Early Epithelial-mesenchymal Transition Research Articles

Genome-Wide CRISPR Screen Identifies an NF2-Adherens Junction Mechanistic Dependency for Cardiac Lineage.

Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear. We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 (MYH6)-GFP (green fluorescence protein) reporter system. After an independent secondary single guide ribonucleic acid validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal NF2 knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays. Transcriptional regulation and trajectory inference from NF2-null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor SOX2 and upregulation of late anticardiac regulators CDX2 and MSX1 in NF2 knockout cells reflect a necessary role for NF2 in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)-LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation. These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.

Feasibility of mass cytometry proteomic characterisation of circulating tumour cells in head and neck squamous cell carcinoma for deep phenotyping

BackgroundCirculating tumour cells (CTCs) are a potential cancer biomarker, but current methods of CTC analysis at single-cell resolution are limited. Here, we describe high-dimensional single-cell mass cytometry proteomic analysis of CTCs in HNSCC.MethodsParsortix microfluidic-enriched CTCs from 14 treatment-naïve HNSCC patients were analysed by mass cytometry analysis using 41 antibodies. Immune cell lineage, epithelial-mesenchymal transition (EMT), stemness, proliferation and immune checkpoint expression was assessed alongside phosphorylation status of multiple signalling proteins. Patient-matched tumour gene expression and CTC EMT profiles were compared. Standard bulk CTC RNAseq was performed as a baseline comparator to assess mass cytometry data.ResultsCTCs were detected in 13/14 patients with CTC counts of 2–24 CTCs/ml blood. Unsupervised clustering separated CTCs into epithelial, early EMT and advanced EMT groups that differed in signalling pathway activation state. Patient-specific CTC cluster patterns separated into immune checkpoint low and high groups. Patient tumour and CTC EMT profiles differed. Mass cytometry outperformed bulk RNAseq to detect CTCs and characterise cell phenotype.DiscussionWe demonstrate mass cytometry allows high-plex proteomic characterisation of CTCs at single-cell resolution and identify common CTC sub-groups with potential for novel biomarker development and immune checkpoint inhibitor treatment stratification.

Pharmacological targeting of netrin-1 inhibits EMT in cancer.

Epithelial-to-mesenchymal transition (EMT) regulates tumour initiation, progression, metastasis and resistance to anti-cancer therapy1-7. Although great progress has been made in understanding the role of EMT and its regulatory mechanisms in cancer, no therapeutic strategy to pharmacologically target EMT has been identified. Here we found that netrin-1 is upregulated in a primary mouse model of skin squamous cell carcinoma (SCC) exhibiting spontaneous EMT. Pharmacological inhibition of netrin-1 by administration of NP137, a netrin-1-blocking monoclonal antibody currently used in clinical trials in human cancer (ClinicalTrials.gov identifier NCT02977195 ), decreased the proportion of EMT tumour cells in skin SCC, decreased the number of metastases and increased the sensitivity of tumour cells to chemotherapy. Single-cell RNA sequencing revealed the presence of different EMT states, including epithelial, early and late hybrid EMT, and full EMT states, in control SCC. By contrast, administration of NP137 prevented the progression of cancer cells towards a late EMT state and sustained tumour epithelial states. Short hairpin RNA knockdown of netrin-1 and its receptor UNC5B in EPCAM+ tumour cells inhibited EMT in vitro in the absence of stromal cells and regulated a common gene signature that promotes tumour epithelial state and restricts EMT. To assess the relevance of these findings to human cancers, we treated mice transplanted with the A549 human cancer cell line-which undergoes EMT following TGFβ1 administration8,9-with NP137. Netrin-1 inhibition decreased EMT in these transplanted A549 cells. Together, our results identify a pharmacological strategy for targeting EMT in cancer, opening up novel therapeutic interventions for anti-cancer therapy.

Single cell meta-analysis of EndMT and EMT state in COVID-19.

