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T cells induce pre-metastatic osteolytic disease and help bone metastases establishment in a mouse model of metastatic breast cancer.
PUBLISHED: 01-01-2013
Bone metastases, present in 70% of patients with metastatic breast cancer, lead to skeletal disease, fractures and intense pain, which are all believed to be mediated by tumor cells. Engraftment of tumor cells is supposed to be preceded by changes in the target tissue to create a permissive microenvironment, the pre-metastatic niche, for the establishment of the metastatic foci. In bone metastatic niche, metastatic cells stimulate bone consumption resulting in the release of growth factors that feed the tumor, establishing a vicious cycle between the bone remodeling system and the tumor itself. Yet, how the pre-metastatic niches arise in the bone tissue remains unclear. Here we show that tumor-specific T cells induce osteolytic bone disease before bone colonization. T cells pro-metastatic activity correlate with a pro-osteoclastogenic cytokine profile, including RANKL, a master regulator of osteoclastogenesis. In vivo inhibition of RANKL from tumor-specific T cells completely blocks bone loss and metastasis. Our results unveil an unexpected role for RANKL-derived from T cells in setting the pre-metastatic niche and promoting tumor spread. We believe this information can bring new possibilities for the development of prognostic and therapeutic tools based on modulation of T cell activity for prevention and treatment of bone metastasis.
Authors: J. Preston Campbell, Alyssa R. Merkel, S. Kathryn Masood-Campbell, Florent Elefteriou, Julie A. Sterling.
Published: 09-04-2012
Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6. This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.
18 Related JoVE Articles!
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Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound
Authors: Tobias Bäuerle, Dorde Komljenovic, Martin R. Berger, Wolfhard Semmler.
Institutions: German Cancer Research Center, Heidelberg, Germany, German Cancer Research Center, Heidelberg, Germany.
Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques. For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US). MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7. In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.
Cancer Biology, Issue 66, Medicine, Physiology, Physics, bone metastases, animal model, angiogenesis, imaging, magnetic resonance imaging, MRI, volumetric computed tomography, ultrasound
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Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI
Authors: Heling Zhou, Dawen Zhao.
Institutions: University of Texas Southwestern Medical Center.
Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 µm in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.
Medicine, Issue 85, breast cancer brain metastasis, intracardiac injection, ultrasound imaging, MRI, MDA-MB231/Br-GFP cells
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A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells
Authors: Said Rahim, Aykut Üren.
Institutions: Georgetown University.
Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells. Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density. The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.
Cellular Biology, Issue 50, Invasion, HUVEC, xCELLigence, impedance, real-time, cell-index
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Experimental Metastasis Assay
Authors: Sonali Mohanty, Lei Xu.
Institutions: University of Rochester Medical Center, University of Rochester Medical Center.
Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.
medicine, Issue 42, cancer, metastasis, experimental, mouse, intravenous injection, lung
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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
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Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model
Authors: Mary Zimmerman, Xiaolin Hu, Kebin Liu.
Institutions: Medical College of Georgia.
Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.
Immunology, Issue 45, Metastasis, CTL adoptive transfer, Lung, Tumor Immunology
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Osteoclast Derivation from Mouse Bone Marrow
Authors: Ruth Tevlin, Adrian McArdle, Charles K.F. Chan, John Pluvinage, Graham G. Walmsley, Taylor Wearda, Owen Marecic, Michael S. Hu, Kevin J. Paik, Kshemendra Senarath-Yapa, David A. Atashroo, Elizabeth R. Zielins, Derrick C. Wan, Irving L. Weissman, Michael T. Longaker.
Institutions: Stanford University School of Medicine, Stanford University.
Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease. As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation. An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.
Cellular Biology, Issue 93, osteoclast, RANKL, culture, resorption assay, bone remodeling, bone turnover, skeletal homeostasis
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A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders
Authors: Dennis J. Wu, Neha Dixit, Erika Suzuki, Thanh Nguyen, Hyun Seock Shin, Jack Davis, Emanual Maverakis, Iannis E. Adamopoulos.
Institutions: University of California, Davis, Shriners Hospitals for Children - Northern California, University of California, Davis.
Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.
