Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and γ-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.
17 Related JoVE Articles!
Induction and Analysis of Epithelial to Mesenchymal Transition
Institutions: R&D Systems, Inc., R&D Systems, Inc..
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.
Molecular Biology, Issue 78, Cellular Biology, Biochemistry, Biomedical Engineering, Stem Cell Biology, Cancer Biology, Medicine, Bioengineering, Anatomy, Physiology, biology (general), Pathological Conditions, Signs and Symptoms, Wounds and Injuries, Neoplasms, Diagnosis, Therapeutics, Epithelial to mesenchymal transition, EMT, cancer, metastasis, cancer stem cell, cell, assay, immunohistochemistry
Isolation and Culture of Avian Embryonic Valvular Progenitor Cells
Institutions: Cornell University.
Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro
models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.
Developmental Biology, Issue 44, quail, chicken, HH14-, HH25, endocardial, explant, mesenchymal, cushion, valve progenitor
Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing
Institutions: Midwestern University, Midwestern University.
Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro
model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.
Developmental Biology, Issue 96, Collective cell migration, reepithelization, zebrafish, keratocyte, epithelial to mesenchymal transition, cell-cell adhesion, cutaneous wound healing
In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells
Institutions: National Cancer Institute.
Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent ‘floater’ cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.
Medicine, Issue 96, Ovarian cancer, tumor-initiating cells, cancer stem cell, tumorigenicity, spheroid, mouse, gynecological cancer
Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice
Institutions: The Walter and Eliza Hall Institute for Medical Research, University of Melbourne, Olivia Newton-John Cancer Research Institute.
Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.
Medicine, Issue 97, cancer, colitis, colon, endoscopy, mucosa, therapy.
Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.
Institutions: Leiden University Medical Center.
Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.
Medicine, Issue 96, Breast Cancer, Tumor growth, Orthotopic Injection, Metastasis
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
Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts
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
Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia
Institutions: University of Dundee, UK.
Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification. Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.
Cellular Biology, Issue 87, Cancer, RNA synthesis, Hypoxia, Microscopy, Click-iT, Open Microscopy Environment, OMERO
Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications
Institutions: London Health Sciences Centre, Western University, London Health Sciences Centre, Lawson Health Research Institute, Western University.
The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo
preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.
Medicine, Issue 84, Metastasis, circulating tumor cells (CTCs), CellSearch system, user defined marker characterization, in vivo, preclinical mouse model, clinical research
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
Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans
Institutions: University of Washington, University of Washington.
Oxygen is essential for all metazoans to survive, with one known exception1
. Decreased O2
availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5
. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans
is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2
concentrations without confounding effects since C. elegans
(and other gasses) by diffusion, as opposed to a facilitated respiratory system6
. Factors known to be involved in the response to hypoxia are conserved in C. elegans
. The actual response to hypoxia depends on the specific concentration of O2
that is available. In C. elegans
, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1
, the highly-conserved hypoxia-inducible transcription factor6-9
. C .elegans
embryos require hif-1
to survive in 5,000-20,000 ppm O27,10
. Hypoxia is a general term for "less than normal O2
". Normoxia (normal O2
) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2
to be normoxia. However, it has been shown that C. elegans
has a behavioral preference for O2
concentrations from 5-12% (50,000-120,000 ppm O2
. In larvae and adults, hif-1
acts to prevent hypoxia-induced diapause in 5,000 ppm O212
. However, hif-1
does not play a role in the response to lower concentrations of O2
(anoxia, operational definition <10 ppm O2
. In anoxia, C. elegans
enters into a reversible state of suspended animation in which all microscopically observable activity ceases10
. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2
. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans
samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14
. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.
Biochemistry, Issue 65, Molecular Biology, Cellular Biology, Genetics, Developmental Biology, C. elegans, hypoxia, hypoxia inducible factor-1 (hif-1), anoxia, oxygen
Tracking Hypoxic Signaling within Encapsulated Cell Aggregates
Institutions: University of South Carolina, University of South Carolina.
In Diabetes mellitus type 1, autoimmune destruction of the pancreatic β-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2
. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3
, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5
which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8
. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7
. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9
, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10
. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact β-cells whose insulin secretory function is highly dependent on oxygen11-13
. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14
. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15
. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15
. The virus is stored in 10% glycerol at -80° C as many 150 μL aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15
. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1α for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.
Bioengineering, Issue 58, Cell encapsulation, PEG, cell aggregation, hypoxia, insulin secretion, fluorescent imaging
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
In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model
Institutions: University of Texas Southwestern Medical Center , University of Texas Southwestern Medical Center , Kyoto University Graduate School of Medicine.
It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo,
tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1α (HIF-1α) to drive transcription of various genes. Therefore, we have used a HIF-1α reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro
HIF-1α bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2
) for 24 hr before BLI, while the cells in normoxia (21% O2
) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1α binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo
bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo
imaging results. Here, we will introduce approaches of in vitro
HIF-1α bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo
bioluminescence imaging to monitor intracranial tumor hypoxia.
Medicine, Issue 56, bioluminescence imaging (BLI), tumor hypoxia dynamics, hypoxia inducible factor-1α (HIF-1α), breast cancer brain metastasis
Induction and Testing of Hypoxia in Cell Culture
Institutions: Baylor College of Medicine.
Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3
, rheumatoid arthritis, atherosclerosis etc… Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4
. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.
Cell Biology, Issue 54, mammalian cell, hypoxia, anoxia, hypoxia inducible factor (HIF), reoxygenation, normoxia
Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche
Institutions: Stanford University School of Medicine, Stanford University School of Medicine.
Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.
We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.
Medicine, Issue 97, Metastatic niche, bone microenvironment, breast cancer metastasis, human bone, osteotropism, ex vivo model, explant culture system, bioluminescence imaging