COVID-19 prognoses suggests that a proportion of patients develop fibrosis, but there is no evidence to indicate whether patients have progression of mesenchymal transition (MT) in the lungs. The role of MT during the COVID-19 pandemic remains poorly understood. Using single-cell RNA sequencing, we profiled the transcriptomes of cells from the lungs of healthy individuals (n = 45), COVID-19 patients (n = 58), and idiopathic pulmonary fibrosis (IPF) patients (n = 64) human lungs to map the entire MT change. This analysis enabled us to map all high-resolution matrix-producing cells and identify distinct subpopulations of endothelial cells (ECs) and epithelial cells as the primary cellular sources of MT clusters during COVID-19. For the first time, we have identied early and late subgroups of endothelial mesenchymal transition (EndMT) and epithelial-mesenchymal transition (EMT) using analysis of public databases for single-cell sequencing. We assessed epithelial subgroups by age, smoking status, and gender, and the data suggest that the proportional changes in EMT in COVID-19 are statistically significant. Further enumeration of early and late EMT suggests a correlation between invasive genes and COVID-19. Finally, EndMT is upregulated in COVID-19 patients and enriched for more inflammatory cytokines. Further, by classifying EndMT as early or late stages, we found that early EndMT was positively correlated with entry factors but this was not true for late EndMT. Exploring the MT state of may help to mitigate the fibrosis impact of SARS-CoV-2 infection.

Pentraxin 3 mediates early inflammatory response and EMT process in human tubule epithelial cells induced by PM2.5

Pentraxin 3 (PTX3) is a multifunctional molecule that mainly expressed in response to proinflammatory stimuli under physiological and pathological conditions. It is produced in tubule epithelial cells that is involved in the innate immune response and inflammatory reactions in the kidney. However, its role in fine particulate matter (PM2.5)-induced renal injury associated with inflammation remains to be investigated. As a result of PM2.5 exposure, the levels of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α levels were increased in HK-2 cells. Notably, the mesenchymal phenotypes with migratory abilities of HK-2 cells were found following PM2.5 exposure. The elevated expressions of PTX3 mRNA and protein in response to PM2.5 were tested by RT-PCR and Western blotting respectively. Further determinate the role of PTX3 by siRNA showed lack of PTX3 could increase IL-6 production and promote epithelial-mesenchymal transition (EMT) process, as evidenced by decreased expressions of E-cadherin, and increased expressions of N-cadherin and α-SMA in HK-2 cells following PM2.5 exposure. Our study indicates that PTX3 mediates early inflammatory response and EMT in PM2.5-exposed HK-2 cells, suggesting a counter-regulatory role of PTX3 in the early course of tubule cell injury induced by PM2.5.

EPEN-29. Spatial transcriptomic analysis of ependymoma implicates unresolved wound healing as a driver of tumor progression

Abstract Ependymoma is a childhood brain tumor that remains incurable in approximately 50 percent of cases, most commonly in posterior fossa subgroup A (PFA). Uncovering how heterogeneous cell types within the tumor microenvironment (TME) interact is crucial to a complete understanding of PFA disease progression. The underlying cellular components of the PFA TME have been revealed by single-cell RNAseq (scRNAseq), identifying divergent epithelial differentiation and epithelial-mesenchymal transition (EMT) lineages. Here, we utilize spatial transcriptomics (Visium) of 14 PFA samples, integrated with scRNAseq, to chart neoplastic and immune cell architecture, with a higher resolution of cellular heterogeneity than scRNAseq alone. At a gross level, all PFA were primarily comprised of neoplastic epithelial and mesenchymal transcriptomic spatial zones, each containing a diversity of hierarchical cellular stages. In all samples we revealed spatially and transcriptomically-distinct mesenchymal zone-associated subclusters, including a quiescent undifferentiated progenitor-like subpopulation and clusters with characteristics of early and late stage EMT. Two early stage EMT clusters were distinguished by signatures of either myeloid cell interaction or hypoxia, and both were demonstrated to be EMT-initiating processes in in-vitro PFA experimental models. Myeloid cell interaction is the predominant initiating stage of EMT in PFA, occurring in zones that are spatially distinct from hypoxia induced EMT. Other mesenchymal clusters represent later EMT stages characterized by wound repair and tissue remodeling. Increased proliferation was a general characteristic of epithelial zone clusters, which included a second undifferentiated progenitor-like population that showed a particularly high mitotic rate and was associated with histologically hypercellular areas. Given the biological parallels with normal wound healing, we propose that mesenchymal and epithelial zones interact to create a cycle of persistent tissue damage response and mitogenic re-epithelialization signals. Unresolved wound repair is therefore a potential driver of PFA progression, a new concept that could provide novel targets for effective therapeutic intervention.

