Demonstrating a Quartz Crystal Microbalance with Dissipation (QCMD) to Enhance the Monitoring and Mechanistic Understanding of Iron Carbonate Crystalline Films.

This paper reports the real time monitoring of siderite deposition, on both Au- and Fe-coated surfaces, using the changes in frequency and dissipation of quartz crystal microbalance with dissipation (QCMD). In an iron chloride solution saturated with carbon dioxide, buffered with sodium bicarbonate to pH 6.8, roughly spherical particles of siderite formed within 15 min, which subsequently deposited on the QCMD crystal surface. Imaging of the surface showed a layer formed from particles ca. < 0.5 µm in diameter. Larger particles are clearly deposited on top of the lower layer; these larger particles are >1 µm in diameter. Monitoring of the frequency clearly differentiates the formation of the lower layer from the larger crystals deposited on top at later times. The elastic moduli calculated from QCMD data showed a progressive dissipation increase; the modeling of the solid-liquid interface using a flat approximation resulted in a poor estimation of elastic and storage moduli. Rather, the impedance modeled as a viscoelastic layer in contact with a semi-infinite liquid, where a random bumpy surface with a Gaussian correlator is used, is much more accurate in determining the elastic and storage moduli as losses from the uneven interface are considered. A further step considers that the film is in fact a composite consisting of hard spherical particles of siderite with water in the vacant spaces. This is treated by considering the individual contributions of the phases to the losses measured, thereby further improving the accuracy of the description of the film and the QCMD data. Collectively, this work presents a new framework for the use of QCMD, paired with traditional approaches, to enhance the understanding of crystal deposition and film formation as well as quantify the often evolving mechanical properties.

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.

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.

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.

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.

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.

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.

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'.

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.

Collective cell migration and metastases induced by an epithelial-to-mesenchymal transition in Drosophila intestinal tumors.

Metastasis underlies the majority of cancer-related deaths yet remains poorly understood due, in part, to the lack of models in vivo. Here we show that expression of the EMT master inducer Snail in primary adult Drosophila intestinal tumors leads to the dissemination of tumor cells and formation of macrometastases. Snail drives an EMT in tumor cells, which, although retaining some epithelial markers, subsequently break through the basal lamina of the midgut, undergo a collective migration and seed polyclonal metastases. While metastases re-epithelialize over time, we found that early metastases are remarkably mesenchymal, discarding the requirement for a mesenchymal-to-epithelial transition for early stages of metastatic growth. Our results demonstrate the formation of metastases in adult flies, and identify a key role for partial-EMTs in driving it. This model opens the door to investigate the basic mechanisms underlying metastasis, in a powerful in vivo system suited for rapid genetic and drug screens.

Contribution of epithelial-mesenchymal transitions to organogenesis and cancer metastasis.

The epithelial-to-mesenchymal transition (EMT) plays crucial roles during development, and inappropriate activation of EMTs are associated with tumor progression and promoting metastasis. In recent years, increasing studies have identified developmental contexts where cells undergo an EMT and transition to a partial-state, downregulating just a subset of epithelial characteristics and increasing only some mesenchymal traits, such as invasive motility. In parallel, recent studies have shown that EMTs are rarely fully activated in tumor cells, generating a diverse array of transition states. As our appreciation of the full spectrum of intermediate phenotypes and the huge diversity in underlying mechanisms grows, cross-disciplinary collaborations investigating developmental-EMTs and cancer-EMTs will be fundamental in order to achieve a full mechanistic understanding of this complex cell process.

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.

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.

A role for E-cadherin in ensuring cohesive migration of a heterogeneous population of non-epithelial cells.

Collective cell migration is a key process underlying the morphogenesis of many organs as well as tumour invasion, which very often involves heterogeneous cell populations. Here we investigated how such populations can migrate cohesively in the Drosophila posterior midgut, comprised of epithelial and mesenchymal cells and show a novel role for the epithelial adhesion molecule E-cadherin (E-Cad) in mesenchymal cells. Despite a lack of junctions at the ultrastructure level, reducing E-Cad levels causes mesenchymal cells to detach from one another and from neighbouring epithelial cells; as a result, coordination between the two populations is lost. Moreover, Bazooka and recycling mechanisms are also required for E-Cad accumulation in mesenchymal cells. These results indicate an active role for E-Cad in mediating cohesive and ordered migration of non-epithelial cells, and discount the notion of E-Cad as just an epithelial feature that has to be switched off to enable migration of mesenchymal cells.

