There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
27 Related JoVE Articles!
Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies
Institutions: University of Oxford.
Kindlins are essential coactivators, with talin, of the cell surface receptors integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin β-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3's role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.
Virology, Issue 85, Heterologous protein expression, insect cells, Spodoptera frugiperda, baculovirus, protein purification, kindlin, cell adhesion
Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications
Institutions: London Health Sciences Centre, Western University, London Health Sciences Centre, Lawson Health Research Institute, Western University.
The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo
preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.
Medicine, Issue 84, Metastasis, circulating tumor cells (CTCs), CellSearch system, user defined marker characterization, in vivo, preclinical mouse model, clinical research
A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies
Institutions: Ottawa Hospital Research Institute, University of Ottawa, University of Ottawa, The Second Hospital of Shandong University, University of Tabuk, Ottawa General Hospital.
Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.
Medicine, Issue 85, mouse, tumor model, surgical stress, immunosuppression, perioperative immunotherapy, metastases
Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells
Institutions: University of Houston.
Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ERα, and ERβ. ERα is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ERα's regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ERα-positive breast cancer cell lines were compared before and after estrogen-activation using both the µParaflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations, and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.
Medicine, Issue 84, breast cancer, microRNA, estrogen, estrogen receptor, microarray, qPCR
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
Voluntary Breath-hold Technique for Reducing Heart Dose in Left Breast Radiotherapy
Institutions: Royal Marsden NHS Foundation Trust, University of Surrey, Institute of Cancer Research, Sutton, UK, Institute of Cancer Research, Sutton, UK.
Breath-holding techniques reduce the amount of radiation received by cardiac structures during tangential-field left breast radiotherapy. With these techniques, patients hold their breath while radiotherapy is delivered, pushing the heart down and away from the radiotherapy field. Despite clear dosimetric benefits, these techniques are not yet in widespread use. One reason for this is that commercially available solutions require specialist equipment, necessitating not only significant capital investment, but often also incurring ongoing costs such as a need for daily disposable mouthpieces. The voluntary breath-hold technique described here does not require any additional specialist equipment. All breath-holding techniques require a surrogate to monitor breath-hold consistency and whether breath-hold is maintained. Voluntary breath-hold uses the distance moved by the anterior and lateral reference marks (tattoos) away from the treatment room lasers in breath-hold to monitor consistency at CT-planning and treatment setup. Light fields are then used to monitor breath-hold consistency prior to and during radiotherapy delivery.
Medicine, Issue 89, breast, radiotherapy, heart, cardiac dose, breath-hold
Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma
Institutions: Southern Illinois University School of Medicine.
A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2
. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5
. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6
. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7
. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9
. Bevacizumab however failed to prolong overall survival in a recent phase III trial26
. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians’ choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12
, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.
Medicine, Issue 92, Tumor Treating Fields, TTF System, TTF Therapy, Recurrent Glioblastoma, Bevacizumab, Brain Tumor
Live Imaging of Drosophila Larval Neuroblasts
Institutions: National Institutes of Health.
Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila
central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.
Neuroscience, Issue 89, live imaging, Drosophila, neuroblast, stem cell, asymmetric division, centrosome, brain, cell cycle, mitosis
Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro
using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro
. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo
. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo
tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro
and in vivo
and may be useful in preclinical drug development for these devastating diseases.
Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins
Institutions: George Mason University, Ceres Nanosciences.
Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.
Bioengineering, Issue 90, biomarker, hydrogel, low abundance, mass spectrometry, nanoparticle, plasma, protein, urine
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
Substernal Thyroid Biopsy Using Endobronchial Ultrasound-guided Transbronchial Needle Aspiration
Institutions: State University of New York, Buffalo, Roswell Park Cancer Institute, State University of New York, Buffalo.
Substernal thyroid goiter (STG) represents about 5.8% of all mediastinal lesions1
. There is a wide variation in the published incidence rates due to the lack of a standardized definition for STG. Biopsy is often required to differentiate benign from malignant lesions. Unlike cervical thyroid, the overlying sternum precludes ultrasound-guided percutaneous fine needle aspiration of STG. Consequently, surgical mediastinoscopy is performed in the majority of cases, causing significant procedure related morbidity and cost to healthcare. Endobronchial Ultrasound-guided Transbronchial Needle Aspiration (EBUS-TBNA) is a frequently used procedure for diagnosis and staging of non-small cell lung cancer (NSCLC). Minimally invasive needle biopsy for lesions adjacent to the airways can be performed under real-time ultrasound guidance using EBUS. Its safety and efficacy is well established with over 90% sensitivity and specificity. The ability to perform EBUS as an outpatient procedure with same-day discharges offers distinct morbidity and financial advantages over surgery. As physicians performing EBUS gained procedural expertise, they have attempted to diversify its role in the diagnosis of non-lymph node thoracic pathologies. We propose here a role for EBUS-TBNA in the diagnosis of substernal thyroid lesions, along with a step-by-step protocol for the procedure.
