Diese Forschung wird von der AACR Henry Shepard Blasenkrebs Research Grant (09-60-30-LI) gefördert.

Verfahren mit tierischen Probanden wurden von der Institutional Animal Care und Use Committee (IACUC) an der University of California, San Francisco genehmigt worden. 1. Zellpräparation <ol><li> Das menschliche Blasenkarzinom-Zelllinie KU-7-luc2-GFP (Caliper Life Sciences, Hopkinton, MA) in RPMI-1640-Medium mit 10% fötalem Rinderserum und 50 ug / ml Gentamicin ergänzt war. </li><li> Die Kulturen werden bei 37 ° C, 5% CO 2 mit Medien wechselt alle zwei bis drei Tage. </li><li> Die Zellen wurden durch Trypsin-Verdau geerntet und unter Verwendung eines Hämozytometers. Eine einzelne Zellsuspension von 2 × 10 6 Zellen in 50 l wurde in Phosphat-gepufferter Salzlösung hergestellt und auf Eis gehalten. </li></ol> 2. Orthotopen Blasentumor Modell <ol><li> Weiblicher Akt (nu / nu) Mäuse 8-10 Wochen alt (Simonsen Laboratory, Gilory, CA) verwendet wurden. </li><li> Setzen Sie ein steriles Tuch über einem Heizkissen dienen, einsa chirurgischen Plattform. Sterile Gleitgel, 24-che Katheter mit Stilett Nadeln, Silbernitrat-Lösung, und steriles Wasser hergestellt. </li><li> Vollnarkose ist bei den Mäusen mit inhalativen Isofluran (3% für Induktion, 1-2% für die Wartung) und-wartung über Nasenkonus induziert. Sterile ophthalmische Salbe wird auf die Augen des Tieres angelegt wird, und ein Heizkissen verwendet wird, um die Körperwärme zu halten. </li><li> Platzieren Sie die Maus in Rückenlage. </li><li> Fassen Sie das Tier Vulva mit einer Pinzette zur Stabilisierung. </li><li> Mit einem 24-che intravenösen Katheter mit der Nadel Stilett entfernt wird, wird die Maus Harnröhre katheterisiert. Eine ausreichende Schmierung verwendet werden sollte. Mit der Maus auf dem Rücken, wird die Harnröhre liegt unmittelbar hinter der Vulva Falten und anterior der Vagina. Eine 45-Grad-Winkel wird vorgelegt, um die Harnröhre kanülieren und unterhalb des Schambeins besteht. Eine flachere Neigung wird dann genommen, um durch die Harnröhre in die Blase besteht. Sanfte Becken-presSie glätten die Harnröhre kann oft helfen. Es ist darauf zu vermeiden, zu hart gegen einen Widerstand, da dies zu urethralen oder Perforation Blase führen kann. Urin im Katheter gesehen bestätigt seine Position innerhalb der Blase Lumen. </li><li> Saugen Urin von den Katheter und Blasenlumens mit der Sondennadel. Die Sondennadel wird für alle nachfolgenden Streben und Injektionen verwendet wird, wobei der Katheter an Ort und Stelle. Dies ermöglicht genaue Volumina Injektion von durch den Wegfall der Totraum innerhalb des Katheters, und es wird wiederholt Katheterisierungen unnötig. </li><li> Um die Glykosaminoglykan Schicht der Blase Urothel beeinträchtigen, wird 10 ul 0,5 M Silbernitrat injiziert und man ließ sie für 10 Sekunden zu verweilen. Dies kann zur Bildung eines weißen Niederschlags mit den restlichen Urin. </li><li> Absaugen und entsorgen Sie die Inhalte Niederschlag und der Blase mit einem Sondennadel. </li><li> Waschen Sie die Blase durch Injektion von 100 ul sterilem Wasser. Absaugen und verwerfen das Wasser. Repebei Waschen mit sterilem Wasser 3-mal für insgesamt 4 Wäschen. </li><li> Legen Sie eine 4-0 Seidenkrawatte rund um die Harnröhre, um die Harnröhre in Vorbereitung auf Entfernung des Katheters zu verschließen. </li><li> Zuvor hergestellten Spritzen 2 x 10 6 KU-7-luc2-GFP-Zellen in 50 ul mit der Sondennadel. </li><li> Gleichzeitig nehmen Sie den Katheter und Sondennadel, so dass die Harnröhre durch die Seidenkrawatte verschlossen. </li><li> Die Maus wird unter Vollnarkose für 2 Stunden gehalten werden, bevor die Seidenkrawatte wird entfernt und das Tier ist erwacht. </li></ol> 3. Ultraschall <ol><li> Vollnarkose mit Isofluran verdampfte und-wartung über Nasenkonus induziert. </li><li> Ein VisualSonics Vevo 770 In Vivo High-Resolution Micro-Imaging System (VisualSonics Inc, Toronto, Ontario, Kanada) mit dem RMV-704 40-MHz-Sonde verwendet wurde. </li><li> Platzieren Sie die Maus in Rückenlage auf dem Ultraschall-Plattform. </li><li> Sichern des Tieres Hinterbeinemit Klebeband übermäßige Bewegung während der Manipulation der Ultraschall-Sonde zu vermeiden. </li><li> Bewerben