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.

Hypoxia as a potential inducer of immune tolerance, tumor plasticity and a driver of tumor mutational burden: Impact on cancer immunotherapy.

In cancer patients, immune cells are often functionally compromised due to the immunosuppressive features of the tumor microenvironment (TME) which contribute to the failures in cancer therapies. Clinical and experimental evidence indicates that developing tumors adapt to the immunological environment and create a local microenvironment that impairs immune function by inducing immune tolerance and invasion. In this context, microenvironmental hypoxia, which is an established hallmark of solid tumors, significantly contributes to tumor aggressiveness and therapy resistance through the induction of tumor plasticity/heterogeneity and, more importantly, through the differentiation and expansion of immune-suppressive stromal cells. We and others have provided evidence indicating that hypoxia also drives genomic instability in cancer cells and interferes with DNA damage response and repair suggesting that hypoxia could be a potential driver of tumor mutational burden. Here, we reviewed the current knowledge on how hypoxic stress in the TME impacts tumor angiogenesis, heterogeneity, plasticity, and immune resistance, with a special interest in tumor immunogenicity and hypoxia targeting. An integrated understanding of the complexity of the effect of hypoxia on the immune and microenvironmental components could lead to the identification of better adapted and more effective combinational strategies in cancer immunotherapy. Clearly, the discovery and validation of therapeutic targets derived from the hypoxic tumor microenvironment is of major importance and the identification of critical hypoxia-associated pathways could generate targets that are undeniably attractive for combined cancer immunotherapy approaches.

Twenty years of EMT: A state of the field from TEMTIA X.

TEMTIA X, the tenth symposium organized by the EMT international Association (TEMTIA) took place in Paris on November 7th-10th, 2022. Similarly to the previous meetings, it reviewed most recent aspects of the epithelial-mesenchymal transition, a cellular process involved during distinct stages of development, but also during wound healing and fibrosis to some level. EMT steps are likewise typically described with various extents during tumor cell progression and metastasis. The meeting emphasized the intermediate stages involved in the process and their potential physiological or pathological importance, taking advantage of the expansion of molecular methods at single cell level. It also introduced new descriptions of EMT occurrences during early embryogenesis. In addition, sessions explored how EMT reflects cell metabolism and how the process can mingle with immune response, particularly during tumor progression, providing new targets, that were discussed, among others, for cancer therapy. Finally, it introduced a new perception of EMT biological meaning based on an evolutionary perspective. The meeting integrated the TEMTIA general assembly , allowing general discussion about the future of the association, starting with the site of the next meeting, now decided to take place in Seattle (US), late 2024.

Designing Organoid Models to Monitor Cancer Progression, Plasticity and Resistance: The Right Set Up for the Right Question.

The recent trend in 3D cell modeling has fostered the emergence of a wide range of models, addressing very distinct goals ranging from the fundamental exploration of cell-cell interactions to preclinical assays for personalized medicine. It is clear that no single model will recapitulate the complexity and dynamics of in vivo situations. The key is to define the critical points, achieve a specific goal and design a model where they can be validated. In this report, we focused on cancer progression. We describe our model which is designed to emulate breast carcinoma progression during the invasive phase. We chose to provide topological clues to the target cells by growing them on microsupports, favoring a polarized epithelial organization before they are embedded in a 3D matrix. We then watched for cell organization and differentiation for these models, adding stroma cells then immune cells to follow and quantify cell responses to drug treatment, including quantifying cell death and viability, as well as morphogenic and invasive properties. We used model cell lines including Comma Dß, MCF7 and MCF10A mammary epithelial cells as well as primary breast cancer cells from patient-derived xenografts (PDX). We found that fibroblasts impacted cell response to Docetaxel and Palbociclib. We also found that NK92 immune cells could target breast cancer cells within the 3D configuration, providing quantitative monitoring of cell cytotoxicity. We also tested several sources for the extracellular matrix and selected a hyaluronan-based matrix as a promising alternative to mouse tumor basement membrane extracts for primary human cancer cells. Overall, we validated a new 3D model designed for breast cancer for preclinical use in personalized medicine.

