JoVE Visualize What is visualize?
Related JoVE Video
Pubmed Article
Systems-level modeling of cancer-fibroblast interaction.
PUBLISHED: 04-01-2009
Cancer cells interact with surrounding stromal fibroblasts during tumorigenesis, but the complex molecular rules that govern these interactions remain poorly understood thus hindering the development of therapeutic strategies to target cancer stroma. We have taken a mathematical approach to begin defining these rules by performing the first large-scale quantitative analysis of fibroblast effects on cancer cell proliferation across more than four hundred heterotypic cell line pairings. Systems-level modeling of this complex dataset using singular value decomposition revealed that normal tissue fibroblasts variably express at least two functionally distinct activities, one which reflects transcriptional programs associated with activated mesenchymal cells, that act either coordinately or at cross-purposes to modulate cancer cell proliferation. These findings suggest that quantitative approaches may prove useful for identifying organizational principles that govern complex heterotypic cell-cell interactions in cancer and other contexts.
Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates >85%, survival rates for stage III/IV disease are <40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF) cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.
24 Related JoVE Articles!
Play Button
Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture
Authors: Kun Xu, Rachel J. Buchsbaum.
Institutions: Tufts Medical Center.
While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis. The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7. Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.
Molecular Biology, Issue 62, Tumor microenvironment, extracellular matrix, three-dimensional, co-culture, spheroid, mixed-cell, cell culture
Play Button
Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts
Authors: Marc D. Bullock, Max Mellone, Karen M. Pickard, Abdulkadir Emre Sayan, Richard Mitter, John N. Primrose, Graham K. Packham, Gareth Thomas, Alexander H. Mirnezami.
Institutions: University of Southampton School of Medicine, University of Southampton School of Medicine, London Research Institute, Cancer Research UK.
Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade. Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques. Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions. Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision. By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.   
Medicine, Issue 86, Colorectal cancer, Cancer metastasis, organotypic culture, laser microdissection, molecular profiling, invasion, tumor microenvironment, stromal tissue, epithelium, fibroblasts
Play Button
MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression
Authors: Mansoureh Sameni, Arulselvi Anbalagan, Mary B. Olive, Kamiar Moin, Raymond R. Mattingly, Bonnie F. Sloane.
Institutions: Wayne State University , Wayne State University .
We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.
Medicine, Issue 60, Immunology, Breast, cancer, extracellular matrix, invasion, proteolysis, tumor microenvironment
Play Button
Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion
Authors: Alimatou M. Tchafa, Arpit D. Shah, Shafei Wang, Melissa T. Duong, Adrian C. Shieh.
Institutions: Drexel University .
The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression. Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 μm s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20. The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.
Biomedical Engineering, Issue 65, Bioengineering, Biophysics, Cancer Biology, Cancer, interstitial fluid flow, invasion, mechanobiology, migration, three-dimensional cell culture, tumor microenvironment
Play Button
Tissue Engineering of a Human 3D in vitro Tumor Test System
Authors: Corinna Moll, Jenny Reboredo, Thomas Schwarz, Antje Appelt, Sebastian Schürlein, Heike Walles, Sarah Nietzer.
Institutions: University Hospital Würzburg.
Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system. Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns). Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow. In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.
Cancer Biology, Issue 78, Biomedical Engineering, Bioengineering, Medicine, Anatomy, Physiology, Molecular Biology, Cellular Biology, Tissue Engineering, Tumor Cells, Cultured, Biotechnology, Culture Techniques, Cell Engineering, Cellular Microenvironment, Equipment and Supplies, Decellularization, BioVaSc, primary cell isolation, tumor test system, dynamic culture conditions, bioreactor, 3D in vitro models, cell culture
Play Button
Detection of Architectural Distortion in Prior Mammograms via Analysis of Oriented Patterns
Authors: Rangaraj M. Rangayyan, Shantanu Banik, J.E. Leo Desautels.
Institutions: University of Calgary , University of Calgary .
We demonstrate methods for the detection of architectural distortion in prior mammograms of interval-cancer cases based on analysis of the orientation of breast tissue patterns in mammograms. We hypothesize that architectural distortion modifies the normal orientation of breast tissue patterns in mammographic images before the formation of masses or tumors. In the initial steps of our methods, the oriented structures in a given mammogram are analyzed using Gabor filters and phase portraits to detect node-like sites of radiating or intersecting tissue patterns. Each detected site is then characterized using the node value, fractal dimension, and a measure of angular dispersion specifically designed to represent spiculating patterns associated with architectural distortion. Our methods were tested with a database of 106 prior mammograms of 56 interval-cancer cases and 52 mammograms of 13 normal cases using the features developed for the characterization of architectural distortion, pattern classification via quadratic discriminant analysis, and validation with the leave-one-patient out procedure. According to the results of free-response receiver operating characteristic analysis, our methods have demonstrated the capability to detect architectural distortion in prior mammograms, taken 15 months (on the average) before clinical diagnosis of breast cancer, with a sensitivity of 80% at about five false positives per patient.