Partial EMT takes the lead in cancer metastasis.

In this issue of Developmental Cell, Lüönd etal. developed a tracing system, using the uncharacterized early epithelial-mesenchymal transition (EMT)-marker Tenascin C, to monitor cells undergoing partial EMT during malignant mammary cancer progression. They find that partial, but not full, EMT contributes to metastasis and that full EMT contributes to chemoresistance.

Cholangiocyte Epithelial to Mesenchymal Transition (EMT) is a potential molecular mechanism driving ischemic cholangiopathy in liver transplantation.

Donation after circulatory death (DCD) has expanded the donor pool for liver transplantation. However, ischemic cholangiopathy (IC) after DCD liver transplantation causes inferior outcomes. The molecular mechanisms of IC are currently unknown but may depend on ischemia-induced genetic reprograming of the biliary epithelium to mesenchymal-like cells. The main objective of this study was to determine if cholangiocytes undergo epithelial to mesenchymal transition (EMT) after exposure to DCD conditions and if this causally contributes to the phenotype of IC. Human cholangiocyte cultures were exposed to periods of warm and cold ischemia to mimic DCD liver donation. EMT was tested by assays of cell migration, cell morphology, and differential gene expression. Transplantation of syngeneic rat livers recovered under DCD conditions were evaluated for EMT changes by immunohistochemistry. Human cholangiocytes exposed to DCD conditions displayed migratory behavior and gene expression patterns consistent with EMT. E-cadherin and CK-7 expressions fell while N-cadherin, vimentin, TGFβ, and SNAIL rose, starting 24 hours and peaking 1–3 weeks after exposure. Cholangiocyte morphology changed from cuboidal (epithelial) before to spindle shaped (mesenchymal) a week after ischemia. These changes were blocked by pretreating cells with the Transforming Growth Factor beta (TGFβ) receptor antagonist Galunisertib (1 μM). Finally, rats with liver isografts cold stored for 20 hours in UW solution and exposed to warm ischemia (30 minutes) at recovery had elevated plasma bilirubin 1 week after transplantation and the liver tissue showed immunohistochemical evidence of early cholangiocyte EMT. Our findings show EMT occurs after exposure of human cholangiocytes to DCD conditions, which may be initiated by upstream signaling from autocrine derived TGFβ to cause mesenchymal specific morphological and migratory changes.

Snail Overexpression Alters the microRNA Content of Extracellular Vesicles Released from HT29 Colorectal Cancer Cells and Activates Pro-Inflammatory State In Vivo.

Simple SummaryKnowledge of the factors that help migration of carcinoma cells is important for prevention of metastasis. Cancer cells release small particles, extracellular vesicles (EVs) that contain such factors. The aim of this study was to assess if the content of EVs changes through different stages of colorectal cancer (CRC) and evaluate how this process affects cancer progression in vivo in mouse CRC model. We found that EVs released from cells that have migratory properties contain different factors then EVs released from original tumor cells. We also show here that EVs can be incorporated into other cells that facilitate metastasis and change their properties depending on the EVs content. The content of cell-released EVs may also serve as a biomarker that denotes the stage of CRC and may be a target to prevent cancer progression.During metastasis, cancer cells undergo phenotype changes in the epithelial-mesenchymal transition (EMT) process. Extracellular vesicles (EVs) released by cancer cells are the mediators of intercellular communication and play a role in metastatic process. Knowledge of factors that influence the modifications of the pre-metastatic niche for the migrating carcinoma cells is important for prevention of metastasis. We focus here on how cancer progression is affected by EVs released from either epithelial-like HT29-cells or from cells that are in early EMT stage triggered by Snail transcription factor (HT29-Snail). We found that EVs released from HT29-Snail, as compared to HT29-pcDNA cells, have a different microRNA profile. We observed the presence of interstitial pneumonias in the lungs of mice injected with HT29-Snail cells and the percent of mice with lung inflammation was higher after injection of HT29-Snail-EVs. Incorporation of EVs released from HT29-pcDNA, but not released from HT29-Snail, leads to the increased secretion of IL-8 from macrophages. We conclude that Snail modifications of CRC cells towards more invasive phenotype also alter the microRNA cargo of released EVs. The content of cell-released EVs may serve as a biomarker that denotes the stage of CRC and EVs-specific microRNAs may be a target to prevent cancer progression.

