Wir danken Harold Varmus, Brian C. Lewis, Douglas Hanahan, Danny Huang, Sharon Pang, Megan Wong und Manasi M. Godbole. Y.C.N.D. stützt sich auf DOD Grant W81XWH-16-1-0619 und NIH Grant 1R01CA204916.

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R., Sambrook, J., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (2012).</li><li>Du, Y. C., Lewis, B. C., Hanahan, D., Varmus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+tumor+progression+factors+by+somatic+gene+transfer+into+a+mouse+model:+Bcl-xL+promotes+islet+tumor+cell+invasion.">Assessing tumor progression factors by somatic gene transfer into a mouse model: Bcl-xL promotes islet tumor cell invasion.</a> PLoS Biol. 5 (10), 2255-2269 (2007).</li><li>Du, Y. C., Chou, C. K., Klimstra, D. 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Die Autoren haben nichts preisgeben.

In dieser Studie haben wir eine mächtige Maus-Modell, RIP-Tag beschrieben; RIP-tva, , somatische Gentherapie Lieferung über Vogelgrippe Retroviren zur Identifizierung und Charakterisierung von metastasierendem Faktoren zu erreichen. Obwohl RIP-Tag; RIP-tva Mäuse entwickeln neuroendokrine Tumoren der Bauchspeicheldrüse, metastasierendem Faktoren identifiziert in diesem Mausmodell können auch Metastasen von anderen Krebsarten fördern.
Unser Ansatz hat den Vorteil der Einf...

Die meisten Krebsarten entstehen durch somatische Mutationen 1. Konventionelle gentechnisch veränderter Mausmodelle (GEMM) haben bedeutende Einblicke in den Beitrag von bestimmten genetischen Veränderungen, die auf Tumorgenese 2zur Verfügung gestellt. Sie haben jedoch mehrere Einschränkungen. Der größte Nachteil dieser Modelle ist, dass sie nicht die sporadische Natur der Tumorbildung im Menschen replizieren, in dem nur einige Zellen innerhalb eines Gewebes genetische Veränderungen erwerben. Die Mutationen in transgenen und Knockout-Mäuse sind auch Keimbahn mit auf die Entwicklung auswirken. Darüber hinaus ist erzeugen diese Maus-Modellen teuer und zeitaufwändig.
Metastasierung ist ein wichtiges Thema im Bereich der Krebs. Modellierung von Metastasen war schwierig in GEMM. Spontane Metastasierung ist selten in der Maus. Penetranz ist variabel und Latenz ist lang in GEMM Metastase 3. Experimentelle Metastasierung Modelle beschäftigen direkte Injektion von Zellen in den Blutkreislauf von Mäusen, so dass die ersten Schritte in der metastasierten Kaskade beseitigt werden.
Um einige der oben genannten Einschränkungen bei der Untersuchung von metastasierendem Faktoren in Mausmodellen zu überwinden, haben wir eine Bitransgenic-Maus-Modell, RIP-Tag entwickelt; RIP-tva4. Die Strategie basiert auf der Kombination der Verwendung von einem Mausmodell hoch synchronisiert Tumor Progression, RIP-Tag 5, und der Rezeptor für Vogelgrippe noch Untergruppe-A-Virus, tva 6,7. Dieses RIP-Tag; RIP-tvaMaus-Modell ermöglicht es Gene somatisch zu einem einzigen Bitransgenic Maus Stamm eingeführt werden. Mit dem SV40 T Antigen unterdrücken die Tumor unterdrückende Funktionen der Rb und p53 entwickeln Mäuse neuroendokrine Pankreastumoren in ähnlicher Weise zu menschlichen Tumorgenese mit Stufen einschließlich Hyperplasie, Adenom, Angiogenese und invasives Karzinom. Diese RIP-Tag -Modell wurde sehr lehrreich für unser Verständnis der Kennzeichen von Krebs, nicht beschränkt auf neuroendokrine Tumoren der Bauchspeicheldrüse. Es hat auch in präklinischen Studien 8eingesetzt.
Wir präsentieren ein Protokoll für somatische Gentransfer durch Injektion von Vogelgrippe Retroviren Intracardiacally in RIP-Tag; RIP-tva Mäuse. Erfolgreiche Infektion mit RCASBP-abgeleitete Geflügelpest Retroviren erfordert aktiv wuchernden Zielzellen. Daher wählten wir RIP-Tag; RIP-tva Mäuse 7 Wochen alt, wann Hyperplasie entwickelt sich bei etwa 50 % von den pankreatischen kleinen Inseln. Linke ventrikuläre intrakardiale Injektion von hohen Titer Viren ist erforderlich, um eine Infektion Wirkungsgrad von 10-20 % 4. Diese Lieferung Methode reduziert die signifikante Verdünnung der Viruspartikel im Umlauf bevor Viren pankreatische kleinen Inseln erreichen.
Mit diesem Ansatz haben wir bereits gezeigt, dass Bcl-xL Krebsmetastasen unabhängig von seinen Anti-Apoptotic Funktion 4,9 fördert. Diese Anti-apoptotische unabhängig metastasierendem Funktion wurde nicht beobachtet, wenn Bcl-xL über ein Transgen in allen pankreatische β-Zellen im gesamten tumorigenic Ontogenese in der RIP-Tag zum Ausdruck kam; RIP-Bcl-xL Maus Modell 10. Deshalb bietet unsere Maus-Modell eine einmalige Gelegenheit, zu identifizieren und zu charakterisieren, Gene Funktionen ausgedrückt in einem späteren Stadium der Tumorgenese. Denn 2-4 % der Inseln entwickeln sich zu Tumoren in der RIP-Tag; RIP-tva Bitransgenic Mäuse ohne Virusinfektion und nicht alle prämaligne Läsionen mit der RCASBP abgeleitete Geflügelpest Retroviren infiziert sind, nur die Faktoren, die einen selektiven Vorteil über den natürlichen Verlauf der Tumorgenese konferieren identifiziert werden können. Insbesondere werden metastasierendem Faktoren am leichtesten durch diese Methode erkannt werden, weil Metastasen in Pankreas Lymphknoten oder anderen Organen nicht normalerweise in RIP-Tag tritt; RIP-tva Mäuse.

