Epithelial-mesenchymal transition (EMT) is a reversible and dynamic process hypothesized to be co-opted by carcinoma during invasion and metastasis. Yet, there is still no quantitative measure to assess the interplay between EMT and cancer progression. Here, we derived a method for universal EMT scoring from cancer-specific transcriptomic EMT signatures of ovarian, breast, bladder, lung, colorectal and gastric cancers. We show that EMT scoring exhibits good correlation with previously published, cancer-specific EMT signatures. This universal and quantitative EMT scoring was used to establish an EMT spectrum across various cancers, with good correlation noted between cell lines and tumours. We show correlations between EMT and poorer disease-free survival in ovarian and colorectal, but not breast, carcinomas, despite previous notions. Importantly, we found distinct responses between epithelial- and mesenchymal-like ovarian cancers to therapeutic regimes administered with or without paclitaxel in vivo and demonstrated that mesenchymal-like tumours do not always show resistance to chemotherapy. EMT scoring is thus a promising, versatile tool for the objective and systematic investigation of EMT roles and dynamics in cancer progression, treatment response and survival.
Cancer metastasis and drug resistance are important malignant tumor phenotypes that cause roughly 90% mortality in human cancers. Current therapeutic strategies, however, face substantial challenges partially due to a lack of applicable pre-clinical models and drug-screening platforms. Notably, microscale and three-dimensional (3D) tissue culture platforms capable of mimicking in vivo microenvironments to replicate physiological conditions have become vital tools in a wide range of cellular and clinical studies. Here, we present a microfluidic device capable of mimicking a configurable tumor microenvironment to study in vivo-like cancer cell migration as well as screening of inhibitors on both parental tumors and migratory cells. In addition, a novel evaporation-based paper pump was demonstrated to achieve adaptable and sustainable concentration gradients for up to 6 days in this model. This straightforward modeling approach allows for fast patterning of a wide variety of cell types in 3D and may be further integrated into biological assays. We also demonstrated cell migration from tumor spheroids induced by an epidermal growth factor (EGF) gradient and exhibited lowered expression of an epithelial marker (EpCAM) compared with parental cells, indicative of partial epithelial-mesenchymal transition (EMT) in this process. Importantly, pseudopodia protrusions from the migratory cells - critical during cancer metastasis - were demonstrated. Insights gained from this work offer new opportunities to achieve active control of in vitro tumor microenvironments on-demand, and may be amenable towards tailored clinical applications.
The ovary surface epithelium (OSE) undergoes ovulatory tear and remodelling throughout life. Resident stem cells drive such tissue homeostasis in many adult epithelia, but their existence in the ovary has not been definitively proven. Lgr5 marks stem cells in multiple epithelia. Here we use reporter mice and single-molecule fluorescent in situ hybridization to document candidate Lgr5(+) stem cells in the mouse ovary and associated structures. Lgr5 is broadly expressed during ovary organogenesis, but becomes limited to the OSE in neonate life. In adults, Lgr5 expression is predominantly restricted to proliferative regions of the OSE and mesovarian-fimbria junctional epithelia. Using in vivo lineage tracing, we identify embryonic and neonate Lgr5(+) populations as stem/progenitor cells contributing to the development of the OSE cell lineage, as well as epithelia of the mesovarian ligament and oviduct/fimbria. Adult Lgr5(+) populations maintain OSE homeostasis and ovulatory regenerative repair in vivo. Thus, Lgr5 marks stem/progenitor cells of the ovary and tubal epithelia.
Three-dimensional (3D) tissue culture platforms that are capable of mimicking in vivo microenvironments to replicate physiological conditions are vital tools in a wide range of cellular and clinical studies. Here, learning from the nature of cilia in lungs - clearing mucus and pathogens from the airway - we develop a 3D culture approach via flexible and kinetic copolymer-based chains (nano-cilia) for diminishing cell-to-substrate adhesion. Multicellular spheroids or colonies were tested for 3-7 days in a microenvironment consisting of generated cells with properties of putative cancer stem cells (CSCs). The dynamic and reversible regulation of epithelial-mesenchymal transition (EMT) was examined in spheroids passaged and cultured in copolymer-coated dishes. The expression of CSC markers, including CD44, CD133, and ABCG2, and hypoxia signature, HIF-1?, was significantly upregulated compared to that without the nano-cilia. In addition, these spheroids exhibited chemotherapeutic resistance in vitro and acquired enhanced metastatic propensity, as verified from microfluidic chemotaxis assay designed to replicate in vivo-like metastasis. The biomimetic nano-cilia approach and microfluidic device may offer new opportunities to establish a rapid and cost-effective platform for the study of anti-cancer therapeutics and CSCs.
Epithelial-mesenchymal transition (EMT) plays a critical role in the early stages of dissemination of carcinoma leading to metastatic tumors, which are responsible for over 90% of all cancer-related deaths. Current therapeutic regimens, however, have been ineffective in the cure of metastatic cancer, thus an urgent need exists to revisit existing protocols and to improve the efficacy of newly developed therapeutics. Strategies based on preventing EMT could potentially contribute to improving the outcome of advanced stage cancers. To achieve this goal new assays are needed to identify targeted drugs capable of interfering with EMT or to revert the mesenchymal-like phenotype of carcinoma to an epithelial-like state. Current assays are limited to examining the dispersion of carcinoma cells in isolation in conventional 2-dimensional (2D) microwell systems, an approach that fails to account for the 3-dimensional (3D) environment of the tumor or the essential interactions that occur with other nearby cell types in the tumor microenvironment. Here we present a microfluidic system that integrates tumor cell spheroids in a 3D hydrogel scaffold, in close co-culture with an endothelial monolayer. Drug candidates inhibiting receptor activation or signal transduction pathways implicated in EMT have been tested using dispersion of A549 lung adenocarcinoma cell spheroids as a metric of effectiveness. We demonstrate significant differences in response to drugs between 2D and 3D, and between monoculture and co-culture.