Ultraschall-Gel auf die Maus Becken. </li><li> Senken Sie eine RMV-704 (40 MHz) Ultraschallsonde auf die Maus, um das Becken quer oder längs Bilder zu erhalten. </li><li> Wenn Tumor gefunden wird, können Bilder für die Messung des Tumors Dimensionen gespeichert werden. </li></ol> 4. Bioluminescent Imaging <ol><li> Führen intraperitoneale Injektion von 200 ul (150 mg / kg) Luciferinsubstrat. </li><li> Induzieren Vollnarkose mit Isofluran und pflegen verdampften über Bugspitze. </li><li> Platzieren Sie Mäuse in Rückenlage in der Biolumineszenz-Imager. </li><li> Fünf Minuten nach der Injektion Luciferin, messen Sie die Biolumineszenz von dem Tier in Photonen pro Sekunde. </li></ol> 5. SÄRNA Vorbereitung <ol><li> Synthetisieren RNA-Oligonukleotiden auf einer Skala von 50 bis 100 mmol. </li><li> Glühen komplementäre einzelsträngige Oligonukleotide iNTO-Duplex saRNAs. </li><li> [1,3]-dioxolan (DLinKC2-DMA), disteroylphosphatidyl Cholin, Cholesterin und PEG-DMG - Lipid-Nanopartikel (LNP)-SÄRNA mit dem ionisierbaren Lipids 1,2-Dilinoleyl-4-(- Dimethylaminoethyl 2) hergestellt Verwendung eines spontanen Vesikelbildung Formulierung Verfahren wie zuvor beschrieben. 1 </li></ol> 6. Intravesikale Verabreichung formuliertes SÄRNA <ol><li> Nach Gründung der orthotopen Blasentumor Modell werden Mäuse mit intravesikale kleine aktivierende RNA (SÄRNA) in Lipid-Nanopartikel (LNPs) (Tabelle 1) formuliert behandelt. </li><li> Vollnarkose wird aufgebaut und mit verdampfte Isofluran aufrechterhalten, und sterile ophthalmische Schmiermittel aufgetragen wird. </li><li> Platzieren Sie das Tier in Rückenlage. </li><li> Führen Katheterisierung der Harnröhre, wie zuvor beschrieben. </li><li> Legen Sie eine 4-0 Seidenkrawatte um die Harnröhre und Katheter. </li><li> Saugen sie die gesamte Urin aus dem Katheter undBlase mit einer leeren Spritze und Sondennadel. </li><li> Spritzen-LNP SÄRNA intravesikal für eine Gesamtdosis von 50 ml (3 mg / kg). Gleichzeitig nehmen Sie den Katheter und Sondennadel, so dass die Harnröhre durch die Seidenkrawatte verschlossen. </li><li> Die Maus bleibt anästhesiert mit der Harnröhre für 2 Stunden verschlossen. </li><li> Die Behandlung mit intravesikale LNP-SÄRNA wurde am Tag 4 nach der Tumorimplantation begonnen. Die Mäuse wurden seriell behandelt alle 3 Tage, für insgesamt 14 Behandlungen. </li></ol> 7. Repräsentative Ergebnisse Blasen, die aus Tumoren KU-7-luc2-GFP-Zellen kann mittels Luciferase-Biolumineszenz-Bildgebung (1A und C) und der Blase Ultraschall (1B und D) überwacht werden. Tumoren sind oft innerhalb von 3-5 Tagen nachweisbar nach Zellinokulation. Wir fanden erfolgreiche Blasentumor Implantation in über 90% der Mäuse. Da die Behandlung mit intravesikale dsRNA erfolgt, kann das Fortschreiten des Tumors wi überwacht werdenth dieser Modalitäten. Nachdem das Tier geopfert wird, kann das Tumorwachstum bestätigte auch mit GFP-Fluoreszenz werden. In der Regel bedeuten, Biolumineszenz mit mittleren Abmessungen für Ultraschall Blasentumoren korreliert, wie visuell in den Abbildungen 1C und 1D gesehen. Jedoch, nachdem die Tiere getötet und Tumor wurde durch GFP bestätigt, fanden wir, dass Biolumineszenz enger mit der Anwesenheit von lebensfähigen Tumor als hat Ultraschallmessungen korreliert. Dies war besonders deutlich bei den Mäusen mit Sarna (als Gegensatz zu den Kontrollen) behandelt, da die verbleibende Narbengewebe im behandelten Mäuse war oft durch Ultraschall sichtbar gemacht, konnte aber nicht von Live-Tumor zu unterscheiden. <img src="/files/ftp_upload/4207/4207fig1.jpg" alt="1" /> Abbildung 1. Das Tumorwachstum Kinetik von Blasentumoren. (A) Biolumineszenz-Bildgebung misst die Luciferaseaktivität innerhalb Blasentumoren zu deren Standort und Wachstumsbedingungen zu bestätigen. (B) In-vivo-Blase ultrasound mit einem 40MHz-Sonde erzeugt Tumor Bilder, deren Abmessungen genau gemessen werden kann. (C) graphische Darstellungen des Tumors progressiver Anstieg der Biolumineszenz-und Ultraschall-Dimensionen. Die Werte stellen Mittelwert (+ / - Standardabweichung) Messungen von 6 Tieren. <a href="http://www.jove.com/files/ftp_upload/4207/4207fig1large.jpg" target="_blank">Klicken Sie hier für eine größere Abbildung anzuzeigen</a> .