Helicobacter pylori induces epithelial-mesenchymal transition in gastric carcinogenesis via the AKT/GSK3β signaling pathway.

Helicobacter pylori (H. pylori) is a main risk factor for gastric cancer (GC). Epithelial-mesenchymal transition (EMT) is involved in the development and progression of H. pylori-associated GC. However, the exact molecular mechanism of this process remains unclear. The AKT/GSK3β signaling pathway has been demonstrated to promote EMT in several types of cancer. The present study investigated whether H. pylori infection induced EMT, and promoted the development and metastasis of cancer in the normal gastric mucosa, and whether this process was dependent on AKT activation. The expression levels of the EMT-associated proteins, including E-cadherin and N-cadherin, were determined in 165 gastric mucosal samples of different disease stages by immunohistochemical analysis. The expression levels of E-cadherin, N-cadherin, AKT, phosphorylated (p-)AKT (Ser473), GSK3β and p-GSK3β (Ser9) were further determined in H. pylori-infected Mongolian gerbil gastric tissues and cells co-cultured with H. pylori by immunohistochemical analysis and western blotting. The results indicated that the expression levels of the epithelial marker E-cadherin were decreased, whereas the expression levels of the mesenchymal marker N-cadherin were increased during gastric carcinogenesis. Their expression levels were associated with H. pylori infection. Furthermore, H. pylori infection resulted in downregulation of E-cadherin expression and upregulation of N-cadherin expression in Mongolian gerbils and GES-1 cells. In addition, an investigation of the associated mechanism of action revealed that p-AKT (Ser473) and p-GSK3β (Ser9) were activated in GES-1 cells following co-culture with H. pylori. Furthermore, following pretreatment of the cells with the AKT inhibitor VIII, the expression levels of E-cadherin, N-cadherin, p-AKT and p-GSK3β did not show significant differences between GES-1 cells that were co-cultured with or without H. pylori. The levels of p-AKT and p-GSK3β were increased in H. pylori-infected Mongolian gerbils. In conclusion, the present study demonstrated that H. pylori infection activated AKT and resulted in the phosphorylation and inactivation of GSK3β, which in turn promoted early stage EMT. These effects were AKT-dependent. This mechanism may serve as a prerequisite for GC development.

Cisplatin prevents breast cancer metastasis through blocking early EMT and retards cancer growth together with paclitaxel.

Cancer growth is usually accompanied by metastasis which kills most cancer patients. Here we aim to study the effect of cisplatin at different doses on breast cancer growth and metastasis.Methods: We used cisplatin to treat breast cancer cells, then detected the migration of cells and the changes of epithelial-mesenchymal transition (EMT) markers by migration assay, Western blot, and immunofluorescent staining. Next, we analyzed the changes of RNA expression of genes by RNA-seq and confirmed the binding of activating transcription factor 3 (ATF3) to cytoskeleton related genes by ChIP-seq. Thereafter, we combined cisplatin and paclitaxel in a neoadjuvant setting to treat xenograft mouse models. Furthermore, we analyzed the association of disease prognosis with cytoskeletal genes and ATF3 by clinical data analysis.Results: When administered at a higher dose (6 mg/kg), cisplatin inhibits both cancer growth and metastasis, yet with strong side effects, whereas a lower dose (2 mg/kg) cisplatin blocks cancer metastasis without obvious killing effects. Cisplatin inhibits cancer metastasis through blocking early steps of EMT. It antagonizes transforming growth factor beta (TGFβ) signaling through suppressing transcription of many genes involved in cytoskeleton reorganization and filopodia formation which occur early in EMT and are responsible for cancer metastasis. Mechanistically, TGFβ and fibronectin-1 (FN1) constitute a positive reciprocal regulation loop that is critical for activating TGFβ/SMAD3 signaling, which is repressed by cisplatin induced expression of ATF3. Furthermore, neoadjuvant administration of cisplatin at 2 mg/kg in conjunction with paclitaxel inhibits cancer growth and blocks metastasis without causing obvious side effects by inhibiting colonization of cancer cells in the target organs.Conclusion: Thus, cisplatin prevents breast cancer metastasis through blocking early EMT, and the combination of cisplatin and paclitaxel represents a promising therapy for killing breast cancer and blocking tumor metastasis.

