Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.
26 Related JoVE Articles!
In vitro Organoid Culture of Primary Mouse Colon Tumors
Institutions: University of Michigan , University of Michigan .
Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1
, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.
Cancer Biology, Issue 75, Medicine, Molecular Biology, Cellular Biology, Biomedical Engineering, Anatomy, Physiology, Genetics, Oncology, Surgery, Organoids, Tumor Cells, Cultured Colonic Neoplasms, Primary Cell Culture, Colon tumor, chelation, collagenase, matrigel, organoid, EGF, colon cancer, cancer, tumor, cell, isolation, immunohistochemistry, mouse, animal model
Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts
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
Three-dimensional Co-culture Model for Tumor-stromal Interaction
Institutions: The University of Tokyo, The University of Tokyo, The University of Tokyo, Nihon University School of Dentistry, Ohu University School of Pharmaceutical Sciences.
Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.
Medicine, Issue 96, Three-dimensional co-culture, cancer, fibroblast, invasion, tumor stroma, collagen
Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model
Institutions: University of Sheffield, University of Sheffield, Sheffield Teaching Hospitals NHS Foundation Trust.
The incidence of both esophageal adenocarcinoma and its precursor, Barrett’s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo
and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia
, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett’s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.
Bioengineering, Issue 99, esophagus, epithelium, tissue engineering, 3D construct, esophageal cancer, Barrett’s Metaplasia
C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays
Institutions: Princeton University, Princeton University.
The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans
navigates its environment through neuron-specific detection of food and chemical odors1, 2
, and can associate nutritive states with chemical odors3
, and the pathogenicity of a food source5
Here, we describe assays of C. elegans
associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6
to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7
). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (<40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6
a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9
Otsu's method is then used to determine a threshold to separate worms from the background.10
Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8
Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans
that are relevant across species.
Neuroscience, Issue 49, memory, associative learning, C. elegans, chemotaxis, spaced training, behavior
Isolation of Myofibroblasts from Mouse and Human Esophagus
Institutions: Keck School of Medicine, University of Southern California.
Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express α-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e.
, the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo
state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.
Cellular Biology, Issue 95, Cellular biology, mouse, human, esophagus, mesenchymal stromal cells, myofibroblasts, primary cells
Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays
Institutions: University Hospital Basel, University Hospital Tübingen.
Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called “cancer stem cells” (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro
is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro
. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.
Medicine, Issue 95, Cancer stem cells, spheres assay, ovarian, single cell, SOX2, in vitro assay, ovarian carcinoma
High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes
Institutions: West Virginia University, West Virginia University, West Virginia University.
High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 µm, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.
Medicine, Issue 101, Ultrasound, cervical lymphnode, mouse, imaging, animal model, anatomy, mapping.
Three-Dimensional (3D) Tumor Spheroid Invasion Assay
Institutions: The Institute of Cancer Research, The Institute of Cancer Research.
Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g
., brain tumors such as glioblastoma multiforme
and squamous cell carcinoma of the head and neck – SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.
Medicine, Issue 99, invasion, metastasis, 3D, tumor spheroids, extracellular matrix, imaging, high-throughput, drug development.
An Immunofluorescent Method for Characterization of Barrett’s Esophagus Cells
Institutions: St. Joseph's Hospital and Medical Center.
Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One of the primary risk factors of EAC is Barrett’s esophagus (BE), a metaplastic change of the normal squamous esophagus in response to chronic heartburn. Despite the well-established connection between EAC and BE, interrogation of the molecular events, particularly altered signaling pathways involving progression of BE to EAC, are poorly understood. Much of this is due to the lack of suitable in vitro
models available to study these diseases. Recently, immortalized BE cell lines have become commercially available allowing for in vitro
studies of BE. Here, we present a method for immunofluorescent staining of immortalized BE cell lines, allowing in vitro
characterization of cell signaling and structure after exposure to therapeutic compounds. Application of these techniques will help develop insight into the mechanisms involved in BE to EAC progression and provide potential avenues for treatment and prevention of EAC.