<ol>
	<li>Semple, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+cationic+lipids+for+siRNA+delivery.">Rational design of cationic lipids for siRNA delivery.</a> Nature. 28, 172-176 (2010).</li><li>Li, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+dsRNAs+induce+transcriptional+activation+in+human+cells.">Small dsRNAs induce transcriptional activation in human cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 17337-17342 (2006).</li><li>Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+effect+of+dsRNA-induced+p21(WAF1/CIP1)+gene+activation+in+human+bladder+cancer+cells.">Antitumor effect of dsRNA-induced p21(WAF1/CIP1) gene activation in human bladder cancer cells.</a> Molecular cancer therapeutics. 7, 698-703 (2008).</li><li>Chancellor, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+results+of+myoblast+injection+into+the+urethra+and+bladder+wall:+a+possible+method+for+the+treatment+of+stress+urinary+incontinence+and+impaired+detrusor+contractility.">Preliminary results of myoblast injection into the urethra and bladder wall: a possible method for the treatment of stress urinary incontinence and impaired detrusor contractility.</a> Neurourology and urodynamics. 19, 279-287 (2000).</li><li>Hadaschik, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+validated+mouse+model+for+orthotopic+bladder+cancer+using+transurethral+tumour+inoculation+and+bioluminescence+imaging.">A validated mouse model for orthotopic bladder cancer using transurethral tumour inoculation and bioluminescence imaging.</a> BJU international. 100, 1377-1384 (2007).</li><li>Fu, C., Apelo, C. A., Torres, B., Thai, K. H., Hsieh, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Bladder+Wall+Injection.">Mouse Bladder Wall Injection.</a> J. Vis. Exp. (53), e2523 (2011).</li><li>Dobek, G. L., Godbey, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Orthotopic+Model+of+Murine+Bladder.">An Orthotopic Model of Murine Bladder.</a> Cancer. J. Vis. Exp. (48), 2535 (2011).</li><li>Dinney, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+metastatic+variants+from+human+transitional+cell+carcinoma+passaged+by+orthotopic+implantation+in+athymic+nude+mice.">Isolation and characterization of metastatic variants from human transitional cell carcinoma passaged by orthotopic implantation in athymic nude mice.</a> The Journal of urology. 154, 1532-1538 (1995).</li></ol>

LCL ist ein benannter Erfinder auf anhängige Patentanmeldungen in Bezug auf SÄRNA, die von der University of California San Francisco eingereicht worden sind und in Lizenz an Alnylam Pharmaceuticals.