Microgravity Induces Transient EMT in Human Keratinocytes by Early Down-Regulation of E-Cadherin and Cell-Adhesion Remodeling

Changes in cell–matrix and cell-to-cell adhesion patterns are dramatically fostered by the microgravity exposure of living cells. The modification of adhesion properties could promote the emergence of a migrating and invasive phenotype. We previously demonstrated that short exposure to the simulated microgravity of human keratinocytes (HaCaT) promotes an early epithelial–mesenchymal transition (EMT). Herein, we developed this investigation to verify if the cells maintain the acquired invasive phenotype after an extended period of weightlessness exposure. We also evaluated cells’ capability in recovering epithelial characteristics when seeded again into a normal gravitational field after short microgravity exposure. We evaluated the ultra-structural junctional features of HaCaT cells by Transmission Electron Microscopy and the distribution pattern of vinculin and E-cadherin by confocal microscopy, observing a rearrangement in cell–cell and cell–matrix interactions. These results are mirrored by data provided by migration and invasion biological assay. Overall, our studies demonstrate that after extended periods of microgravity, HaCaT cells recover an epithelial phenotype by re-establishing E-cadherin-based junctions and cytoskeleton remodeling, both being instrumental in promoting a mesenchymal–epithelial transition (MET). Those findings suggest that cytoskeletal changes noticed during the first weightlessness period have a transitory character, given that they are later reversed and followed by adaptive modifications through which cells miss the acquired mesenchymal phenotype.

Glypican-1 Level Is Elevated in Extracellular Vesicles Released from MC38 Colon Adenocarcinoma Cells Overexpressing Snail.

The transcription factor Snail triggers epithelial-to-mesenchymal transition (EMT), endowing cancer cells with invasive properties during tumor progression. Extracellular vesicles (EVs) released from cancer cells at various stages of cancer progression are known to influence the tumor pre-metastatic niche and metastatic potential. The aim of this study was to analyze the effect of Snail on murine colon adenocarcinoma cells (MC38 line) and on the characteristics of their EVs. Stable clones of Snail-overexpressing MC38 cells were investigated in vitro versus Mock cells. Increased expression of matrix metalloproteinase MMP-14 and augmented activity of MMP-9 and -14 were observed in Snail-MC38 cells. There was no change in the transcriptomic profile of proteoglycans in Snail-MC38 cells; however, the protein level of Glypican-1 (GPC1) was enhanced in EVs released from those cells. Our finding that GPC1 protein level was enhanced in EVs released from MC38 cells that overexpressed Snail and were in an early EMT stage might explain the specificity of the GPC1 biomarker in colon cancer diagnosis. Further, our data suggest that Snail, by changing the level of GPC1 on EVs released by colon cancer cells, may affect the generation of a distant premetastatic niche and metastatic organotropism in colon adenocarcinoma.

Early Events in Actin Cytoskeleton Dynamics and E-Cadherin-Mediated Cell-Cell Adhesion during Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) plays an important role in development and also in initiation of metastasis during cancer. Disruption of cell-cell contacts during EMT allowing cells to detach from and migrate away from their neighbors remains poorly understood. Using immunofluorescent staining and live-cell imaging, we analyzed early events during EMT induced by epidermal growth factor (EGF) in IAR-20 normal epithelial cells. Control cells demonstrated stable adherens junctions (AJs) and robust contact paralysis, whereas addition of EGF caused rapid dynamic changes at the cell-cell boundaries: fragmentation of the circumferential actin bundle, assembly of actin network in lamellipodia, and retrograde flow. Simultaneously, an actin-binding protein EPLIN was phosphorylated, which may have decreased the stability of the circumferential actin bundle. Addition of EGF caused gradual replacement of linear E-cadherin–based AJs with dynamic and unstable punctate AJs, which, unlike linear AJs, colocalized with the mechanosensitive protein zyxin, confirming generation of centripetal force at the sites of cell-cell contacts during EMT. Our data show that early EMT promotes heightened dynamics at the cell-cell boundaries—replacement of stable AJs and actin structures with dynamic ones—which results in overall weakening of cell-cell adhesion, thus priming the cells for front-rear polarization and eventual migration.

