cadherin, EMT, Metastasis, TGF-β, Vimentin,
• MeSH
• buněčná adheze genetika   MeSH
• epitelo-mezenchymální tranzice účinky léků   genetika   MeSH
• genový knockdown MeSH
• kadheriny metabolismus   MeSH
• lidé MeSH
• mukoproteiny MeSH
• nádorové buněčné linie MeSH
• onkogenní proteiny 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
• Názvy látek
• AGR2 protein, human MeSH Prohlížeč
• kadheriny MeSH
• mukoproteiny MeSH
• onkogenní proteiny MeSH
• proteiny Smad MeSH
• proteiny MeSH
• transformující růstový faktor beta MeSH
• vimentin MeSH

Triple-negative breast cancer (TNBC) is a subtype of breast carcinoma known for its unusually aggressive behavior and poor clinical outcome. Besides the lack of molecular targets for therapy and profound intratumoral heterogeneity, the relatively quick overt metastatic spread remains a major obstacle in effective clinical management. The metastatic colonization of distant sites by primary tumor cells is affected by the microenvironment, epigenetic state of particular subclones, and numerous other factors. One of the most prominent processes contributing to the intratumoral heterogeneity is an epithelial-mesenchymal transition (EMT), an evolutionarily conserved developmental program frequently hijacked by tumor cells, strengthening their motile and invasive features. In response to various intrinsic and extrinsic stimuli, malignant cells can revert the EMT state through the mesenchymal-epithelial transition (MET), a process that is believed to be critical for the establishment of macrometastasis at secondary sites. Notably, cancer cells rarely undergo complete EMT and rather exist in a continuum of E/M intermediate states, preserving high levels of plasticity, as demonstrated in primary tumors and, ultimately, in circulating tumor cells, representing a simplified element of the metastatic cascade. In this review, we focus on cellular drivers underlying EMT/MET phenotypic plasticity and its detrimental consequences in the context of TNBC cancer.

• Klíčová slova
• epithelial–mesenchymal transition, mesenchymal–epithelial transition, metastasis, plasticity, triple-negative breast cancer,
• Publikační typ
• časopisecké články MeSH
• přehledy 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.

• Klíčová slova
• Molecular diagnosis, Nephrology, Organ transplantation, Transplantation,
• 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

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
• Názvy látek
• interleukin-1beta MeSH
• látky znečišťující vzduch MeSH
• pevné částice MeSH
• výfukové emise vozidel MeSH

In a large family of Czech origin, we mapped a locus for an autosomal-dominant corneal endothelial dystrophy, posterior polymorphous corneal dystrophy 4 (PPCD4), to 8q22.3-q24.12. Whole-genome sequencing identified a unique variant (c.20+544G>T) in this locus, within an intronic regulatory region of GRHL2. Targeted sequencing identified the same variant in three additional previously unsolved PPCD-affected families, including a de novo occurrence that suggests this is a recurrent mutation. Two further unique variants were identified in intron 1 of GRHL2 (c.20+257delT and c.20+133delA) in unrelated PPCD-affected families. GRHL2 is a transcription factor that suppresses epithelial-to-mesenchymal transition (EMT) and is a direct transcriptional repressor of ZEB1. ZEB1 mutations leading to haploinsufficiency cause PPCD3. We previously identified promoter mutations in OVOL2, a gene not normally expressed in the corneal endothelium, as the cause of PPCD1. OVOL2 drives mesenchymal-to-epithelial transition (MET) by directly inhibiting EMT-inducing transcription factors, such as ZEB1. Here, we demonstrate that the GRHL2 regulatory variants identified in PPCD4-affected individuals induce increased transcriptional activity in vitro. Furthermore, although GRHL2 is not expressed in corneal endothelial cells in control tissue, we detected GRHL2 in the corneal "endothelium" in PPCD4 tissue. These cells were also positive for epithelial markers E-Cadherin and Cytokeratin 7, indicating they have transitioned to an epithelial-like cell type. We suggest that mutations inducing MET within the corneal endothelium are a convergent pathogenic mechanism leading to dysfunction of the endothelial barrier and disease.

• Klíčová slova
• GRHL2, PPCD, corneal dystrophy, corneal edema, corneal endothelium, ectopic expression, epithelial-to-mesenchymal transition, mesenchymal-to-epithelial transition, non-coding mutation, regulatory region,
• MeSH
• dědičné dystrofie rohovky genetika   MeSH
• DNA vazebné proteiny genetika   MeSH
• genetická transkripce MeSH
• genetické lokusy MeSH
• HEK293 buňky MeSH
• intergenová DNA genetika   MeSH
• introny genetika   MeSH
• lidé MeSH
• modely genetické MeSH
• mutace genetika   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• rodina MeSH
• rodokmen MeSH
• rohovkový endotel patologie   MeSH
• sekvence nukleotidů MeSH
• sekvenování celého genomu MeSH
• transkripční faktory genetika   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• GRHL2 protein, human MeSH Prohlížeč
• intergenová DNA MeSH
• transkripční faktory MeSH

Epithelial-mesenchymal transition (EMT) is a crucial process with significance in the metastasis of malignant tumors. It is through the acquisition of plasticity that cancer cells become more mobile and gain the ability to metastasize to other tissues. The mesenchymal-epithelial transition (MET) is the return to an epithelial state, which allows for the formation of secondary tumors. Both processes, EMT and MET, are regulated by different pathways and different mediators, which affects the sophistication of the overall tumorigenesis process. Not insignificant are also cancer stem cells and their participation in the angiogenesis, which occur very intensively within tumors. Difficulties in effectively treating cancer are primarily dependent on the potential of cancer cells to rapidly expand and occupy secondarily vital organs. Due to the ability of these cells to spread, the concept of the circulating tumor cell (CTC) has emerged. Interestingly, CTCs exhibit molecular diversity and stem-like and mesenchymal features, even when derived from primary tumor tissue from a single patient. While EMT is necessary for metastasis, MET is required for CTCs to establish a secondary site. A thorough understanding of the processes that govern the balance between EMT and MET in malignancy is crucial.

• Klíčová slova
• Cancer stem Cells, Circulating Tumor cell (CTC), Epithelial-mesenchymal Transition (EMT), Mesenchymal-epithelial Transition (MET),
• MeSH
• epitelo-mezenchymální tranzice * MeSH
• fenotyp MeSH
• kmenové buňky metabolismus   cytologie   patologie   MeSH
• lidé MeSH
• nádorové cirkulující buňky * patologie   metabolismus   MeSH
• nádorové kmenové buňky * patologie   metabolismus   MeSH
• nádory patologie   metabolismus   MeSH
• patologická angiogeneze * patologie   MeSH
• proliferace buněk genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, U.S. Gov't, Non-P.H.S. 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.

• Klíčová slova
• EGF signaling, FGF signaling, branching morphogenesis, epithelial–stromal interactions, stromal microenvironment,
• 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
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• amfiregulin MeSH
• erbB receptory MeSH
• extracelulárním signálem regulované MAP kinasy MeSH
• fosfoproteiny MeSH
• kolagen MeSH
• ligandy MeSH
• membránové proteiny MeSH
• protoonkogenní proteiny c-akt MeSH
• Spry1 protein, mouse MeSH Prohlížeč
• transformující růstový faktor alfa 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.

• Klíčová slova
• H&E, U-Net, artificial intelligence, deep learning, digital pathology, immunohistochemistry, tissue registration,
• 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
• Názvy látek
• eosin MeSH
• hematoxylin MeSH
• keratiny * MeSH