Evaluating immune response in vitro in a relevant microenvironment: a high-throughput microfluidic model for clinical screening.

Aim: Functional screening of new pharmaceutical compounds requires clinically relevant models to monitor essential cellular and immune responses during cancer progression, with or without treatment. Beyond survival, the emergence of resistant tumor cell clones should also be considered, including specific properties related to plasticity, such as invasiveness, stemness, escape from programmed cell death, and immune response. Numerous pathways are involved in these processes. Defining the relevant ones in the context of a specific tumor type will be key to designing an appropriate combination of inhibitors. However, the diversity and potential redundancy of these pathways remain a challenge for therapy. Methods: A new microfluidic device developed by Okomera was dedicated to the screening of drug treatment for breast cancer. This microchip includes 150 droplet-trapping microwells, offering multi-chip settings and multiple treatment choices. Results: After validating the system with established cell lines and a panel of drugs used clinically at Gustave Roussy, preclinical experiments were initiated including patient-derived xenograft (PDX) and primary tumor cells-derived tumoroids with the collaboration of Gustave Roussy clinicians. Tumor-isolated lymphocytes were also added to the tumoroids, using secondary droplets in proof-of-concept experiments. Conclusions: These results show the relevance of the methodology for screening large numbers of drugs, a wide range of doses, and multiple drug combinations. This methodology will be used for two purposes: 1) new drug screening from the compound library, using the high throughput potential of the chip; and 2) pre-clinical assay for a two-weeks response for personalized medicine, allowing evaluation of drug combinations to flag an optimized treatment with potential clinical application.

Regulation of p63 isoforms by snail and slug transcription factors in human squamous cell carcinoma.

TP63 is a p53-related gene that contains two alternative promoters, which give rise to transcripts that encode proteins with (TAp63) or without (DeltaNp63) an amino-transactivating domain. Whereas the expression of p63 is required for proper development of epithelial structures, the role of p63 in tumorigenesis remains unclear. Here, we investigated the role of Snail and Slug transcription factors, known to promote epithelial-to-mesenchymal transitions during development and cancer, in the regulation of p63 isoforms in human squamous cell carcinoma (SCC). In the present study, we observed that the expressions of DeltaN and TAp63 isoforms were, respectively, down- and up-regulated by both Snail and Slug. However, the induction of TAp63 was not directly caused by these two transcription factors but resulted from the loss of DeltaNp63, which acts as dominant-negative inhibitor of TAp63. In SCC cell lines and cancer tissues, high expression of Snail and Slug was also significantly associated with altered p63 expression. Finally, we showed that DeltaNp63 silencing reduced cell-cell adhesion and increased the migratory properties of cancer cells. These data suggest that the disruption of p63 expression induced by Snail and Slug plays a crucial role in tumor progression. Therefore, p63 and its regulating factors could constitute novel prognosis markers in patients with SCC and attractive targets for the therapeutic modulation of neoplastic cell invasiveness.

Snail family regulation and epithelial mesenchymal transitions in breast cancer progression.

Since its initial description, the interconversion between epithelial and mesenchymal cells (designed as epithelial-mesenchymal or mesenchymal-epithelial transition, EMT or MET, respectively) has received special attention since it provides epithelial cells with migratory features. Different studies using cell lines have identified cytokines, intercellular signaling elements and transcriptional factors capable of regulating this process. Particularly, the identification of Snail family members as key effectors of EMT has opened new ways for the study of this cellular process. In this article we discuss the molecular pathways that control EMT, showing a very tight and interdependent regulation. We also analyze the contribution of EMT and Snail genes in the process of tumorigenesis using the mammary gland as cellular model.