Conserved mechanisms of tumorigenesis in the Drosophila adult midgut.

Whereas the series of genetic events leading to colorectal cancer (CRC) have been well established, the precise functions that these alterations play in tumor progression and how they disrupt intestinal homeostasis remain poorly characterized. Activation of the Wnt/Wg signaling pathway by a mutation in the gene APC is the most common trigger for CRC, inducing benign lesions that progress to carcinomas due to the accumulation of other genetic alterations. Among those, Ras mutations drive tumour progression in CRC, as well as in most epithelial cancers. As mammalian and Drosophila's intestines share many similarities, we decided to explore the alterations induced in the Drosophila midgut by the combined activation of the Wnt signaling pathway with gain of function of Ras signaling in the intestinal stem cells. Here we show that compound Apc-Ras clones, but not clones bearing the individual mutations, expand as aggressive intestinal tumor-like outgrowths. These lesions reproduce many of the human CRC hallmarks such as increased proliferation, blockade of cell differentiation and cell polarity and disrupted organ architecture. This process is followed by expression of tumoral markers present in human lesions. Finally, a metabolic behavioral assay shows that these flies suffer a progressive deterioration in intestinal homeostasis, providing a simple readout that could be used in screens for tumor modifiers or therapeutic compounds. Taken together, our results illustrate the conservation of the mechanisms of CRC tumorigenesis in Drosophila, providing an excellent model system to unravel the events that, upon mutation in Apc and Ras, lead to CRC initiation and progression.

Mesenchymal-to-epithelial transition of intercalating cells in Drosophila renal tubules depends on polarity cues from epithelial neighbours.

The intercalation of mesenchymal cells into epithelia, through mesenchymal-to-epithelial transition (MET), underlies organogenesis, for example, in nephrogenesis, and tissue regeneration, during cell renewal and wound repair. Despite its importance, surprisingly little is known about the mechanisms that bring about MET in comparison with the related and much-studied, reverse process, epithelial-to-mesenchymal transition (EMT). We analyse the molecular events that regulate MET as stellate cells integrate into the established epithelium of the developing renal tubules in Drosophila. We show that stellate cells polarise as they integrate between epithelial principal cells and that the normal, localised expression of cell polarity proteins in principal cells is required for stellate cells to become epithelial. While the basolateral and apical membranes act as cues for stellate cell polarity, adherens junction integrity is required to regulate their movement through the epithelium; in the absence of these junctions stellate cells continue migrating into the tubule lumen. We also show that expression of basolateral proteins in stellate cells is a prerequisite for their ingression between principal cells. We present a model in which the contacts with successive principal cell membrane domains made by stellate cells as they integrate between them act as a cue for the elaboration of stellate cell polarity. We suggest that the formation of zonula adherens junctions between new cell neighbours establishes their apico-basal positions and stabilises them in the epithelium.

Crumbs stabilises epithelial polarity during tissue remodelling.

The apicobasal polarity of epithelia depends on the integrated activity of apical and basolateral proteins, and is essential for tissue integrity and body homeostasis. Yet these tissues are frequently on the move as they are sculpted by active morphogenetic cell rearrangements. How does cell polarity survive these stresses? We analyse this question in the renal tubules of Drosophila, a tissue that undergoes dramatic morphogenetic change as it develops. Here we show that, whereas the Bazooka and Scribble protein groups are required for the establishment of tubule cell polarity, the key apical determinant, Crumbs, is required for cell polarity in the tubules only from the time when morphogenetic movements start. Strikingly, if these movements are stalled, polarity persists in the absence of Crumbs. Similar rescue of the ectodermal phenotype of the crumbs mutant when germ-band extension is reduced suggests that Crumbs has a specific, conserved function in stabilising cell polarity during tissue remodelling rather than in its initial stabilisation. We also identify a requirement for the exocyst component Exo84 during tissue morphogenesis, which suggests that Crumbs-dependent stability of epithelial polarity is correlated with a requirement for membrane recycling and targeted vesicle delivery.