Medicine, Issue 93, substernal thyroid, retrosternal thyroid, intra-thoracic thyroid, goiter, endobronchial ultrasound, EBUS, transbronchial needle aspiration, TBNA, biopsy, needle biopsy
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
In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration
Institutions: Harvard Medical School, MGH for Children, Massachusetts General Hospital.
Mucosal surfaces serve as protective barriers against pathogenic organisms. Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils. This process has the potential to be destructive to tissues if excessive or held in an unresolved state. Cocultured in vitro
models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration. This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil. Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface. The in vitro
model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa
(PAO1). Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli
(MC1000). The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls. This in vitro
model system can be manipulated and applied to other mucosal surfaces. Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections. A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro
coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.
Infection, Issue 83, Cellular Biology, Epithelium, Neutrophils, Pseudomonas aeruginosa, Respiratory Tract Diseases, Neutrophils, epithelial barriers, pathogens, transmigration
Experimental Metastasis Assay
Institutions: University of Rochester Medical Center, University of Rochester Medical Center.
Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro
and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro
as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.
medicine, Issue 42, cancer, metastasis, experimental, mouse, intravenous injection, lung
Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model
Institutions: Medical College of Georgia.
Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.
Immunology, Issue 45, Metastasis, CTL adoptive transfer, Lung, Tumor Immunology
High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT
Institutions: Weizmann Institute of Science, Weizmann Institute of Science, Weizmann Institute of Science.
Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (μCT).
High resolution μCT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2
. As opposed to electron tomography, the μCT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, μCT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4
, which are both time consuming and destructive. Sample conditioning and positioning in μCT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180° or 360°between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7
. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.
Bioengineering, Issue 52, 3D imaging, tomography, x-ray, non invasive, ex-vivo
Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry
Institutions: University of British Columbia .
Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2
. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3
. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism.
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6
. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9
. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging.
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10
. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.
Cellular Biology, Issue 63, cell cycle, kinetics, metabolic labeling, flow cytometry, biomedical, genetics, DNA replication
Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue
Institutions: The Johns Hopkins University School of Medicine, The Johns Hopkins University School of Medicine, The Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine, The Johns Hopkins University School of Medicine, Yale School of Medicine, The Johns Hopkins University School of Medicine, The Johns Hopkins University School of Medicine.
B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) 1,2
. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity3-8
Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10
. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response11
. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12
In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8
, but staining on paraffin-embedded slides had been a challenge until recently13-18
. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.
Cancer Biology, Issue 71, Medicine, Immunology, Biochemistry, Molecular Biology, Cellular Biology, Chemistry, Oncology, immunohistochemistry, B7-H1 (PD-L1), pancreatic adenocarcinoma, pancreatic cancer, pancreas, tumor, T-cell immunity, cancer
Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry
Institutions: Sanford-Burnham Medical Research Institute, University of California, San Diego , VA San Diego Healthcare Center, University of California, San Diego .
Although human saliva proteome and peptidome have been revealed 1-2
they were majorly identified from tryptic digests of saliva proteins. Identification of indigenous peptidome of human saliva without prior digestion with exogenous enzymes becomes imperative, since native peptides in human saliva provide potential values for diagnosing disease, predicting disease progression, and monitoring therapeutic efficacy. Appropriate sampling is a critical step for enhancement of identification of human indigenous saliva peptidome. Traditional methods of sampling human saliva involving centrifugation to remove debris 3-4
may be too time-consuming to be applicable for clinical use. Furthermore, debris removal by centrifugation may be unable to clean most of the infected pathogens and remove the high abundance proteins that often hinder the identification of low abundance peptidome.
Conventional proteomic approaches that primarily utilize two-dimensional gel electrophoresis (2-DE) gels in conjugation with in-gel digestion are capable of identifying many saliva proteins 5-6
. However, this approach is generally not sufficiently sensitive to detect low abundance peptides/proteins. Liquid chromatography-Mass spectrometry (LC-MS) based proteomics is an alternative that can identify proteins without prior 2-DE separation. Although this approach provides higher sensitivity, it generally needs prior sample pre-fractionation 7
and pre-digestion with trypsin, which makes it difficult for clinical use.