The Most Common VHL Point Mutation R167Q in Hereditary VHL Disease Interferes with Cell Plasticity Regulation.

Von Hippel-Lindau disease (VHL) is a rare hereditary syndrome due to mutations of the VHL tumor suppressor gene. Patients harboring the R167Q mutation of the VHL gene have a high risk of developing ccRCCs. We asked whether the R167Q mutation with critical aspects of pseudo-hypoxia interferes with tumor plasticity. For this purpose, we used wild-type VHL (WT-VHL) and VHL-R167Q reconstituted cells. We showed that WT-VHL and VHL-R167Q expression had a similar effect on cell morphology and colony formation. However, cells transfected with VHL-R167Q display an intermediate, HIF2-dependent, epithelial-mesenchymal phenotype. Using RNA sequencing, we showed that this mutation upregulates the expression of genes involved in the hypoxia pathway, indicating that such mutation is conferring an enhanced pseudo-hypoxic state. Importantly, this hypoxic state correlates with the induction of genes belonging to epithelial-mesenchymal transition (EMT) and stemness pathways, as revealed by GSEA TCGA analysis. Moreover, among these deregulated genes, we identified nine genes specifically associated with a poor patient survival in the TCGA KIRC dataset. Together, these observations support the hypothesis that a discrete VHL point mutation interferes with tumor plasticity and may impact cell behavior by exacerbating phenotypic switching. A better understanding of the role of this mutation might guide the search for more effective treatments to combat ccRCCs.

Epithelial-mesenchymal transition: a cancer researcher's conceptual friend and foe.

Epithelial-mesenchymal transition (EMT) describes a series of rapid changes in cellular phenotype. During EMT, epithelial cells down-modulate cell-cell adhesion structures, alter their polarity, reorganize their cytoskeleton, and become isolated, motile, and resistant to anoikis. The term EMT is often applied to distinct biological events as if it were a single conserved process, but in fact EMT-related processes can vary in intensity from a transient loss of cell polarity to the total cellular reprogramming, as found by transcriptional analysis. Based on clinical observations, it is more appropriate in most cases to describe the emergence of an EMT-like phenotype during tumor progression. Although EMT implies complete trans-differentiation, EMT-like emphasizes the intermediary phenotype associated with tumor cell renewal and adaptation to specific microenvironments. Here, we categorize the various EMT-like phenotypes found in human carcinomas that, depending on the tumor type, may or not represent analogous stages in tumor progression. We based these categories on the global tumor phenotype. The tumor microenvironment, which is associated with stromal reactions, hypoxia, paucity of nutrients, impaired differentiation, and activation of various EMT-associated pathways, modulates overall tumor phenotype and leads to tumor heterogeneity.

Transforming growth factor-beta1-mediated Slug and Snail transcription factor up-regulation reduces the density of Langerhans cells in epithelial metaplasia by affecting E-cadherin expression.

Epithelial metaplasia (EpM) is an acquired tissue abnormality resulting from the transformation of epithelium into another tissue with a different structure and function. This adaptative process is associated with an increased frequency of (pre)cancerous lesions. We propose that EpM is involved in cancer development by altering the expression of adhesion molecules important for cell-mediated antitumor immunity. Langerhans cells (LCs) are intraepithelial dendritic cells that initiate immune responses against viral or tumor antigens on both skin and mucosal surfaces. In the present study, we showed by immunohistology that the density of CD1a(+) LCs is reduced in EpM of the uterine cervix compared with native squamous epithelium and that the low number of LCs observed in EpM correlates with the down-regulation of cell-surface E-cadherin. We also demonstrated that transforming growth factor-beta1 is not only overexpressed in metaplastic tissues but also reduces E-cadherin expression in keratinocytes in vitro by inducing the promoter activity of Slug and Snail transcription factors. Finally, we showed that in vitro-generated LCs adhere poorly to keratinocytes transfected with either Slug or Snail DNA. These data suggest that transforming growth factor-beta1 indirectly reduces antigen-presenting cell density in EpM by affecting E-cadherin expression, which might explain the increased susceptibility of abnormal tissue differentiation to the development of cancer by the establishment of local immunodeficiency responsible for EpM tumorigenesis.

