Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.
15 Related JoVE Articles!
Reconstitution Of β-catenin Degradation In Xenopus Egg Extract
Institutions: Vanderbilt University Medical Center, Cincinnati Children's Hospital Medical Center, Vanderbilt University School of Medicine.
egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus
egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3
. Herein, a method is described for isolating Xenopus
egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, β-catenin. Two different methods are described to assess β-catenin protein degradation in Xenopus
egg extract. One method is visually informative ([35
S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess β-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus
egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of β-catenin degradation.
Molecular Biology, Issue 88, Xenopus laevis, Xenopus egg extracts, protein degradation, radiolabel, luciferase, autoradiography, high-throughput screening
An Immunofluorescent Method for Characterization of Barrett’s Esophagus Cells
Institutions: St. Joseph's Hospital and Medical Center.
Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One of the primary risk factors of EAC is Barrett’s esophagus (BE), a metaplastic change of the normal squamous esophagus in response to chronic heartburn. Despite the well-established connection between EAC and BE, interrogation of the molecular events, particularly altered signaling pathways involving progression of BE to EAC, are poorly understood. Much of this is due to the lack of suitable in vitro
models available to study these diseases. Recently, immortalized BE cell lines have become commercially available allowing for in vitro
studies of BE. Here, we present a method for immunofluorescent staining of immortalized BE cell lines, allowing in vitro
characterization of cell signaling and structure after exposure to therapeutic compounds. Application of these techniques will help develop insight into the mechanisms involved in BE to EAC progression and provide potential avenues for treatment and prevention of EAC.
Cellular Biology, Issue 89, Barrett's Esophagus, Immunofluorescence, adenocarcinoma, morphology, gastroesophageal reflux disease, immortalized BE cell lines
Detection of Alternative Splicing During Epithelial-Mesenchymal Transition
Institutions: Northwestern University Feinberg School of Medicine.
Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and γ-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.
Cellular Biology, Issue 92, alternative splicing, EMT, RNA, primer design, real time PCR, splice isoforms
In vitro Organoid Culture of Primary Mouse Colon Tumors
Institutions: University of Michigan , University of Michigan .
Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1
, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.
Cancer Biology, Issue 75, Medicine, Molecular Biology, Cellular Biology, Biomedical Engineering, Anatomy, Physiology, Genetics, Oncology, Surgery, Organoids, Tumor Cells, Cultured Colonic Neoplasms, Primary Cell Culture, Colon tumor, chelation, collagenase, matrigel, organoid, EGF, colon cancer, cancer, tumor, cell, isolation, immunohistochemistry, mouse, animal model
Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)
Institutions: University of British Columbia .
Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1
]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2
, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3
H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4
, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1
): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.
Neuroscience, Issue 72, Biochemistry, Neurobiology, Molecular Biology, Cellular Biology, Physiology, Proteins, synapse, cultured hippocampal neurons, palmitoylation, lipid, immunoprecipitation, western blotting, biotin, Acyl-Biotin Exchange, ABE, neuron, brain, cell culture, rat, mouse, animal model
Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection
Institutions: University of Maine, University of Maine.
Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo
, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans
, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans
. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.
Immunology, Issue 93, Zebrafish, mucosal candidiasis, mucosal infection, epithelial barrier, epithelial cells, innate immunity, swimbladder, Candida albicans, in vivo.
Chicken Embryo Spinal Cord Slice Culture Protocol
Institutions: University College London.
Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo
. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.
Developmental Biology, Issue 73, Neurobiology, Neuroscience, Medicine, Cellular Biology, Molecular Biology, Anatomy, Physiology, Biomedical Engineering, Genetics, Surgery, Cells, Animal Structures, Embryonic Structures, Nervous System, spinal cord, embryo, development, Slice-Culture, motor neuron, neurons, immunostaining, chick, imaging, animal model
Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice: A Model for Immunotherapy Development
Institutions: University of California, Davis, University of California, Davis, Merck KGaA, Darmstadt, Germany.
A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-γ)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.
Medicine, Issue 80, Urinary Bladder, Animals, Genetically Modified, Cancer Vaccines, Immunotherapy, Animal Experimentation, Models, Neoplasms Bladder Cancer, C57BL/6 Mouse, MUC1, Immunotherapy, Preclinical Model
Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells
Institutions: University of Michigan Comprehensive Cancer Center, University of Michigan Comprehensive Cancer Center.
Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1
. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex vivo
. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4
. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3
. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7
. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.
Medicine, Issue 86, pathological conditions, signs and symptoms, neoplasms, three dimensional cultures, Matrigel, breast cells, malignant phenotype, signaling
Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow
Institutions: Sanquin Research and Landsteiner Laboratory, AMC at University of Amsterdam.
During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e.,
through the cell-cell junctions and the transcellular route, i.e.,
through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro
assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.
Immunology, Issue 90, Leukocytes, Human Umbilical Vein Endothelial Cells (HUVECs), transmigration, VE-cadherin, PECAM-1, endothelium, transcellular, paracellular
Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics
Institutions: Memorial Sloan Kettering Cancer Center, Memorial Sloan Kettering Cancer Center, Weill Cornell Medical College, Memorial Sloan Kettering Cancer Center, Memorial Sloan Kettering Cancer Center, Memorial Sloan Kettering Cancer Center.
The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e.
, soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo
(fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.
Cancer Biology, Issue 92, Ex vivo sectioning, Treatment response, Sensitivity/Resistance, Drug development, Patient tumors, Preclinical and Clinical
Modified Mouse Embryonic Stem Cell based Assay for Quantifying Cardiogenic Induction Efficiency
Institutions: Vanderbilt University School of Medicine, Vanderbilt University School of Medicine, Vanderbilt University School of Medicine, Veterans Administration TVHS.
Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/β-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/β-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.
Cellular Biology, Issue 50, Embryonic stem cells (ES) cells, embryonic bodies (EB), signaling pathways, modulators, 96-round bottom well microtiter plates and hanging droplets.
Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling
Institutions: Mount Sinai School of Medicine .
Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2
. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5
. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus
embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus
embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7
is visualized in Xenopus
ectodermal cells to study Wnt signal transduction8
. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9
. This ex vivo
protocol should be a useful addition to in vitro
studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.
Developmental Biology, Issue 51, Xenopus embryo, ectoderm, Diversin, Frizzled, membrane recruitment, polarity, Wnt
Immunostaining of Dissected Zebrafish Embryonic Heart
Institutions: Mayo Clinic College of Medicine.
Zebrafish embryo becomes a popular in vivo
vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2
. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero
make them ideal for assessing cardiac defects 3
. The expression of any gene can be manipulated via morpholino technology or RNA injection 4
. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6
. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies.
Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope.
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.
Developmental Biology, Issue 59, Zebrafish, Danio rerio, Embryonic Heart, Cardiology, Dissection, Immunostaining
Labeling and Imaging Cells in the Zebrafish Hindbrain
Institutions: University of Maryland, Baltimore County, Children's National Medical Center.
Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1
. We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2
. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.
JoVE Neuroscience, Issue 41, development, zebrafish, embryo, brain, neural tube, microinjection, sectioning, time-lapse microscopy, confocal microscopy