Metabolic switch and epithelial-mesenchymal transition cooperate to regulate pluripotency.

Both metabolic switch from oxidative phosphorylation to glycolysis (OGS) and epithelial–mesenchymal transition (EMT) promote cellular reprogramming at early stages. However, their connections have not been elucidated. Here, when a chemically defined medium was used to induce early EMT during mouse reprogramming, a facilitated OGS was also observed at the same time. Additional investigations suggested that the two events formed a positive feedback loop via transcriptional activation, cooperated to upregulate epigenetic factors such as Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5, and accelerated pluripotency induction at the early stage. However, at late stages, by over‐inducing glycolysis and preventing the necessary mesenchymal–epithelial transition, the two events trapped the cells at a new pluripotency state between naïve and primed states and inhibited further reprogramming toward the naïve state. In addition, the pluripotent stem cells at the new state have high similarity to epiblasts from E4.5 and E5.5 embryos, and have distinct characteristics from the previously reported epiblast‐like or formative states. Therefore, the time‐dependent cooperation between OGS and EMT in regulating pluripotency should extend our understanding of related fields.

Tendon Biomimetic Electrospun PLGA Fleeces Induce an Early Epithelial-Mesenchymal Transition and Tenogenic Differentiation on Amniotic Epithelial Stem Cells.

Background. The design of tendon biomimetic electrospun fleece with Amniotic Epithelial Stem Cells (AECs) that have shown a high tenogenic attitude may represent an alternative strategy to overcome the unsatisfactory results of conventional treatments in tendon regeneration. Methods. In this study, we evaluated AEC-engineered electrospun poly(lactide-co-glycolide) (PLGA) fleeces with highly aligned fibers (ha-PLGA) that mimic tendon extracellular matrix, their biocompatibility, and differentiation towards the tenogenic lineage. PLGA fleeces with randomly distributed fibers (rd-PLGA) were generated as control. Results. Optimal cell infiltration and biocompatibility with both PLGA fleeces were shown. However, only ha-PLGA fleeces committed AECs towards an Epithelial-Mesenchymal Transition (EMT) after 48 h culture, inducing their cellular elongation along the fibers’ axis and the upregulation of mesenchymal markers. AECs further differentiated towards tenogenic lineage as confirmed by the up-regulation of tendon-related genes and Collagen Type 1 (COL1) protein expression that, after 28 days culture, appeared extracellularly distributed along the direction of ha-PLGA fibers. Moreover, long-term co-cultures of AEC-ha-PLGA bio-hybrids with fetal tendon explants significantly accelerated of half time AEC tenogenic differentiation compared to ha-PLGA fleeces cultured only with AECs. Conclusions. The fabricated tendon biomimetic ha-PLGA fleeces induce AEC tenogenesis through an early EMT, providing a potential tendon substitute for tendon engineering research.

Epithelial-mesenchymal transition markers screened in a cell-based model and validated in lung adenocarcinoma

BackgroundRe-capture of the differences between tumor and normal tissues observed at the patient level in cell cultures and animal models is critical for applications of these cancer-related differences. The epithelial-mesenchymal transition (EMT) process is essential for tumor migratory and invasive capabilities. Although plenty of EMT markers are revealed, molecular features during the early stages of EMT are poorly understood.MethodsA cell-based model to induce lung cell (A549) EMT using conditioned medium of in vitro cancer activated fibroblast (WI38) was established. High-throughput sequencing methods, including RNA-seq and miRNA-seq, and advanced bioinformatics methods were used to explore the transcriptome profile transitions accompanying the progression of EMT. We validated our findings with experimental techniques including transwell and immunofluorescence assay, as well as the TCGA data.ResultsWe have constructed an in vitro cell model to mimic the EMT in patients. We discovered that several new transcription factors were among the early genes (3 h) to respond to cancer micro-environmental cues which could play critical roles in triggering further EMT signals. The early EMT markers also included genes encoding membrane transporters and blood coagulation function. Three of the nine-examined early EMT hallmark genes, GALNT6, SPARC and HES7, were up-regulated specifically in the early stages of lung adenocarcinoma (LUAD) and confirmed by TCGA patient transcriptome data. In addition, we showed that miR-3613, a regulator of EGFR pathway genes, was constantly repressed during EMT progress and indicative of an epithelial miRNA marker.ConclusionsThe CAF-stimulated EMT cell model may recapture some of the molecular changes during EMT progression in clinical patients. The identified early EMT hallmark genes GALNT6, SPARC and HES7and miR-3613 provide new markers and therapeutic targets for LUAD for the further clinical diagnosis and drug screening.

