Guidelines and definitions for research on epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) encompasses dynamic changes in cellular organization from epithelial to mesenchymal phenotypes, which leads to functional changes in cell migration and invasion. EMT occurs in a diverse range of physiological and pathological conditions and is driven by a conserved set of inducing signals, transcriptional regulators and downstream effectors. With over 5,700 publications indexed by Web of Science in 2019 alone, research on EMT is expanding rapidly. This growing interest warrants the need for a consensus among researchers when referring to and undertaking research on EMT. This Consensus Statement, mediated by 'the EMT International Association' (TEMTIA), is the outcome of a 2-year-long discussion among EMT researchers and aims to both clarify the nomenclature and provide definitions and guidelines for EMT research in future publications. We trust that these guidelines will help to reduce misunderstanding and misinterpretation of research data generated in various experimental models and to promote cross-disciplinary collaboration to identify and address key open questions in this research field. While recognizing the importance of maintaining diversity in experimental approaches and conceptual frameworks, we emphasize that lasting contributions of EMT research to increasing our understanding of developmental processes and combatting cancer and other diseases depend on the adoption of a unified terminology to describe EMT.

PLA2G12A as a Novel Biomarker for Colorectal Cancer with Prognostic Relevance.

Metastasis is the leading cause of colorectal cancer (CRC)-related deaths. Therefore, the identification of accurate biomarkers predictive of metastasis is needed to better stratify high-risk patients to provide preferred management and reduce mortality. In this study, we identified 13 new genes that modified circulating tumor cell numbers using a genome-wide genetic screen in a whole animal CRC model. Candidate genes were subsequently evaluated at the gene expression level in both an internal human CRC cohort of 153 patients and an independent cohort from the TCGA including 592 patients. Interestingly, the expression of one candidate, PLA2G12A, significantly correlated with both the time to recurrence and overall survival in our CRC cohort, with its low expression being an indicator of a poor clinical outcome. By examining the TCGA cohort, we also found that low expression of PLA2G12A was significantly enriched in epithelial-mesenchymal transition signatures. Finally, the candidate functionality was validated in vitro using three different colon cancer cell lines, revealing that PLA2G12A deficiency increases cell proliferation, migration, and invasion. Overall, our study identifies PLA2G12A as a prognostic biomarker of early-stage CRC, providing evidence that its deficiency promotes tumor growth and dissemination.

The endoderm: a divergent cell lineage with many commonalities.

The endoderm is a progenitor tissue that, in humans, gives rise to the majority of internal organs. Over the past few decades, genetic studies have identified many of the upstream signals specifying endoderm identity in different model systems, revealing them to be divergent from invertebrates to vertebrates. However, more recent studies of the cell behaviours driving endodermal morphogenesis have revealed a surprising number of shared features, including cells undergoing epithelial-to-mesenchymal transitions (EMTs), collective cell migration, and mesenchymal-to-epithelial transitions (METs). In this Review, we highlight how cross-organismal studies of endoderm morphogenesis provide a useful perspective that can move our understanding of this fascinating tissue forward.

Epithelial-Mesenchymal Transition (EMT) as a Therapeutic Target.

Metastasis is the spread of cancer cells from the primary tumour to distant sites and organs throughout the body. It is the primary cause of cancer morbidity and mortality, and is estimated to account for 90% of cancer-related deaths. During the initial steps of the metastatic cascade, epithelial cancer cells undergo an epithelial-mesenchymal transition (EMT), and as a result become migratory and invasive mesenchymal-like cells while acquiring cancer stem cell properties and therapy resistance. As EMT is involved in such a broad range of processes associated with malignant transformation, it has become an increasingly interesting target for the development of novel therapeutic strategies. Anti-EMT therapeutic strategies could potentially not only prevent the invasion and dissemination of cancer cells, and as such prevent the formation of metastatic lesions, but also attenuate cancer stemness and increase the effectiveness of more classical chemotherapeutics. In this review, we give an overview about the pros and cons of therapies targeting EMT and discuss some already existing candidate drug targets and high-throughput screening tools to identify novel anti-EMT compounds.

Differential roles of the Drosophila EMT-inducing transcription factors Snail and Serpent in driving primary tumour growth.