Cellular Biology, Issue 89, Barrett's Esophagus, Immunofluorescence, adenocarcinoma, morphology, gastroesophageal reflux disease, immortalized BE cell lines
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
Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene
Institutions: University of California, Davis.
Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1
. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2
. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1
that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.
Medicine, Issue 94, oncogene, focus formation, foci, retrovirus, NIH 3T3, Platinum-E, ecotropic, pBABEpuro
In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes
Institutions: Wayne State University School of Medicine, Wayne State University School of Medicine, Wayne State University School of Medicine.
Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3
. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4
and secretory diarrhea5
. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6
. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g.
, by cholera toxin and heat stable E. coli
enterotoxin) that stimulates cAMP or cGMP production in the gut7
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro
and also in vivo8-19
. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27
. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e.
, 1477-DTRL-1480 in human CFTR)20
. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22
. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29
. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro
, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29
Biochemistry, Issue 66, Molecular Biology, Chemistry, CFTR, macromolecular complex, protein interaction, PDZ scaffold protein, epithelial cell, cystic fibrosis
In vivo Dual Substrate Bioluminescent Imaging
Institutions: Case Western Reserve University .
Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo
bioluminescent imaging technologies 1-3
. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i)
evolve within primary tumors, (ii)
disseminate throughout the body, and (iii)
reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via
the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis
) and sea pansy (Renilla reniformis
), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro
cell-based reporter gene assays 4
. Here we demonstrate the in vivo
utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo
activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6
. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9
. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.
Medicine, Issue 56, firefly luciferase, Renilla Luciferase, breast cancer, metastasis, Smad
A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion
Institutions: Roswell Park Cancer Institute, D'Youville College.
Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted “X”). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.
Medicine, Issue 98, hormone, cytokine, growth factor, migration, invasion, collagen, cancer
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
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations
Institutions: Wake Forest University Health Sciences, R.J. Reynolds Tobacco Company.
Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to increased susceptibility of smokers to microbial infections and tumor incidence. Ex vivo
suppression of receptor-mediated immune responses in human peripheral blood mononuclear cells (PBMCs) treated with smoke constituents is an attractive approach to study mechanisms and evaluate the likely long-term effects of exposure to tobacco products. Here, we optimized methods to perform ex vivo
assays using PBMCs stimulated by bacterial lipopolysaccharide, a Toll-like receptor-4 ligand. The effects of whole smoke-conditioned medium (WS-CM), a combustible tobacco product preparation (TPP), and nicotine were investigated on cytokine secretion and target cell killing by PBMCs in the ex vivo
assays. We show that secreted cytokines IFN-γ, TNF, IL-10, IL-6, and IL-8 and intracellular cytokines IFN-γ, TNF-α, and MIP-1α were suppressed in WS-CM-exposed PBMCs. The cytolytic function of effector PBMCs, as determined by a K562 target cell killing assay was also reduced by exposure to WS-CM; nicotine was minimally effective in these assays. In summary, we present a set of improved assays to evaluate the effects of TPPs in ex vivo
assays, and these methods could be readily adapted for testing other products of interest.
Immunology, Issue 95, Tobacco product preparation, whole smoke-conditioned medium, human peripheral blood mononuclear cells, PBMC, lipopolysaccharide, cell death, secreted cytokines, intracellular cytokines, K562 killing assay.
Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis
Institutions: Mayo Clinic College of Medicine, Center for Individualized Medicine, Tufts University School of Medicine, Mayo Clinic.
Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers. To robustly and efficiently identify oncogenic “drivers” among these tumors and define their complex relationships with concurrent genetic alterations during tumor pathogenesis remains a daunting task. Recently, zebrafish have emerged as an important animal model for studying human diseases, largely because of their ease of maintenance, high fecundity, obvious advantages for in vivo
imaging, high conservation of oncogenes and their molecular pathways, susceptibility to tumorigenesis and, most importantly, the availability of transgenic techniques suitable for use in the fish. Transgenic zebrafish models of cancer have been widely used to dissect oncogenic pathways in diverse tumor types. However, developing a stable transgenic fish model is both tedious and time-consuming, and it is even more difficult and more time-consuming to dissect the cooperation of multiple genes in disease pathogenesis using this approach, which requires the generation of multiple transgenic lines with overexpression of the individual genes of interest followed by complicated breeding of these stable transgenic lines. Hence, use of a mosaic transient transgenic approach in zebrafish offers unique advantages for functional genomic analysis in vivo
. Briefly, candidate transgenes can be coinjected into one-cell-stage wild-type or transgenic zebrafish embryos and allowed to integrate together into each somatic cell in a mosaic pattern that leads to mixed genotypes in the same primarily injected animal. This permits one to investigate in a faster and less expensive manner whether and how the candidate genes can collaborate with each other to drive tumorigenesis. By transient overexpression of activated ALK
in the transgenic fish overexpressing MYCN
, we demonstrate here the cooperation of these two oncogenes in the pathogenesis of a pediatric cancer, neuroblastoma that has resisted most forms of contemporary treatment.
Developmental Biology, Issue 97, zebrafish, animal model, mosaic transgenesis, coinjection, functional genomics, tumor initiation
Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies
Institutions: Spanish National Cancer Research Center, Institute for Research in Biomedicine (IRB Barcelona), Queen Mary University of London.
Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2
. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo
tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g.,
NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro
sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro
and in vivo
validation to eliminate false discoveries.
Medicine, Issue 100, Pancreatic ductal adenocarcinoma, cancer stem cells, spheres, metformin (met), metabolism
Analysis of Cell Migration within a Three-dimensional Collagen Matrix
Institutions: Witten/Herdecke University.
The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.
Bioengineering, Issue 92, cell migration, 3D collagen matrix, cell tracking
Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke
Institutions: Philip Morris Products S.A..
Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo
exposure situation. Recently developed 3D tissue culture models better reflect the in vivo
situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo
. In this work, the utilization of an in vitro
exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.
Bioengineering, Issue 96, human organotypic bronchial epithelial, 3D culture, in vitro exposure system, cigarette smoke, cilia beating, xenobiotic metabolism, network models, systems toxicology
Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis
Institutions: Johns Hopkins University Bloomberg School of Public Health, Chinese University of Hong Kong, Johns Hopkins University School of Medicine.
Anastasis (Greek for “rising to life”) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c
into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor for identifying and tracking anastasis in mammalian cells.
Cellular Biology, Issue 96, Anastasis, apoptosis, apoptotic bodies, caspase, cell death, cell shrinkage, cell suicide, cytochrome c, DNA damage, genetic alterations, mitochondrial outer membrane permeabilization (MOMP), programmed cell death, reversal of apoptosis
A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke
Institutions: CDCF-AOX Lab, Cornell University.
Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12
. Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens9,10
. Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung3
. Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke4
. A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke14
. However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine8
. In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy1,2,14
. We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively11,13
Bioengineering, Issue 59, Cigarette smoke, free radical, spin-trap, ESR
A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells
Institutions: University of Massachusetts, University of Massachusetts, University of Massachusetts.
Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5
. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8
. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9
. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13
. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11
. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.
Cancer Biology, Issue 55, tumor, vascular, endothelial, tube formation, Matrigel, in vitro
Isolation and Characterization of Neutrophils with Anti-Tumor Properties
Institutions: Hebrew University Medical School, Hadassah-Hebrew University Medical Center.
Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or naïve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a > 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro
and in vivo
functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo
. We further describe techniques to label the neutrophils for in vivo
tracking, and to determine their anti-metastatic capacity in vivo
. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.
Immunology, Issue 100, Neutrophil isolation, tumor-entrained neutrophils, high-density neutrophils, low-density neutrophils, anti-tumor cytotoxicity, BrdU labeling, CFSE labeling, luciferase assay, neutrophil depletion, anti-metastatic activity, lung metastatic seeding assay, neutrophil adoptive transfer.