Regulation of miRNAs by Snail during epithelial-to-mesenchymal transition in HT29 colon cancer cells

Epithelial-to-mesenchymal transition (EMT) in cancer cells, represents early stages of metastasis and is a promising target in colorectal cancer (CRC) therapy. There have been many attempts to identify markers and key pathways induced throughout EMT but the process is complex and depends on the cancer type and tumour microenvironment. Here we used the colon cancer cell line HT29, which stably overexpressed Snail, the key transcription factor in early EMT, as a model for colorectal adenocarcinoma cells with a pro-metastatic phenotype. We investigated miRNA expression regulation during that phenotypic switching. We found that overexpression of Snail in HT29 cells triggered significant changes in individual miRNA levels but did not change the global efficiency of miRNA processing. Snail abundance repressed the expression of miR-192 and miR-194 and increased miR-205, let-7i and SNORD13 levels. These identified changes correlated with the reported transcriptomic alterations in Snail-overexpressing HT29 cells. We also investigated how Snail affected the miRNA content of extracellular vesicles (EVs) released from HT29 cells. Our data suggest that the presence of Snail significantly alters the complex mRNA/miRNA interactions in the early steps of metastasis and also has an impact on the content of EVs released from HT29 cells.

SnapshotDx Quiz: December 2018

SnapshotDx Quiz: December 2018

Effect of Wnt/β-catenin signal regulating EMT level on the differentiation of mouse ESC to liver tissue

Objective: To study embryonic stem cell (ESC) differentiation into liver tissue structure from the perspective of epithelial mesenchymal transition (EMT). Methods: ESC of Balb/c mice was selected to induced into hepatic cell using hepatocyte growth factor (HGF), fibroblast growth factor (FGF) in vitro, and at the time points of metaphase (13 d) and maturity (17 d) of differentiation, dynamic inhibition of Wnt/β-catenin signal was made to reduce the level of EMT. Finally, three-dimensional organization structure growth of the differentiation cells was observed in the differentiation system.Expressions of the liver cells vascular markers[albumin (Alb) and vascular endothelial growth factor (VEGFR)]were detected. Results: During the differentiation of ESC, the level of early EMT in the experimental group and the control group was not significantly different. The level of mid-late EMT in the experiment group was significantly lower than the control group. On the day 18 and 20 of differentiation, the relative mRNA expression level of E-cadherin was 0.61±0.15 and 0.47±0.05 in the experimental group, and 0.07±0.05 and 0 in the control group, respectively.The expression level of ALB/AFP/CK8/CK19 in the experimental group was generally higher than that of the control group in the same period, while CD31/VEGFR1 markers in the experimental group decreased more slowly in the late period of differentiation compared with the control group. In the supernatant of ESC culture, the Alb of the experimental group could be detected onday 7, and the concentration was (0.32±0.02) mg/L, while Alb in the control group was (0.19±0.05) mg/L. Urea in the experimental group could be detected on the day 13, and the concentration was (8.7 ±1.0) μmol/L, and the urea concentration of the control group was (3.1±1.2) μmol/L. The concentration of Alb and urea in the culture supernatant of ESC differentiation system increased significantly with the prolongation of the differentiation time, and the Alb and urea concentrations in the experimental group were significantly higher than those in the control group at the same time period. In addition, the differentiated cells in the experimental group could maintain the growth of three-dimensional tissue, while the differentiated cells in the control group eventually showed a single cell state. The expression of hepatic and vascular cell markers could be detected in the experimental group. Immunofluorescence results showed that the hepatocytes and vascular structures were tightly arranged. HE staining showed the formation of hepatic lobular structure, while the control group had no vascular component markers. Conclusion: The differentiation of ESC into liver tissue can be effectively promoted by decreasing the level of EMT at the mid-late stage of ESC differentiation.