Several transcription factors have been identified that activate an epithelial-to-mesenchymal transition (EMT), which endows cells with the capacity to break through basement membranes and migrate away from their site of origin. A key program in development, in recent years it has been shown to be a crucial driver of tumour invasion and metastasis. However, several of these EMT-inducing transcription factors are often expressed long before the initiation of the invasion-metastasis cascade as well as in non-invasive tumours. Increasing evidence suggests that they may promote primary tumour growth, but their precise role in this process remains to be elucidated. To investigate this issue we have focused our studies on two Drosophila transcription factors, the classic EMT inducer Snail and the Drosophila orthologue of hGATAs4/6, Serpent, which drives an alternative mechanism of EMT; both Snail and GATA are specifically expressed in a number of human cancers, particularly at the invasive front and in metastasis. Thus, we recreated conditions of Snail and of Serpent high expression in the fly imaginal wing disc and analysed their effect. While either Snail or Serpent induced a profound loss of epithelial polarity and tissue organisation, Serpent but not Snail also induced an increase in the size of wing discs. Furthermore, the Serpent-induced tumour-like tissues were able to grow extensively when transplanted into the abdomen of adult hosts. We found the differences between Snail and Serpent to correlate with the genetic program they elicit; while activation of either results in an increase in the expression of Yorki target genes, Serpent additionally activates the Ras signalling pathway. These results provide insight into how transcription factors that induce EMT can also promote primary tumour growth, and how in some cases such as GATA factors a 'multi hit' effect may be achieved through the aberrant activation of just a single gene.

Sensitive High-Throughput Assays for Tumour Burden Reveal the Response of a Drosophila melanogaster Model of Colorectal Cancer to Standard Chemotherapies.

Drosophila melanogaster (Drosophila) models of cancer are emerging as powerful tools to investigate the basic mechanisms underlying tumour progression and identify novel therapeutics. Rapid and inexpensive, it is possible to carry out genetic and drug screens at a far larger scale than in vertebrate organisms. Such whole-organism-based drug screens permits assessment of drug absorption and toxicity, reducing the possibility of false positives. Activating mutations in the Wnt and Ras signalling pathways are common in many epithelial cancers, and when driven in the adult Drosophila midgut, it induces aggressive intestinal tumour-like outgrowths that recapitulate many aspects of human colorectal cancer (CRC). Here we have taken a Drosophila CRC model in which tumourous cells are marked with both GFP and luciferase reporter genes, and developed novel high-throughput assays for quantifying tumour burden. Leveraging these assays, we find that the Drosophila CRC model responds rapidly to treatment with standard CRC-drugs, opening the door to future rapid genetic and drug screens.

A common framework for EMT and collective cell migration.

During development, cells often switch between static and migratory behaviours. Such transitions are fundamental events in development and are linked to harmful consequences in pathology. It has long been considered that epithelial cells either migrate collectively as epithelial cells, or undergo an epithelial-to-mesenchymal transition and migrate as individual mesenchymal cells. Here, we assess what is currently known about in vivo cell migratory phenomena and hypothesise that such migratory behaviours do not fit into alternative and mutually exclusive categories. Rather, we propose that these categories can be viewed as the most extreme cases of a general continuum of morphological variety, with cells harbouring different degrees or combinations of epithelial and mesenchymal features and displaying an array of migratory behaviours.

Model for Interpreting Surface Crystallization Using Quartz Crystal Microbalance: Theory and Experiments.

