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Exploring the gain of function contribution of AKT to mammary tumorigenesis in mouse models.
PLoS ONE
PUBLISHED: 01-27-2010
Elevated expression of AKT has been noted in a significant percentage of primary human breast cancers, mainly as a consequence of the PTEN/PI3K pathway deregulation. To investigate the mechanistic basis of the AKT gain of function-dependent mechanisms of breast tumorigenesis, we explored the phenotype induced by activated AKT transgenes in a quantitative manner. We generated several transgenic mice lines expressing different levels of constitutively active AKT in the mammary gland. We thoroughly analyzed the preneoplastic and neoplastic mammary lesions of these mice and correlated the process of tumorigenesis to AKT levels. Finally, we analyzed the impact that a possible senescent checkpoint might have in the tumor promotion inhibition observed, crossing these lines to mammary specific p53(R172H) mutant expression, and to p27 knock-out mice. We analyzed the benign, premalignant and malignant lesions extensively by pathology and at molecular level analysing the expression of proteins involved in the PI3K/AKT pathway and in cellular senescence. Our findings revealed an increased preneoplastic phenotype depending upon AKT signaling which was not altered by p27 or p53 loss. However, p53 inactivation by R172H point mutation combined with myrAKT transgenic expression significantly increased the percentage and size of mammary carcinoma observed, but was not sufficient to promote full penetrance of the tumorigenic phenotype. Molecular analysis suggest that tumors from double myrAKT;p53(R172H) mice result from acceleration of initiated p53(R172H) tumors and not from bypass of AKT-induced oncogenic senescence. Our work suggests that tumors are not the consequence of the bypass of senescence in MIN. We also show that AKT-induced oncogenic senescence is dependent of pRb but not of p53. Finally, our work also suggests that the cooperation observed between mutant p53 and activated AKT is due to AKT-induced acceleration of mutant p53-induced tumors. Finally, our work shows that levels of activated AKT are not essential in the induction of benign or premalignant tumors, or in the cooperation of AKT with other tumorigenic signal such as mutant p53, once AKT pathway is activated, the relative level of activity seems not to determine the phenotype.
Authors: Melanie R. Rutkowski, Michael J. Allegrezza, Nikolaos Svoronos, Amelia J. Tesone, Tom L. Stephen, Alfredo Perales-Puchalt, Jenny Nguyen, Paul J. Zhang, Steven N. Fiering, Julia Tchou, Jose R. Conejo-Garcia.
Published: 03-26-2014
ABSTRACT
Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of αβ and γδ T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.
21 Related JoVE Articles!
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Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells
Authors: Anupama Pal, Celina G. Kleer.
Institutions: University of Michigan Comprehensive Cancer Center, University of Michigan Comprehensive Cancer Center.
Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.
Medicine, Issue 86, pathological conditions, signs and symptoms, neoplasms, three dimensional cultures, Matrigel, breast cells, malignant phenotype, signaling
51311
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Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer
Authors: Rebecca E. Nakles, Sarah L. Millman, M. Carla Cabrera, Peter Johnson, Susette Mueller, Philipp S. Hoppe, Timm Schroeder, Priscilla A. Furth.
Institutions: Georgetown University, Georgetown University, Helmholtz Zentrum München - German Research Center for Environmental Health, Georgetown University, Dankook University.
Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 μm x 700 μm fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.
Cancer Biology, Issue 72, Medicine, Cellular Biology, Molecular Biology, Anatomy, Physiology, Oncology, Mammary Glands, Animal, Epithelial Cells, Mice, Genetically Modified, Primary Cell Culture, Time-Lapse Imaging, Early Detection of Cancer, Models, Genetic, primary cell culture, preneoplastic mammary epithelial cells, genetically engineered mice, time-lapse imaging, BRCA1, animal model
50198
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Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)
Authors: Yoray Sharon, Lina Alon, Sarah Glanz, Charlotte Servais, Neta Erez.
Institutions: Tel Aviv University.
Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker α-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFRα as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFRα is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to α-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.
Cancer Biology, Issue 71, Cellular Biology, Molecular Biology, Medicine, Oncology, Pathology, Bioengineering, Biomedical Engineering, Cancer-Associated Fibroblasts, fibroblast, FACS sorting, PDGFRalpha, Breast cancer, Skin carcinoma, stroma, tumor, cancer, tissue, cell, culture, human, mouse, animal model
4425
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In vivo Dual Substrate Bioluminescent Imaging
Authors: Michael K. Wendt, Joseph Molter, Christopher A. Flask, William P. Schiemann.