To circumvent the hindrance in mass spectrometry due to sample preparation, we have developed a technique called capillary ultrafiltration (CUF) probes 8-11
. Data from our laboratory demonstrated that the CUF probes are capable of capturing proteins in vivo
from various microenvironments in animals in a dynamic and minimally invasive manner 8-11
. No centrifugation is needed since a negative pressure is created by simply syringe withdrawing during sample collection. The CUF probes combined with LC-MS have successfully identified tryptic-digested proteins 8-11
. In this study, we upgraded the ultrafiltration sampling technique by creating a lollipop-like ultrafiltration (LLUF) probe that can easily fit in the human oral cavity. The direct analysis by LC-MS without trypsin digestion showed that human saliva indigenously contains many peptide fragments derived from various proteins. Sampling saliva with LLUF probes avoided centrifugation but effectively removed many larger and high abundance proteins. Our mass spectrometric results illustrated that many low abundance peptides became detectable after filtering out larger proteins with LLUF probes. Detection of low abundance saliva peptides was independent of multiple-step sample separation with chromatography. For clinical application, the LLUF probes incorporated with LC-MS could potentially be used in the future to monitor disease progression from saliva.
Medicine, Issue 66, Molecular Biology, Genetics, Sampling, Saliva, Peptidome, Ultrafiltration, Mass spectrometry
Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells
Institutions: Balgrist University Hospital, Zurich.
Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1
, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2
. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo
, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4
. We are currently optimizing these techniques for in vivo
monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo
analysis of metastasis beside classical methods like qPCR5
or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ
gene encoding the bacterial enzyme β-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7
and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo
down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8
in studies assessing new anticancer therapies9-11
. A limiting factor of X-gal staining is the low contrast to e.g.
blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ
lung perfusion, a technique that was recently established by Borsig et al.12
who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ
under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-
expressing tumor cells and in-situ
perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13
, which was originally described to be non-metastatic14
Cancer Biology, Issue 66, Medicine, Molecular Biology, Cellular Biology, lung metastasis, lacZ-tagging, 5-Bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) staining, in-situ lung perfusion, metastases, imaging
RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells
Institutions: Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City.
The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g.
drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2
. RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4
in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.
Genetics, Issue 72, Molecular Biology, Immunology, Medicine, Genomics, Proteins, RNA-seq, Next Generation DNA Sequencing, Transcriptome, Transcription, Thrombin, Endothelial cells, high-throughput, DNA, genomic DNA, RT-PCR, PCR
Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence
Institutions: Albert Einstein College of Medicine , Albert Einstein College of Medicine , Albert Einstein College of Medicine .
Telomeres are repeating DNA sequences at the tip ends of the chromosomes that are diverse in length and in humans can reach a length of 15,000 base pairs. The telomere serves as a bioprotective mechanism of chromosome attrition at each cell division. At a certain length, telomeres become too short to allow replication, a process that may lead to chromosome instability or cell death. Telomere length is regulated by two opposing mechanisms: attrition and elongation. Attrition occurs as each cell divides. In contrast, elongation is partially modulated by the enzyme telomerase, which adds repeating sequences to the ends of the chromosomes. In this way, telomerase could possibly reverse an aging mechanism and rejuvenates cell viability. These are crucial elements in maintaining cell life and are used to assess cellular aging. In this manuscript we will describe an accurate, short, sophisticated and cheap method to assess telomere length in multiple tissues and species. This method takes advantage of two key elements, the tandem repeat of the telomere sequence and the sensitivity of the qRT-PCR to detect differential copy numbers of tested samples. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.
Genetics, Issue 75, Molecular Biology, Cellular Biology, Medicine, Biomedical Engineering, Genomics, Telomere length, telomerase activity, telomerase, telomeres, telomere, DNA, PCR, polymerase chain reaction, qRT-PCR, sequencing, aging, telomerase assay
Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung
Institutions: National Institutes of Health.
The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.
Medicine, Issue 81, mouse, rodents, intratracheal intubation, delivery of exogenous substances, lung, study of airway pathology and physiology, pulmonary fibrosis
Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer
Institutions: Joint Unit Hospices de Lyon-bioMérieux, BioMérieux, Hospices Civils de Lyon, Lyon 1 University, BioMérieux, Hospices Civils de Lyon, Hospices Civils de Lyon.
The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1
. ‘How to use PSA' remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2
or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4
. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g.
PCA3 in prostate cancer5,6
and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10
. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1
Medicine, Issue 81, Cancer Biology, Genetics, Molecular Biology, Prostate, Retroviridae, Biomarkers, Pharmacological, Tumor Markers, Biological, Prostatectomy, Microarray Analysis, Gene Expression, Diagnosis, Human Endogenous Retroviruses, HERV, microarray, Transcriptome, prostate cancer, Affymetrix
Establishing Primary Adult Fibroblast Cultures From Rodents
Institutions: University of Rochester.
The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.
Cellular Biology, Issue 44, Primary cells, fibroblasts, rodents, cell culture
Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation
Institutions: La Jolla Institute for Allergy and Immunology.
Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens. Central to these studies is CD1d - the antigen presenting molecule that presents glycolipids to NKT cells. The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells. Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy. Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.
Immunology, Issue 10, Natural Killer T cells, NKT cells, CD1 Tetramers, antigen presentation, glycolipid antigens, CD1d, Mucosal Immunity, Translational Research