Surface crystallization of calcium sulfate was investigated using a dissipation crystal quartz microbalance (QCM-D) together with microscopy to understand the mechanical property changes occurring during the growth process. The use of optical microscopy and SEM revealed that needle-shaped crystals grow as clusters on the QCM sensor's surface, not in uniform layers. As crystallization growth progressed, QCM-D revealed inversions between negative and positive frequency shifts. This behavior, a function of the growth of crystals in clusters, is not adequately predicted by existing models. As such, a new mass-to-frequency conversion model is proposed herein to explain the observed frequency inversions. This model is derived from a lumped element approach with point-contact loading and Mason equivalent circuit theory. Critically, the physical phenomena occurring form the basis of the model, particularly addressing the three sources of impedance. When a crystal nucleates and grows, its inertial impedance is considered along with a Kelvin-Voigt link with a hydration layer. A comparison between the proposed model and experimental data, of both frequency and dissipation data for the first four harmonics, shows good agreement for the supersaturations (S = C/C*) of S = 3.75, S = 3.48, and S = 3.22. Additionally, significant improvements over existing models for the case of surface crystallization are observed. The proposed model was therefore able to explain that frequency inversions are caused by a shift from inertia-dominated to elastic-dominated impedance, occurring as a result of crystal growth. Using the nucleation induction time and nucleation rates, determined with imaging, an additional understanding of the crystals' mechanical properties (stiffness and dampening) was obtained.

Modelling Cancer Metastasis in Drosophila melanogaster.

Cancer metastasis, the process by which tumour cells spread throughout the body and form secondary tumours at distant sites, is the leading cause of cancer-related deaths. The metastatic cascade is a highly complex process encompassing initial dissemination from the primary tumour, travel through the blood stream or lymphatic system, and the colonisation of distant organs. However, the factors enabling cells to survive this stressful process and adapt to new microenvironments are not fully characterised. Drosophila have proven a powerful system in which to study this process, despite important caveats such as their open circulatory system and lack of adaptive immune system. Historically, larvae have been used to model cancer due to the presence of pools of proliferating cells in which tumours can be induced, and transplanting these larval tumours into adult hosts has enabled tumour growth to be monitored over longer periods. More recently, thanks largely to the discovery that there are stem cells in the adult midgut, adult models have been developed. We focus this review on the development of different Drosophila models of metastasis and how they have contributed to our understanding of important factors determining metastatic potential, including signalling pathways, the immune system and the microenvironment.

Effects of the Ionic Liquid Structure on Porosity of Lignin-Derived Carbon Materials.

Converting lignin into advanced porous carbon materials, with desirable surface functionalities, can be challenging. While lignin-derived carbons produced by pyrolysis at >600 °C develop porosity, they also simultaneously lose nearly all their surface functional groups. By contrast, pyrolysis of lignin at lower temperatures (e.g., <400 °C) results in the formation of nonporous char that retains some surface functionalities. However, copyrolysis of lignin with some ionic liquids (ILs) at lower temperatures offers an opportunity to produce porous carbon materials with both large surface areas and an abundance of surface functional groups. This study investigates the effects of IL properties (solubility, thermal, and ionic size) on the specific surface areas of lignin-derived carbons produced by copyrolysis of lignin and ILs at 350-400 °C for 20 min. It was found that ILs that have bulky anions and small cation sizes can induce porosity in lignin-derived carbons with large surface areas. Among 16 ILs that were tested, [C2MIm][NTF2] demonstrated the best performance; the inclusion of it in the copyrolysis process resulted in lignin-derived carbons with ∼528 m2 g-1 and 0.48 cm3 g-1. Lignin-derived carbons produced using no IL, [C2MIm][NTF2], and [C4MIm][OTF] were further characterized for morphology, interfacial chemical, and elemental properties. The copyrolysis of lignin and [C2MIm][NTF2], and [C4MIm][OTF] resulted in doping of heteroatoms (N and S) on the porous carbon materials during pyrolysis reaction. The present findings contribute to a better understanding of the main property of ILs responsible for creating porosity in lignin carbon during pyrolysis.

Controlling simonkolleite crystallisation via metallic Zn oxidation in a betaine hydrochloride solution.