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
3245
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Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats
Authors: Sonia de Assis, Anni Warri, M. Idalia Cruz, Leena Hilakivi-Clarke.
Institutions: Georgetown University, University of Turku Medical Faculty.
Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.
Medicine, Issue 44, mammary gland morphology, terminal end buds, mammary cancer, maternal dietary exposures, pregnancy, prepubertal dietay exposures
2260
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Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice
Authors: Nikki Cheng, Diana L. Lambert.
Institutions: University of Kansas Medical Center.
The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.
Medicine, Issue 54, transplantation, mammary, fibroblast, PyVmT carcinoma, collagen type-I , tumor
2716
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Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy
Authors: Caroline Bonnans, Marja Lohela, Zena Werb.
Institutions: INSERM U661, Functional Genomic Institute, University of California.
Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.
Cancer Biology, Issue 92, intravital imaging, spinning disk confocal, ApcMin/+ mice, colorectal cancer, tumor, myeloid cells
51916
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Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands
Authors: Rushin Shojaii, Stephanie Bacopulos, Wenyi Yang, Tigran Karavardanyan, Demetri Spyropoulos, Afshin Raouf, Anne Martel, Arun Seth.
Institutions: University of Toronto, Sunnybrook Research Institute, University of Toronto, Sunnybrook Research Institute, Medical University of South Carolina, University of Manitoba.
Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution. This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.
Bioengineering, Issue 89, Histology Volume Reconstruction, Transgenic Mouse Model, Image Registration, Digital Histology, Image Processing, Mouse Mammary Gland
51325
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Evaluation of Mammary Gland Development and Function in Mouse Models
Authors: Isabelle Plante, Michael K.G. Stewart, Dale W. Laird.
Institutions: University of Western Ontario.
The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.
Developmental Biology, Issue 53, mammary gland, whole mount, mouse model, mammary gland development, milk ejection
2828
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A Sensitive Method to Quantify Senescent Cancer Cells
Authors: Julie Cahu, Brigitte Sola.
Institutions: Université de Caen Basse-Normandie.
Human cells do not indefinitely proliferate. Upon external and/or intrinsic cues, cells might die or enter a stable cell cycle arrest called senescence. Several cellular mechanisms, such as telomere shortening and abnormal expression of mitogenic oncogenes, have been shown to cause senescence. Senescence is not restricted to normal cells; cancer cells have also been reported to senesce. Chemotherapeutical drugs have been shown to induce senescence in cancer cells. However, it remains controversial whether senescence prevents or promotes tumorigenesis. As it might eventually be patient-specific, a rapid and sensitive method to assess senescence in cancer cell will soon be required. To this end, the standard β-galactosidase assay, the currently used method, presents major drawbacks: it is time consuming and not sensitive. We propose here a flow cytometry-based assay to study senescence on live cells. This assay offers the advantage of being rapid, sensitive, and can be coupled to the immunolabeling of various cellular markers.
Cancer Biology, Issue 78, Medicine, Cellular Biology, Anatomy, Physiology, Genetics, Oncology, Tumor Cells, Cultured, Early Detection of Cancer, senescence, cancer, cells, flow cytometry, C12FDG, cell culture, clinical applications
50494
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Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy
Authors: Esra Güç, Manuel Fankhauser, Amanda W. Lund, Melody A. Swartz, Witold W. Kilarski.
Institutions: École Polytechnique Fédérale de Lausanne, Oregon Health & Science University.
Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.
Bioengineering, Issue 86, Intravital imaging, epifluorescence, two-photon imaging, Tumor matrix, Matrix remodeling
51388
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Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Authors: Robert S. McNeill, Ralf S. Schmid, Ryan E. Bash, Mark Vitucci, Kristen K. White, Andrea M. Werneke, Brian H. Constance, Byron Huff, C. Ryan Miller.
Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.
Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
51763
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In Vivo Modeling of the Morbid Human Genome using Danio rerio
Authors: Adrienne R. Niederriter, Erica E. Davis, Christelle Golzio, Edwin C. Oh, I-Chun Tsai, Nicholas Katsanis.
Institutions: Duke University Medical Center, Duke University, Duke University Medical Center.
Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.
Molecular Biology, Issue 78, Genetics, Biomedical Engineering, Medicine, Developmental Biology, Biochemistry, Anatomy, Physiology, Bioengineering, Genomics, Medical, zebrafish, in vivo, morpholino, human disease modeling, transcription, PCR, mRNA, DNA, Danio rerio, animal model
50338
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Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?