Wir präsentieren ein Protokoll für die Errichtung eines Maus-Xenograft orthotopen Tumormodell Blase durch intravesikale Behandlung der Tumoren mit LNP SÄRNA formuliert, die den Promotor von p21 CIP1/WAF1 (p21) Zielen für die Aktivierung der Transkription gefolgt. 2, 3 Die entstehenden Tumoren können bestätigt und mit Biolumineszenz-Bildgebung und Blase Ultraschall überwacht. Orthotopische Modellen überlegen sein kann intraperitoneale, subkutane oder intravenöse Modellen durch ein Milieu vo...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> KU-7-Luc-GFP-Zellen </td><td> Caliper </td><td> 128091 </td><td></td></tr><tr><td> Thymus Nacktmäusen (nu / nu) </td><td> Simonsen Laboratories </td><td> 6-7 Wochen alt </td><td> Sim: (NCR) nu / nu fisol </td></tr><tr><td> Ultraschall-Gerät </td><td> VisualSonics </td><td> Vevo 770 </td><td> RMV-704-Sonde </td></tr><tr><td> Biolumineszenz-Einheit </td><td> Caliper </td><td> IVIS Spectrum </td><td></td></tr><tr><td> Exel safelet Katheter </td><td> Fischer </td><td> 14-841-21 </td><td> 24G x ¾ &quot; </td></tr><tr><td> Silbernitrat </td><td> Sigma </td><td> S8157 </td><td></td></tr><tr><td> Hamilton-Spritze </td><td> Hamilton CoHat Ihr Unternehmen </td><td> 80301 </td><td></td></tr><tr><td> LNP-SÄRNA </td><td> Alnylam Pharmaceuticals </td><td></td><td></td></tr></tbody></table> Tabelle 1. Spezifischen Reagenzien und Geräte.

an orthotopic bladder tumor model and the evaluation of intravesical sarna treatment

Wir stellen eine neue Methode zur Behandlung von Blasenkrebs mit intravesikal geliefert kleine aktivierende RNA (SÄRNA) in einem orthotopen Xenograft Maus Blasentumor Modell. Das Maus-Modell wird durch Katheterisierung der Harnröhre unter inhalativen Vollnarkose eingerichtet. Verätzung wird dann auf der Blasenschleimhaut mit intravesikale Silbernitratlösung, um die Blase eingeführt Glycosaminoglycan Schicht zu stören und erlaubt den Zellen zu befestigen. Nach mehrmaligem Waschen mit sterilem Wasser, die menschliche Blasenkarzinom KU-7-luc2-GFP-Zellen durch den Katheter in die Blase instilliert für 2 Stunden zu verweilen. Anschließende Wachstum von Blasentumoren wird bestätigt und überwacht durch In-vivo-Blase Ultraschall und Biolumineszenz Bildgebung. Die Tumoren werden dann mit intravesikal SÄRNA in Lipid-Nanopartikel (LNPs) formuliert behandelt. Das Tumorwachstum wird mit Ultraschall und Biolumineszenz überwacht. Alle Schritte dieses Verfahrens sind in der beigefügten Video demonstriert.

Watch this Scientific Journal Video about An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment at JoVE.com

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

Gründung einer orthotopen Tumormodell Blase zu Antitumor-Wirkung von intravesikal geliefert Sarna und Überwachung des Tumorwachstums durch Ultraschall und Biolumineszenz-Bildgebung zu beurteilen.

Einem orthotopen Blasentumor Modell und der Auswertung der intravesikalen Behandlung SÄRNA

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

an-orthotopic-bladder-tumor-model-evaluation-intravesical-sarna

Research

JoVE Journal

Medicine

14.7K Aufrufe.  University of California, San Francisco. Gründung einer orthotopen Tumormodell Blase zu Antitumor-Wirkung von intravesikal geliefert Sarna und Überwachung des Tumorwachstums durch Ultraschall und Biolumineszenz-Bildgebung zu beurteilen.

Serie: An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

Einem orthotopen Modell des Murine Blasenkrebs

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Forschung

Medizin

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-Photonen-Imaging der Tumorzellinvasion in einem orthotopen Mausmodell des oralen Plattenepithelkarzinoms

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Kombination Strahlentherapie in einem orthotopen Maus Brain Tumor-Modell

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Ein orthotopen Mausmodell des Anaplastic Schilddrüsenkarzinom

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Ein orthotopen Blasenkrebs-Modell für Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

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  ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Minimal Invasive Gründung von murinen orthotopen Blasen Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

Bewertung der Tumor-infiltrierenden Leukozyten-Untergruppen in einer subkutanen Tumormodell

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

Bewertung der Wirksamkeit der<em&gt; H. pylori</em&gt; Protein HP-NAP als therapeutisches Werkzeug zur Behandlung von Blasenkrebs in einem orthotopen Mausmodell

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

Ein &quot;Patient-Like&quot; Orthotope Syngene Maus-Modell der Leberzellkarzinom Metastasen

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...