Zinc oxide nanoparticles, with a hexagonal flake structure, are of significant interest across a range of applications including photocatalysis and biomedicine. Simonkolleite (Zn5(OH)8Cl2·H2O), a layered double hydroxide, is a precursor for ZnO. Most simonkolleite synthesis routes require precise pH adjustment of Zn-containing salts in alkaline solution, and still produce some undesired morphologies along with the hexagonal one. Additionally, liquid-phase synthesis routes, based on conventional solvents, are environmentally burdensome. Herein aqueous ionic liquid, betaine hydrochloride (betaine·HCl), solutions are used to directly oxidise metallic Zn, producing pure simonkolleite nano/microcrystals (X-ray diffraction analysis, thermogravimetric analysis). Imaging (scanning electron microscopy) showed regular and uniform hexagonal simonkolleite flakes. Morphological control, as a function of reaction conditions (betaine·HCl concentration, reaction time, and reaction temperature), was achieved. Different growth mechanisms were observed as a function of the concentration of betaine·HCl solution, both traditional classical growth of individual crystals and non-traditional growth patterns; the latter included examples of Ostwald ripening and oriented attachment. After calcination, simonkolleite's transformation into ZnO retains its hexagonal skeleton; this produces a nano/micro-ZnO with a relatively uniform shape and size through a convenient reaction route.

Effective pretreatment of lignin-rich coconut wastes using a low-cost ionic liquid.

Coconut husks and shells are underutilised agricultural feedstocks in the bio-based industry. These biomass wastes have a higher lignin content than other woody biomass and have excellent potential as raw materials for the production of lignin-based materials. This work demonstrates the performance of a low-cost protic ionic liquid, N,N,N-dimethylbutylammonium hydrogen sulfate ([DMBA][HSO4]), for ionoSolv pretreatment of coconut husk and shell at 150 °C for 45-90 min and 170 °C for 15-60 min. Optimum pretreatment conditions were observed at 170 °C and 45 min for both feedstocks. At these conditions, [DMBA][HSO4] was able to remove almost 77 wt% of the lignin from the husk; leaving a cellulosic rich pulp behind, which released 82 % of the theoretical maximum glucose after enzymatic saccharification. The pretreated shell, by comparison, achieved 82 wt% lignin removal and 89 % glucose yield and these higher values could be attributed to the highly porous structure of coconut shell cell walls. The cleavage of the ß-O-4 aryl ether linkages of lignin followed by extensive C-C condensation in the lignin at longer pretreatment times was shown by HSQC NMR analysis. This extensive condensation was evidenced by molecular weights > 10,000 g/mol exhibited by lignin precipitated after pretreatment at high temperature and long times. The high degree of lignin removal and high glucose release from both feedstocks demonstrate that [DMBA][HSO4] is an excellent ionic liquid for fractionation of very lignin-rich biomass.

Exploring the role of crystal habit in the Ostwald rule of stages.

The crystallization of calcium carbonate is shown to be dictated by the Ostwald rule of stages (ORS), for high relative initial supersaturations ( S CaCO 3 = [ C a 2 + ] [ CO 3 2 - ] / K SP, Calcite > 2500 ), under sweet (carbon dioxide saturated) and anoxic (oxygen depleted) solution conditions. Rhombohedral calcite crystals emerge after the sequential crystallization and dissolution of the metastable polymorphs: vaterite (snowflake-shaped) and aragonite (needle-shaped). However, the presence of certain cations, which can form trigonal carbonates (e.g. Fe2+ and Ni2+), in concentrations as low as 1.5 mM, triggers the emergence of calcite crystals, with a star-shaped crystal habit, first. These star-shaped crystals dissolve to yield needle-shaped aragonite crystals, which in turn dissolve to give the rhombohedral calcite crystals. The star-shaped crystals, formed at high SCaCO3 , possess higher surface free energy (therefore higher apparent solubility) than their rhombohedral counterparts. This sequence of dissolution and recrystallization demonstrates that the ORS does not only drive the crystal towards its thermodynamically most stable polymorph but also towards its most stable crystal habit.

Precursor engineering of hydrotalcite-derived redox sorbents for reversible and stable thermochemical oxygen storage.

Chemical looping processes based on multiple-step reduction and oxidation of metal oxides hold great promise for a variety of energy applications, such as CO2 capture and conversion, gas separation, energy storage, and redox catalytic processes. Copper-based mixed oxides are one of the most promising candidate materials with a high oxygen storage capacity. However, the structural deterioration and sintering at high temperatures is one key scientific challenge. Herein, we report a precursor engineering approach to prepare durable copper-based redox sorbents for use in thermochemical looping processes for combustion and gas purification. Calcination of the CuMgAl hydrotalcite precursors formed mixed metal oxides consisting of CuO nanoparticles dispersed in the Mg-Al oxide support which inhibited the formation of copper aluminates during redox cycling. The copper-based redox sorbents demonstrated enhanced reaction rates, stable O2 storage capacity over 500 redox cycles at 900 °C, and efficient gas purification over a broad temperature range. We expect that our materials design strategy has broad implications on synthesis and engineering of mixed metal oxides for a range of thermochemical processes and redox catalytic applications.

