We would like to thank Pascal DuBois for proof-reading the article. The authors would also like to thank the personnel of the Animal Facility at Hunter College of The City University of New York. This work was supported by a NIH RCMI grant to Hunter College.

Ethics Statement Alle tierexperimentellen Studien wurden von der Institutional Animal Care und Verwenden Committee (IACUC) des Hunter College der City University of New York zugelassen. Anmerkung: acht BALB / c-Mäuse wurden in diesem experimentellen Verfahren verwendet. Fünf BALB / c-Mäuse wurden mit 5 × 10 6 BNL 1ME A.7R.1 Maus hepatozelluläre Karzinomzellen implantiert werden, um die primären Tumoren zu erzeugen. Drei BALB / c-Mäuse keine Implantationen wurden als Kontrollen verwendet. BNL 1ME A.7R.1 ist eine murine Leberzellkarzinom-Zelllinie, die durch chemische Umwandlung aus dem murinen nicht-tumor Hepatozyten-Zellinie BNL CL.2 abgeleitet wurde. BNL 1ME A.7R.1 wurde von der American Type Culture Collection (ATCC) erhalten. 1. Pre-Chirurgie <ol><li> Stellen Sie sicher, dass Mäuse, für alle Überlebens Operationen sind gesund. Um sicherzustellen, dass Mäusen gesund sind, inspizieren Anzeichen von Austrocknung durch Ausbeulung der Haut. Verwenden Sie keine Mäuse, die mit jedem Anzeichen von Krankheit oder distress in diesen Experimenten. </li><li> Wiegen Mäusen, um sicherzustellen, sie sind nicht untergewichtig. Sicherzustellen, dass das Gewicht mindestens 16-20 Gramm. </li><li> Vorbereitung 5 x 10 6 BNL 1ME A.7R.1 Maus HCC-Zellen für die Implantation per Maus: <ol><li> Saugen Sie Medien aus einem 10 ml 60% - 70% konfluente Zellkulturschale BNL 1ME A.7R.1 Maus HCC-Zellen. Waschen zweimal mit 5 ml PBS. 2 ml Trypsin, in die Schale und bei 37 ° C in einem befeuchteten Inkubator mit Luft und 5% CO 2 für 5 min. </li><li> Anschließend trypsiniert Pipette 10 ul Zellen in einer manuellen Zählkammer zur Zellzählung unter dem Mikroskop bei einer Vergrößerung von 10x. Zählen Sie die Gesamtzahl der Zellen in den vier großen Eckquadrate gefunden und multiplizieren Sie den Verdünnungsfaktor durch die Gesamtzahl der Zellen. Dann dividieren durch die Anzahl der Quadrate gezählt und mit 10 multiplizieren, 6 für die Zellkonzentration (Zellen pro ml). </li><li> Mit den Programmen, sammeln 5 Millionen Zellen pro Maus in sterile1,5 ml Mikrozentrifugenröhrchen. Beschriften von 1,5 ml Mikrozentrifugenröhrchen. </li></ol></li><li> Resuspendieren der 5 x 10 6 BNL 1ME A.7R.1 Maus HCC-Zellen in 100 ul phosphatgepufferter Salzlösung (PBS). Bewahren Sie diese Zellen auf Eis, bis die Implantation in der Chirurgie. </li><li> Führen Sie Aktivitäten für die Vorbereitung von Mäusen für die Operation, wie Narkose und Haarschneide weg vom sterilen Geräten und Materialien, um eine Kontamination zu vermeiden. </li><li> Induzieren Narkose bei Mäusen durch Inhalation von Isofluran mit einem Isofluran Verdampfer zusammen mit Sauerstoff bei einer konstanten Konzentration Flussrate von 1 l pro min (ca. 2,1%). Anmerkung: Das System ist selbstständig und enthält eine Präzisions-Verdampfer, eine Sauerstoffversorgung und einen Aktivkohlebehälter, die mit einem Atmungskreislauf für Gas / Anästhetikum Abfangen benötigte verknüpft ist. </li><li> Sicherzustellen, dass der Anästhesie geeignet ist, um das Tier unterzogen Schmerzen während der Operation zu verhindern. Führen Zehen Prise und beobachten Tier für nicht-Verantsiveness Anästhesie zu bestätigen. </li><li> Zu dieser Zeit, schalten Sie die Ventilleitung, Siegel an die Box und offen für die Nasenkegel. </li><li> Sobald die Maus betäubt, tragen Sie eine kleine Menge einer Augensalbe auf die Hornhaut als vorbeugende Maßnahme vor dem Austrocknen und Schäden. </li><li> Anschließend bewegen Sie den Tier zum Vorbereitungsbereich, mit dem Nasenkegel ordnungsgemäß aufgestellt und angeschlossen (zur Aufrechterhaltung der Narkose). Bereiten Sie die Website des chirurgischen Einschnitt