Medicine, Issue 78, Anatomy, Physiology, Cancer Biology, angular spread, architectural distortion, breast cancer, Computer-Assisted Diagnosis, computer-aided diagnosis (CAD), entropy, fractional Brownian motion, fractal dimension, Gabor filters, Image Processing, Medical Informatics, node map, oriented texture, Pattern Recognition, phase portraits, prior mammograms, spectral analysis
Play Button
The In ovo CAM-assay as a Xenograft Model for Sarcoma
Authors: Gwen M.L. Sys, Lore Lapeire, Nikita Stevens, Herman Favoreel, Ramses Forsyth, Marc Bracke, Olivier De Wever.
Institutions: Ghent University Hospital, Ghent University, Ghent University, Pathlicon.
Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions. This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.
Cancer Biology, Issue 77, Medicine, Cellular Biology, Molecular Biology, Biomedical Engineering, Bioengineering, Developmental Biology, Anatomy, Physiology, Oncology, Surgery, Adipose Tissue, Connective Tissue, Neoplasm, Muscle Tissue, Sarcoma, Animal Experimentation, Cell Culture Techniques, Neoplasms, Experimental, Neoplasm Transplantation, Biological Assay, Sarcomas, CAM-assay, CAM, assay, xenograft, proliferation, invasion, cancer, tumor, in ovo, animal model
Play Button
Cell Co-culture Patterning Using Aqueous Two-phase Systems
Authors: John P. Frampton, Joshua B. White, Abin T. Abraham, Shuichi Takayama.
Institutions: University of Michigan , University of Michigan .
Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.
Bioengineering, Issue 73, Biomedical Engineering, Microbiology, Molecular Biology, Cellular Biology, Biochemistry, Biotechnology, Cell Migration Assays, Culture Techniques, bioengineering (general), Patterning, Aqueous Two-Phase System, Co-Culture, cell, Dextran, Polyethylene glycol, media, PEG, DEX, colonies, cell culture
Play Button
Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus
Authors: In Young Choi, HoTae Lim, Gabsang Lee.
Institutions: Johns Hopkins University School of Medicine.
A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases. Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.
Stem Cell Biology, Issue 86, Induced pluripotent stem cells, Human embryonic stem cells, Sendai-virus
Play Button
Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles
Authors: Jing Xu, Mansoor Amiji.
Institutions: Northeastern University.
More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8 Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.
Bioengineering, Issue 59, Gelatin Nanoparticle, Gene Therapy, Targeted Delivery, Pancreatic Cancer, Epidermal Growth Factor Receptor, EGFR
Play Button
Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)
Authors: Yoray Sharon, Lina Alon, Sarah Glanz, Charlotte Servais, Neta Erez.
Institutions: Tel Aviv University.
Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker α-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFRα as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFRα is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to α-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.
Cancer Biology, Issue 71, Cellular Biology, Molecular Biology, Medicine, Oncology, Pathology, Bioengineering, Biomedical Engineering, Cancer-Associated Fibroblasts, fibroblast, FACS sorting, PDGFRalpha, Breast cancer, Skin carcinoma, stroma, tumor, cancer, tissue, cell, culture, human, mouse, animal model
Play Button
Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale
Authors: Valentina Gandin, Kristina Sikström, Tommy Alain, Masahiro Morita, Shannon McLaughlan, Ola Larsson, Ivan Topisirovic.
Institutions: McGill University, Karolinska Institutet, McGill University.
mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed “anota” algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.
Biochemistry, Issue 87, Cells, Eukaryota, Nutritional and Metabolic Diseases, Neoplasms, Metabolic Phenomena, Cell Physiological Phenomena, mRNA translation, ribosomes, protein synthesis, genome-wide analysis, translatome, mTOR, eIF4E, 4E-BP1
Play Button
Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen
Authors: Dennis Ma, Jonathan Collins, Tomas Hudlicky, Siyaram Pandey.
Institutions: University of Windsor, Brock University.
Breast cancer is one of the most common cancers amongst women in North America. Many current anti-cancer treatments, including ionizing radiation, induce apoptosis via DNA damage. Unfortunately, such treatments are non-selective to cancer cells and produce similar toxicity in normal cells. We have reported selective induction of apoptosis in cancer cells by the natural compound pancratistatin (PST). Recently, a novel PST analogue, a C-1 acetoxymethyl derivative of 7-deoxypancratistatin (JCTH-4), was produced by de novo synthesis and it exhibits comparable selective apoptosis inducing activity in several cancer cell lines. Recently, autophagy has been implicated in malignancies as both pro-survival and pro-death mechanisms in response to chemotherapy. Tamoxifen (TAM) has invariably demonstrated induction of pro-survival autophagy in numerous cancers. In this study, the efficacy of JCTH-4 alone and in combination with TAM to induce cell death in human breast cancer (MCF7) and neuroblastoma (SH-SY5Y) cells was evaluated. TAM alone induced autophagy, but insignificant cell death whereas JCTH-4 alone caused significant induction of apoptosis with some induction of autophagy. Interestingly, the combinatory treatment yielded a drastic increase in apoptotic and autophagic induction. We monitored time-dependent morphological changes in MCF7 cells undergoing TAM-induced autophagy, JCTH-4-induced apoptosis and autophagy, and accelerated cell death with combinatorial treatment using time-lapse microscopy. We have demonstrated these compounds to induce apoptosis/autophagy by mitochondrial targeting in these cancer cells. Importantly, these treatments did not affect the survival of noncancerous human fibroblasts. Thus, these results indicate that JCTH-4 in combination with TAM could be used as a safe and very potent anti-cancer therapy against breast cancer and neuroblastoma cells.