Microwell technology has revolutionized many aspects of in vitro cellular studies from 2D traditional cultures to 3D in vivo-like functional assays. However, existing lithography-based approaches are often costly and time-consuming. This study presents a rapid, low-cost prototyping method of CO2 laser ablation of a conventional untreated culture dish to create concave microwells used for generating multicellular aggregates, which can be readily available for general laboratories. Polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), and polystyrene (PS) microwells are investigated, and each produces distinctive microwell features. Among these three materials, PS cell culture dishes produce the optimal surface smoothness and roundness. A549 lung cancer cells are grown to form cancer aggregates of controllable size from ?40 to ?80 ?m in PS microwells. Functional assays of spheroids are performed to study migration on 2D substrates and in 3D hydrogel conditions as a step towards recapitulating the dissemination of cancer cells. Preclinical anti-cancer drug screening is investigated and reveals considerable differences between 2D and 3D conditions, indicating the importance of assay type as well as the utility of the present approach.
Epithelial ovarian cancer (EOC) is hallmarked by a high degree of heterogeneity. To address this heterogeneity, a classification scheme was developed based on gene expression patterns of 1538 tumours. Five, biologically distinct subgroups - Epi-A, Epi-B, Mes, Stem-A and Stem-B - exhibited significantly distinct clinicopathological characteristics, deregulated pathways and patient prognoses, and were validated using independent datasets. To identify subtype-specific molecular targets, ovarian cancer cell lines representing these molecular subtypes were screened against a genome-wide shRNA library. Focusing on the poor-prognosis Stem-A subtype, we found that two genes involved in tubulin processing, TUBGCP4 and NAT10, were essential for cell growth, an observation supported by a pathway analysis that also predicted involvement of microtubule-related processes. Furthermore, we observed that Stem-A cell lines were indeed more sensitive to inhibitors of tubulin polymerization, vincristine and vinorelbine, than the other subtypes. This subtyping offers new insights into the development of novel diagnostic and personalized treatment for EOC patients.
Epithelial-mesenchymal transition (EMT) is a fundamental mechanism in development driving body plan formation. EMT describes a transition process wherein polarized epithelial cells lose their characteristics and acquire a mesenchymal phenotype. The apico-basal polarity of epithelial cells is replaced by a front-rear polarity in mesenchymal cells which favor cell-extracellular matrix than intercellular adhesion. These events serve as a prerequisite to the context-dependent migratory and invasive functions of mesenchymal cells. In solid tumors, carcinoma cells undergoing EMT not only invade and metastasize but also exhibit cancer stem cell-like properties, providing resistance to conventional and targeted therapies. In cardiovascular systems, epicardial cells engaged in EMT contribute to myocardial regeneration. Conversely, cardiovascular endothelial cells undergoing EMT cause cardiac fibrosis. Growing evidence has shed light on the potential development of novel therapeutics that target cell movement by applying the EMT concept, and this may provide new therapeutic strategies for the treatment of cancer and heart diseases.
Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Discovery of novel therapeutic opportunities for EOC is important for the improvement of clinical outcome of the patients. Emerging evidence is suggesting that epithelial-mesenchymal transition (EMT) plays a crucial role in the aggressiveness in EOC including increasing migration and invasion ability, contributing to chemoresistance and cancer stem cell populations. Targeting EMT in EOC thus offers an attractive therapeutic option.
LPA (lysophosphatidic acid) is a natural phospholipid that plays important roles in promoting cancer cell proliferation, invasion and metastases. We previously reported that LPA induces ovarian cancer cell dispersal and disruption of AJ (adherens junction) through the activation of SFK (Src family kinases). In this study, we have investigated the regulatory mechanisms during the early phase of LPA-induced cell dispersal. An in vitro model of the ovarian cancer cell line SKOV3 for cell dispersal was used. LPA induces rapid AJ disruption by increasing the internalization of N-cadherin-?-catenin. By using immunoprecipitations, LPA was shown to induce increased tyrosine phosphorylation of ?-catenin and alter the balance of ?-catenin-bound SFK and PTP1B (phosphotyrosine phosphatase 1B). The altered balance of tyrosine kinase/phosphatase correlated with a concomitant disintegration of the ?-catenin-?-catenin, but not the ?-catenin-N-cadherin complex. This disintegration of ?-catenin from ?-catenin and the cell dispersal caused by LPA can be rescued by blocking SFK activity with the chemical inhibitor, PP2. More importantly, PP2 also restores the level of PTP1B bound to ?-catenin. We propose that LPA signalling alters AJ stability by changing the dynamics of tyrosine kinase/phosphatase bound to AJ proteins. This work provides further understanding of the early signalling events regulating ovarian cancer cell dispersal and AJ disruption induced by LPA.
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