Medicine, Issue 88, osteoclast, arthritis, minicircle DNA, macrophages, cell culture, hydrodynamic delivery
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A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions
Authors: Valentina Goncharova, Sophia K. Khaldoyanidi.
Institutions: Torrey Pines Institute for Molecular Studies, Cascade LifeSciences Inc..
Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.
Bioengineering, Issue 77, Cellular Biology, Biophysics, Physiology, Molecular Biology, Biomedical Engineering, Immunology, Cells, Biological Factors, Equipment and Supplies, Cell Physiological Phenomena, Natural Science Disciplines, Life Sciences (General), circulating cells, extravasation, physiological shear stress, endothelial cells, microenvironment, chemokine gradient, flow, chamber, cell culture, assay
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Bioluminescent Orthotopic Model of Pancreatic Cancer Progression
Authors: Ming G. Chai, Corina Kim-Fuchs, Eliane Angst, Erica K. Sloan.
Institutions: Monash University, University of Bern, University of California Los Angeles .
Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.
Cancer Biology, Issue 76, Medicine, Molecular Biology, Cellular Biology, Genetics, Biomedical Engineering, Surgery, Neoplasms, Pancreatic Cancer, Cancer, Orthotopic Model, Bioluminescence, In Vivo Imaging, Matrigel, Metastasis, pancreas, tumor, cancer, cell culture, laparotomy, animal model, imaging
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An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth
Authors: Dalit Barkan, Jeffrey E. Green.
Institutions: University of Haifa, National Cancer Institute.
Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4. The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems. in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.
Medicine, Issue 54, Tumor dormancy, cancer recurrence, metastasis, reconstituted basement membrane extract (BME), 3D culture, breast cancer
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Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics
Authors: Adriana D. Corben, Mohammad M. Uddin, Brooke Crawford, Mohammad Farooq, Shanu Modi, John Gerecitano, Gabriela Chiosis, Mary L. Alpaugh.
Institutions: Memorial Sloan Kettering Cancer Center, Memorial Sloan Kettering Cancer Center, Weill Cornell Medical College, Memorial Sloan Kettering Cancer Center, Memorial Sloan Kettering Cancer Center, Memorial Sloan Kettering Cancer Center.
The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.
Cancer Biology, Issue 92, Ex vivo sectioning, Treatment response, Sensitivity/Resistance, Drug development, Patient tumors, Preclinical and Clinical
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A Preclinical Murine Model of Hepatic Metastases
Authors: Kevin C. Soares, Kelly Foley, Kelly Olino, Ashley Leubner, Skye C. Mayo, Ajay Jain, Elizabeth Jaffee, Richard D. Schulick, Kiyoshi Yoshimura, Barish Edil, Lei Zheng.
Institutions: The Johns Hopkins University School of Medicine, The Johns Hopkins University School of Medicine, University of Colorado Anschutz Medical Campus.
Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.
Medicine, Issue 91, Pancreatic Neoplasms, Immunotherapy, Hemispleen, Hepatic Metastases, Pancreatic Cancer, Liver, Preclinical Model, Metastatic, Murine
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A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies
Authors: Lee-Hwa Tai, Christiano Tanese de Souza, Shalini Sahi, Jiqing Zhang, Almohanad A Alkayyal, Abhirami Anu Ananth, Rebecca A.C. Auer.
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
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An Orthotopic Murine Model of Human Prostate Cancer Metastasis
Authors: Janet Pavese, Irene M. Ogden, Raymond C. Bergan.
Institutions: Northwestern University, Northwestern University, Northwestern University.
Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.
Medicine, Issue 79, Urogenital System, Male Urogenital Diseases, Surgical Procedures, Operative, Life Sciences (General), Prostate Cancer, Metastasis, Mouse Model, Drug Discovery, Molecular Biology
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Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications
Authors: Lori E. Lowes, Benjamin D. Hedley, Michael Keeney, Alison L. Allan.
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
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Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats
Authors: Gert Blueschke, Gabi Hanna, Andrew N. Fontanella, Gregory M. Palmer, Alina Boico, Hooney Min, Mark W. Dewhirst, David C. Irwin, Yulin Zhao, Thies Schroeder.
Institutions: Duke University Medical Center, Duke University Medical Center, University of Colorado Denver, University of Mainz.
Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.
Cancer Biology, Issue 93, Lung metastases, intravital microscopy, tumor blood flow, tumor vasculature, blood flow velocity, sarcoma metastasis, breast cancer metastasis
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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
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What is Visualize?

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

How does it work?

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

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