Authors: Julia Y. Ljubimova, Hui Ding, Jose Portilla-Arias, Rameshwar Patil, Pallavi R. Gangalum, Alexandra Chesnokova, Satoshi Inoue, Arthur Rekechenetskiy, Tala Nassoura, Keith L. Black, Eggehard Holler.
Institutions: Cedars-Sinai Medical Center.
Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.
Chemistry, Issue 88, Cancer treatment, personalized medicine, polymalic acid, nanodrug, biopolymer, targeting, host compatibility, biodegradability
50668
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Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells
Authors: Ziming Cheng, Ting Zhou, Azhar Merchant, Thomas J. Prihoda, Brian L. Wickes, Guogang Xu, Christi A. Walter, Vivienne I. Rebel.
Institutions: UT Health Science Center at San Antonio, UT Health Science Center at San Antonio, UT Health Science Center at San Antonio, UT Health Science Center at San Antonio, UT Health Science Center at San Antonio.
In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases. LacI transgenic mice carry a recoverable λ phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of β-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation. The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.
Infection, Issue 84, In vivo mutagenesis, hematopoietic stem/progenitor cells, LacI mouse model, DNA mutations, E. coli
50752
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Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer
Authors: Elizabeth S. Nakasone, Hanne A. Askautrud, Mikala Egeblad.
Institutions: Watson School of Biological Sciences, Cold Spring Harbor Laboratory, University of Oslo and Oslo University Hospital.
The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.
Cancer Biology, Issue 73, Medicine, Molecular Biology, Cellular Biology, Biomedical Engineering, Genetics, Oncology, Pharmacology, Surgery, Tumor Microenvironment, Intravital imaging, chemotherapy, Breast cancer, time-lapse, mouse models, cancer cell death, stromal cell migration, cancer, imaging, transgenic, animal model
50088
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Analysis of Cell Migration within a Three-dimensional Collagen Matrix
Authors: Nadine Rommerswinkel, Bernd Niggemann, Silvia Keil, Kurt S. Zänker, Thomas Dittmar.
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
51963
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A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster
Authors: Ajay Srivastava.
Institutions: Western Kentucky University .
Drosophila has illuminated our understanding of the genetic basis of normal development and disease for the past several decades and today it continues to contribute immensely to our understanding of complex diseases 1-7. Progression of tumors from a benign to a metastatic state is a complex event 8 and has been modeled in Drosophila to help us better understand the genetic basis of this disease 9. Here I present a simple protocol to genetically induce, observe and then analyze the progression of tumors in Drosophila larvae. The tumor induction technique is based on the MARCM system 10 and exploits the cooperation between an activated oncogene, RasV12 and loss of cell polarity genes (scribbled, discs large and lethal giant larvae) to generate invasive tumors 9. I demonstrate how these tumors can be visualized in the intact larvae and then how these can be dissected out for further analysis. The simplified protocol presented here should make it possible for this technique to be utilized by investigators interested in understanding the role of a gene in tumor invasion.
Medicine, Issue 79, Imaginal Discs, Drosophila melanogaster, Neoplasm Metastasis, Drosophila, Invasive Tumors, Benign Tumors, Cephalic Complex, Mosaic Analysis with a Repressible Cell Marker technique
50624
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Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging
Authors: Ed Lim, Kshitij Modi, Anna Christensen, Jeff Meganck, Stephen Oldfield, Ning Zhang.
Institutions: Caliper Life Sciences.
Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location. Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.
Medicine, Issue 50, osteolytic lesions, micro CT, tumor, bioluminescence, in vivo, imaging, IVIS, luciferase, low dose, co-registration, 3D reconstruction
2775
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In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum
Authors: Ed Lim, Kshitij D Modi, JaeBeom Kim.
Institutions: Caliper Life Sciences.
4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds. The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result. Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures. Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents. Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.
Cellular Biology, Issue 26, tumor, mammary, mouse, bioluminescence, in vivo, imaging, IVIS, luciferase, luciferin
1210
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Mammary Epithelial Transplant Procedure
Authors: Karen A. Dunphy, Luwei Tao, D. Joseph Jerry.
Institutions: University of Massachussetts, Pioneer Valley Life Sciences institute, University of Massachussetts.
This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8. Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.
Cellular Biology, Issue 40, transplantation, mammary, epithelium, cleared fat pad
1849
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