Autoregulation of E-cadherin expression by cadherin-cadherin interactions: the roles of beta-catenin signaling, Slug, and MAPK.

Transcriptional repression of E-cadherin, characteristic of epithelial to mesenchymal transition, is often found also during tumor cell invasion. At metastases, migratory fibroblasts sometimes revert to an epithelial phenotype, by a process involving regulation of the E-cadherin-beta-catenin complex. We investigated the molecular basis of this regulation, using human colon cancer cells with aberrantly activated beta-catenin signaling. Sparse cultures mimicked invasive tumor cells, displaying low levels of E-cadherin due to transcriptional repression of E-cadherin by Slug. Slug was induced by beta-catenin signaling and, independently, by ERK. Dense cultures resembled a differentiated epithelium with high levels of E-cadherin and beta-catenin in adherens junctions. In such cells, beta-catenin signaling, ErbB-1/2 levels, and ERK activation were reduced and Slug was undetectable. Disruption of E-cadherin-mediated contacts resulted in nuclear localization and signaling by beta-catenin, induction of Slug and inhibition of E-cadherin transcription, without changes in ErbB-1/2 and ERK activation. This autoregulation of E-cadherin by cell-cell adhesion involving Slug, beta-catenin and ERK could be important in tumorigenesis.

[Epithelio-mesenchymal transition and cutaneous wound healing]. / Transition épithélio-mésenchymateuse et réparation des blessures cutanées.

Successful cutaneous wound repair involves in a series of tightly coordinated and overlapping phases, including inflammation and clot formation, keratinocyte activation and migration (re-epithelialization), basement membrane and ECM remodeling, followed by dermal and epidermal maturation. We examine here the process of wound re-epithelialization, emphasizing the similarity between re-epithelialization and developmental epithelial-mesenchymal transition (EMT), based on morphological and molecular criteria. Changes in cell-cell and cell-substrate adhesion during re-epithelialization are also reminiscent of pathological processes described during malignant tumor progression, another situation involving partial or total EMT. We therefore propose that wound re-epithelialization represents a partial and reversible form of EMT.

Snail and slug play distinct roles during breast carcinoma progression.

PURPOSE: Carcinoma progression is linked to a partially dedifferentiated epithelial cell phenotype. As previously suggested, this regulation could involve transcription factors, Snail and Slug, known to promote epithelial-mesenchymal transitions during development. Here, we investigate the role of Snail and Slug in human breast cancer progression. EXPERIMENTAL DESIGN: We analyzed Snail, Slug, and E-cadherin RNA expression levels and protein localization in large numbers of transformed cell lines and breast carcinomas, examined the correlation with tumor histologic features, and described, at the cellular level, Snail and Slug localization in carcinomas using combined in situ hybridization and immunolocalization. RESULTS: In contrast with transformed cell lines, Slug was found to colocalize with E-cadherin at the cellular level in normal mammary epithelial cells and all tested carcinomas. Snail also colocalized at the cellular level with E-cadherin in tumors expressing high levels of Snail RNA. In addition, Snail was significantly expressed in tumor stroma, varying with tumors. Slug and Snail genes were significantly overexpressed in tumors associated with lymph node metastasis. Finally, the presence of semidifferentiated tubules within ductal carcinomas was linked to Slug expression levels similar to or above normal breast samples. CONCLUSIONS: These results suggest that Snail or Slug expression in carcinoma cells does not generally preclude significant E-cadherin expression. They emphasize a link between Snail, Slug, and lymph node metastasis in a large sampling of mammary carcinomas, and suggest a role for Slug in the maintenance of semidifferentiated structures. Snail and Slug proteins seem to support distinct tumor invasion modes and could provide new therapeutic targets.