<table><tbody><tr><td>RCASBP-Y DV plasmid</td><td>Addgene</td><td>11478</td><td></td></tr><tr><td>RCAS-RNAi plasmid</td><td>Addgene</td><td>15182</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>fetal bovine serum</td><td>Atlanta Biologicals</td><td>25-005-CI</td><td></td></tr><tr><td>L-glutamine, 100x</td><td>Corning</td><td>25-005-CI</td><td></td></tr><tr><td>Penicillin-Streptomycin solution, 100x</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>PBS&lt;sup&gt;-/-&lt;/sup&gt;, 1x</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Superfect</td><td>Qiagen</td><td>301305</td><td></td></tr><tr><td>Polyallomer centrifuge tube</td><td>Beckman Coulter</td><td>326823</td><td></td></tr><tr><td>0.45 mm Nalgene&lt;br/&gt; Syringe Filters with PES Membrane</td><td>Thermo Scientific</td><td>194-2545</td><td></td></tr><tr><td>Insulin Syringes</td><td>BD</td><td>329461</td><td></td></tr><tr><td>synaptophysin</td><td>Vector Laboratories</td><td>VP-S284</td><td></td></tr><tr><td>VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG)</td><td>Vector Laboratories</td><td>PK-6101</td><td></td></tr><tr><td>AmpliTaq DNA Polymerase with Buffer II</td><td>Life Technologies</td><td>N8080153</td><td></td></tr><tr><td>MyTaq DNA Polymerase</td><td>Bioline</td><td>BIO-21106</td><td></td></tr></tbody></table>

identification and characterization of metastatic factors by gene transfer into the novel rip-tag; rip-tva murine model

Metastasiertem Krebs entfallen 90 % der Todesfälle bei Patienten mit soliden Tumoren. Es ist dringend notwendig, die Treiber von Krebsmetastasen besser zu verstehen und neue therapeutische Targets zu identifizieren. Um molekularen Ereignisse zu untersuchen, die das Fortschreiten von primären Krebs, Metastasen zu fahren, haben wir eine Bitransgenic-Maus-Modell, RIP-Tag entwickelt; RIP-tva. In diesem Mausmodell treibt die Ratte Insulin Promotor (RIP) Ausdruck der SV40 T-Antigen (Tag) und der Rezeptor für Untergruppe ein Virus der Vogelgrippe noch (tva) in pankreatische β-Zellen. Die Mäuse entwickeln neuroendokrine Tumoren der Bauchspeicheldrüse mit 100 % Penetranz durch klar definierte Stadien, die ähnlich wie menschliche Tumorgenese mit Stufen einschließlich Hyperplasie, Adenom, Angiogenese und invasives Karzinom. Weil RIP-Tag; RIP-tva Mäuse entwickeln keine Metastasen, genetische Veränderung, die Metastasierung fördern leicht identifizierbar. Somatische Gentransfer in tva exprimierenden, wuchernden pankreatische β prämaligne Läsionen durch intrakardiale Injektion von Vogelgrippe Retroviren beherbergen die gewünschte genetische Veränderung erreicht wird. Ein Titer von &#62; 1 x 108 infektiösen Einheiten pro ml als angemessen für in-Vivo -Infektion angesehen. Darüber hinaus können Vogelgrippe Retroviren infizieren Zelllinien aus Tumoren in RIP-Tag; RIP-tva Mäuse mit hohem Wirkungsgrad. Die Zell-Linien können auch verwendet werden, metastasierendem Faktoren charakterisieren. Hier zeigen wir wie Sie nutzen dieses Mausmodell und Zell-Linien, um die Funktionen von Kandidatengenen im Tumor Metastasen zu beurteilen.