Cancer Biology, Issue 63, Medicine, Biochemistry, Breast adenocarcinoma, neuroblastoma, tamoxifen, combination therapy, apoptosis, autophagy
Play Button
Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts
Authors: Urszula M. Polanska, Ahmet Acar, Akira Orimo.
Institutions: University of Manchester, Juntendo University.
Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3. The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of α-smooth muscle actin (α-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients. Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model. In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11. We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.
Medicine, Issue 56, cancer, stromal myofibroblasts, experimentally generated carcinoma-associated fibroblasts (exp-CAFs), fibroblast, human mammary carcinomas, tumour xenografts
Play Button
Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context
Authors: Paul Timpson, Ewan J. Mcghee, Zahra Erami, Max Nobis, Jean A. Quinn, Mike Edward, Kurt I. Anderson.
Institutions: University of Glasgow, University of Glasgow.
Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.
Bioengineering, Issue 56, Organotypic culture, cell migration, invasion, 3-dimensional matrix, Collagen I, second harmonic generation, host-tumor interaction, microenvironment
Play Button
Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Authors: Robert S. McNeill, Ralf S. Schmid, Ryan E. Bash, Mark Vitucci, Kristen K. White, Andrea M. Werneke, Brian H. Constance, Byron Huff, C. Ryan Miller.
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
Play Button
Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer
Authors: Elizabeth S. Nakasone, Hanne A. Askautrud, Mikala Egeblad.
Institutions: Watson School of Biological Sciences, Cold Spring Harbor Laboratory, University of Oslo and Oslo University Hospital.
The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.
Cancer Biology, Issue 73, Medicine, Molecular Biology, Cellular Biology, Biomedical Engineering, Genetics, Oncology, Pharmacology, Surgery, Tumor Microenvironment, Intravital imaging, chemotherapy, Breast cancer, time-lapse, mouse models, cancer cell death, stromal cell migration, cancer, imaging, transgenic, animal model
Play Button
A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions
Authors: Valentina Goncharova, Sophia K. Khaldoyanidi.
Institutions: Torrey Pines Institute for Molecular Studies, Cascade LifeSciences Inc..
Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.
Bioengineering, Issue 77, Cellular Biology, Biophysics, Physiology, Molecular Biology, Biomedical Engineering, Immunology, Cells, Biological Factors, Equipment and Supplies, Cell Physiological Phenomena, Natural Science Disciplines, Life Sciences (General), circulating cells, extravasation, physiological shear stress, endothelial cells, microenvironment, chemokine gradient, flow, chamber, cell culture, assay
Play Button
From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data
Authors: Wen-Ting Tsai, Ahmed Hassan, Purbasha Sarkar, Joaquin Correa, Zoltan Metlagel, Danielle M. Jorgens, Manfred Auer.
Institutions: Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory.
Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g., signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation. The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.
Bioengineering, Issue 90, 3D electron microscopy, feature extraction, segmentation, image analysis, reconstruction, manual tracing, thresholding
Play Button
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Authors: James Smadbeck, Meghan B. Peterson, George A. Khoury, Martin S. Taylor, Christodoulos A. Floudas.
Institutions: Princeton University.
The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity. To disseminate these methods for broader use we present Protein WISDOM (, a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Play Button
Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles
Authors: Eva Wagner, Sören Brandenburg, Tobias Kohl, Stephan E. Lehnart.
Institutions: Heart Research Center Goettingen, University Medical Center Goettingen, German Center for Cardiovascular Research (DZHK) partner site Goettingen, University of Maryland School of Medicine.
In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.
Bioengineering, Issue 92, cardiac myocyte, atria, ventricle, heart, primary cell isolation, fluorescence microscopy, membrane tubule, transverse-axial tubule system, image analysis, image processing, T-tubule, collagenase
Play Button
Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer
Authors: Philippe Pérot, Valérie Cheynet, Myriam Decaussin-Petrucci, Guy Oriol, Nathalie Mugnier, Claire Rodriguez-Lafrasse, Alain Ruffion, François Mallet.
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
Play Button
High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Authors: Subarna Bhattacharya, Paul W. Burridge, Erin M. Kropp, Sandra L. Chuppa, Wai-Meng Kwok, Joseph C. Wu, Kenneth R. Boheler, Rebekah L. Gundry.
Institutions: Medical College of Wisconsin, Stanford University School of Medicine, Medical College of Wisconsin, Hong Kong University, Johns Hopkins University School of Medicine, Medical College of Wisconsin.
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.
Cellular Biology, Issue 91, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
Play Button
Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation
Authors: Mitchell Kronenberg.
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
Copyright © JoVE 2006-2015. All Rights Reserved.
Policies | License Agreement | ISSN 1940-087X
simple hit counter

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.