New insights into the role of EMT in tumor immune escape.

Novel immunotherapy approaches have provided durable remission in a significant number of cancer patients with cancers previously considered rapidly lethal. Nonetheless, the high degree of nonresponders, and in some cases the emergence of resistance in patients who do initially respond, represents a significant challenge in the field of cancer immunotherapy. These issues prompt much more extensive studies to better understand how cancer cells escape immune surveillance and resist immune attacks. Here, we review the current knowledge of how cellular heterogeneity and plasticity could be involved in shaping the tumor microenvironment (TME) and in controlling antitumor immunity. Indeed, recent findings have led to increased interest in the mechanisms by which cancer cells undergoing epithelial-mesenchymal transition (EMT), or oscillating within the EMT spectrum, might contribute to immune escape through multiple routes. This includes shaping of the TME and decreased susceptibility to immune effector cells. Although much remains to be learned on the mechanisms at play, cancer cell clones with mesenchymal features emerging from the TME seem to be primed to face immune attacks by specialized killer cells of the immune system, the natural killer cells, and the cytotoxic T lymphocytes. Recent studies investigating patient tumors have suggested EMT as a candidate predictive marker to be explored for immunotherapy outcome. Promising data also exist on the potential utility of targeting these cancer cell populations to at least partly overcome such resistance. Research is now underway which may lead to considerable progress in optimization of treatments.

An in situ hybridization technique to detect low-abundance slug mRNA in adherent cultured cells.

In this chapter, we describe a simple and relatively rapid technique for detecting low-abundance slug mRNA in cultured cells. The procedure uses nonradioactive digoxigenin-labeled RNA probes that are more sensitive than deoxyribonucleic probes and simpler to detect than radioactively labeled probes. Cells are grown in glass chamber slides, fixed in an acidic fixative, dehydrated through ethanol and xylene, permeabilized in pepsin, and post-fixed. Slides are then incubated overnight at 37 degrees C in a buffer containing 50% formamide and 5-10 ng/gL probe. Excess probe is removed by washing at high temperature in low-salt buffer and by treatment with RNase. Probe is detected immunohistochemically with an anti-digoxigenin Fab fragment, using a tyramide amplification kit to enhance signal and Fast Red for visualization. The technique has the advantages of probe stability and sensitivity, hybridization at low temperature, rapidity and sensitivity of probe detection, and the production of permanent specimens.

Epithelial-mesenchymal transitions: from cell plasticity to concept elasticity.

Epithelial-mesenchymal transition (EMT) is a developmental cellular process occurring during early embryo development, including gastrulation and neural crest cell migration. It can be broken down in distinct functional steps: (1) loss of baso-apical polarization characterized by cytoskeleton, tight junctions, and hemidesmosomes remodeling; (2) individualization of cells, including a decrease in cell-cell adhesion forces, (3) emergence of motility, and (4) invasive properties, including passing through the subepithelial basement membrane. These phases occur in an uninterrupted process, without requiring mitosis, in an order and with a degree of completion dictated by the microenvironment. The whole process reflects the activation of specific transcription factor families, called EMT transcription factors. Several mechanisms can combine to induce EMT. Some are reversible, involving growth factors and cytokines and/or environmental signals including extracellular matrix and local physical conditions. Others are irreversible, such as genomic alterations during carcinoma progression, along a selective and irreversible clonal drift. In carcinomas, these signals can converge to initiate a metastable phenotype. In this state, similarly to activated keratinocytes during re-epithelialization, cells can initiate a cohort migration and engage into a transient and reversible EMT controlled by the local environment prior to efficient intravasation and metastasis. EMT transcription factors also participate in cancer progression by inducing apoptosis resistance and maintaining stem-like properties exposed in tumor recurrences. These properties, very important on a clinical point of view, are not intrinsically linked to EMT, but can share common pathways.