Die in Vivo und in Vitro Infektionsrate von RIP-Tag; RIP-tva Tumorzellen durch RCASBP-basierte Viren sind ~ 20 % ~ 80 % bzw. 20. Im RIP-Tag; RIP-tva Mausmodell, etwa 4 % der 400 Pankreas Inseln in jedem Mausklick entwickeln sich natürlich in Tumoren 20; Daher ist die ausreichende Menge an Tumorzellen in jeder Maus für histologische und phänotypische Analyse der möglichen Wirkung der Gene, die durch die...

Watch this Scientific Journal Video about Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model at JoVE.com

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

Wir präsentieren ein Protokoll, um eine neuartige somatische Gentherapie-Transfer-System unter Verwendung RIP-Tag zu demonstrieren; RIP-tva Maus-Modell für die Funktion von Genen in Metastasen zu untersuchen. Die Vogelgrippe Retroviren liefern wir Intracardiacally um Gentransfer in Pre-maligne, nicht-invasive Läsionen des Pankreas β-Zellen bei Erwachsenen Mäusen zu gewährleisten.

Identifizierung und Charakterisierung von metastasierendem Faktoren durch Gentransfer in die neuartige RIP-Tag; RIP-tva Mausmodell

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis ...

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Cancer Research

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

8.7K Aufrufe.  Weill Cornell Medicine. Wir präsentieren ein Protokoll, um eine neuartige somatische Gentherapie-Transfer-System unter Verwendung RIP-Tag zu demonstrieren; RIP-tva Maus-Modell für die Funktion von Genen in Metastasen zu untersuchen. Die Vogelgrippe Retroviren liefern wir Intracardiacally um Gentransfer in Pre-maligne, nicht-invasive Läsionen des Pankreas β-Zellen bei Erwachsenen Mäusen zu gewährleisten.

Serie: Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Radionuklid-Fluoreszenz-Reporter-Gen Imaging zu Tumorprogression in Nagetier Tumormodellen verfolgen

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Forschung

Krebsforschung

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

Murine Modell der metastasierenden Lebertumoren in der Einstellung der Ischämie Reperfusionsverletzung

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Kombinierte Verwendung von Tail Vein Metastasis Assays und Real-Time In Vivo Imaging zur Quantifizierung der metastasierenden Kolonisation und Belastung von Brustkrebs in der Lunge

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

Entdeckung metastasierender Regulatoren mit einem schnellen und quantitativen intravitalen Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

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.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Ein orthotopen Mausmodell der menschlichen Prostatakrebs-Metastasen

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  ...

Medizin

A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was established using robust, reproducible in vivo models and standardized methods to identify novel players in melanoma metastasis. This approach allows for data inference at specific experimental stages to precisely characterize a gene's role in metastasis. Models are established by introducing genetically modified melanoma cells via intracardiac, intradermal, or subcutaneous injections into mice, followed by monitoring with serial in vivo imaging. Once preestablished endpoints are reached, primary tumors and/or metastases-bearing organs are harvested and processed for various analyses. Tumor cells can be sorted and subjected to any of several 'omics' platforms, including single-cell RNA sequencing. Organs undergo imaging and immunohistopathological analyses to quantify the overall burden of metastases and map their specific anatomic location. This optimized pipeline, including standardized protocols for engraftment, monitoring, tissue harvesting, processing, and analysis, can be adopted for patient-derived, short-term cultures and established human and murine cell lines of various solid cancer types.

This article describes a workflow of techniques employed for testing novel candidate mediators of melanoma metastasis and their mechanism(s) of action.

Eine robuste Entdeckungsplattform zur Identifizierung neuartiger Mediatoren der Melanommetastasierung

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

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 &#945;&#946; and &#947;&#948; 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.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Initiation Metastasierende Mammakarzinom durch Targeting des duktalen Epithel mit Adenovirus-Cre: Eine neue transgene Maus-Modell der Brustkrebs

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Beurteilung von Lungenmetastasen in Maus-Mammatumormodelle durch quantitative Echtzeit-PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

Ex-vivo-Live-Imaging von Lungenmetastasen und deren Mikromilieu

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

Untersuchung der Rolle von Alveolarmakrophagen in Metastasierung von Brustkrebs

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...