Methods to Generate and Assay for Distinct Stages of Cancer Metastasis in Adult Drosophila melanogaster.

Metastasis underlies the majority of cancer-related deaths. Until recently, research on this complex multi-step process has been hindered by a lack of genetically tractable experimental models amenable to high-throughput analyses. This was recently overcome with the development of a model of metastatic colorectal cancer (CRC) in adult flies, which relies on the activation of a partial-epithelial-to-mesenchymal transition (EMT) in intestinal tumors. In this model, tumor cells are labeled with both GFP and luciferase reporters, enabling high-throughput analyses. We report here the detailed protocol for generating the model, and assaying for primary tumor burden and distinct stages of metastasis, including the number of circulating tumor cells and secondary metastases.

Mesenchymal-to-epithelial transitions require tissue-specific interactions with distinct laminins.

Mesenchymal-to-epithelial transition (MET) converts cells from migratory mesenchymal to polarized epithelial states. Despite its importance for both normal and pathological processes, very little is known about the regulation of MET in vivo. Here we exploit midgut morphogenesis in Drosophila melanogaster to investigate the mechanisms underlying MET. We show that down-regulation of the EMT transcription factor Serpent is required for MET, but not sufficient, as interactions with the surrounding mesoderm are also essential. We find that midgut MET relies on the secretion of specific laminins via the CopII secretory pathway from both mesoderm and midgut cells. We show that secretion of the laminin trimer containing the Wingblister α-subunit from the mesoderm is an upstream cue for midgut MET, leading to basal polarization of αPS1 integrin in midgut cells. Polarized αPS1 is required for the formation of a monolayered columnar epithelium and for the apical polarization of αPS3, Baz, and E-Cad. Secretion of a distinct LamininA-containing trimer from midgut cells is required to reinforce the localization of αPS1 basally, and αPS3 apically, for robust repolarization. Our data suggest that targeting these MET pathways, in conjunction with therapies preventing EMT, may present a two-pronged strategy toward blocking metastasis in cancer.

Τhe role of surface energy in the apparent solubility of two different calcite crystal habits.

The interplay between polymorphism and facet-specific surface energy on the dissolution of crystals is examined in this work. It is shown that, using cationic additives, it is possible to produce star-shaped calcite crystals at very high supersaturations. In crystallization processes following the Ostwald rule of stages these star-shaped crystals appear to have higher solubility than both their rhombohedral counterparts and needle-shaped aragonite crystals. The vapour pressures of vaterite, aragonite, star-shaped calcite and rhombohedral calcite crystals are measured using thermogravimetric analysis and the corresponding enthalpies of melting are obtained. Using inverse gas chromatography, the surface energy of the aforementioned crystals is measured as well and the surface energy of the main crystal facets is calculated. Combining the effect of facet-specific surface energies and the enthalpies of melting on a modified version of the classical solubility equation for regular solutions, it is proved that the star-shaped calcite crystals can indeed have higher apparent solubility than aragonitecrystals.

Epithelial-mesenchymal plasticity: emerging parallels between tissue morphogenesis and cancer metastasis.

Many cells possess epithelial-mesenchymal plasticity (EMP), which allows them to shift reversibly between adherent, static and more detached, migratory states. These changes in cell behaviour are driven by the programmes of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET), both of which play vital roles during normal development and tissue homeostasis. However, the aberrant activation of these processes can also drive distinct stages of cancer progression, including tumour invasiveness, cell dissemination and metastatic colonization and outgrowth. This review examines emerging common themes underlying EMP during tissue morphogenesis and malignant progression, such as the context dependence of EMT transcription factors, a central role for partial EMTs and the nonlinear relationship between EMT and MET. This article is part of a discussion meeting issue 'Contemporary morphogenesis'.