für die Chirurgie durch Depilation an diesem Vorbereitungsbereich. </li><li> Sicherzustellen, dass der Bereich der Operation ist an einer Stelle frei von anderen Aktivitäten. </li><li> Stellen Sie sicher, dass die Arbeitsoberfläche stabil und angemessen desinfiziert, unverschmutzten und aufgeräumten vor der Operation ist. Legen Sie eine sterile Stück Leinen oder OP-Tray darüber. </li><li> Stellen Sie sicher, dass das Operationsfeld sind nur verfügbar für Personen, die Durchführung der Operation. Stellen Sie sicher, dass die chirurgische Raum wird gereinigt und mit einem geeigneten Reinigungsmittel gewischt und mit einem geeigneten Desi gesprühtctant. </li><li> Stellen Sie sicher, dass es auch von Türen und Fenstern, wie Luftströmungen positioniert kann Staub, was zu Kontamination der Website zu verbreiten. </li><li> Führen Sie die Überlebenszeit der Operation mit sterilen Instrumenten. Entweder genießen Instrumente in einem Desinfektionsmittel nach Empfehlung oder Autoklav-Herstellers. Verwenden Sie sterile Grundversorgung, die betriebsbereit sind. </li><li> Separate Instrumente nach Funktion, um zu verhindern Verunreinigung der sterilen Bereichen. </li><li> Rasur ungefähr 1 Zoll rund um die Seite des Einschnitts (Mittelschnitt), um die Leber des Tieres gelangen. Entfernen der Haare von dem Tier entfernt von der Stelle der Operation. </li><li> Nach der Rasur, reiben Sie die Fläche mit einem seifigen Desinfektionsmittel (betadine chirurgische Macchia) und waschen Sie die überschüssige Desinfektions weg mit sterilem PBS-Lösung. Führen Sie dies mehrmals, mit dem gesamten Prozess Peeling Dauer ca. 5 min. Entfernen und die verwendeten Handschuhe verworfen. </li><li> Danach, um Schutzkleidung zu ändern, wie Surgical Kleid, Gestrüpp top und Gesichtsmaske. Als nächstes desinfizieren Hände mit der richtigen Peeling-Lösung vor, die sich OP-Handschuhe. Anmerkung: Dies ist ein notwendiger Schritt Abwurf und Verunreinigung der Operationsstelle mit möglichen Verunreinigungen zu verhindern. </li></ol> 2. Chirurgie <ol><li> Geben Sie eine externe Wärmequelle für Wärme während der gesamten Dauer der Anästhesie. Das Tier kann eine Unterkühlung zu erleben, weil Anästhesie verursacht Störungen der Thermoregulation; Daher halten die Körpertemperatur. </li><li> Legen Sie die Maus auf dem Rücken in der gesamten Operation. </li><li> Befestigen Sie die Vorderbeine, Hinterbeine und der Schwanz der Maus auf dem OP-Tisch mit Stücken von chirurgische Bänder. </li><li> Stellen Sie eine gerade Linie Einschnitt unmittelbar schlechter als die xyphisternum. </li><li> Verwenden Sie eine sterile selbsthaltender Retraktor, die Leber zu schützen. </li><li> Mit einer Spritze mit einer 20G Nadel langsam und vorsichtig zu injizieren 100 ul PBS, enthaltend 5 × 10 6 BNL 1ME A.7R.1Maus HCC-Zellen in den rechten Leberlappen. </li><li> Nach dem Entfernen der Nadel, tragen Sie eine sterile Wattestäbchen für mindestens 1 min, um eine mögliche Blutung zu stoppen wegen der hohen Durchblutung der Leber. </li><li> Schließen Sie das subkutane Gewebe mit einer absorbierbaren Naht, gefolgt von Hautverschluss mit chirurgischen Klammern. Anschließend reinigen Sie die Wunde noch einmal mit Betadin-Peeling. Anmerkung: Dies wird den Verschluß der Operationswunde zu gewährleisten. </li><li> Subkutan geben 100 ul physiologischer Kochsalzlösung. </li></ol> 3. Post-Chirurgie <ol><li> Unmittelbar nach der Operation, übertragen Sie die Maus, um einen sauberen Käfig mit einem postoperativen Identifikationskarte, um Hausmeister des Maus-Zustand zu informieren. Geben Sie Nahrung und Wasser. Hinweis: Nach unserer Erfahrung, eine Maus, die gerade durchgemacht hat Chirurgie am besten abschneiden, wenn allein mit nassen Nahrung und Wasser für die erste 24-Stunden-Periode nach der Operation untergebracht. </li><li> Halten Sie das Tier warm mit einer Wärmelampe, die Hitze abstrahlen nicht übertraf 38 ºC. USE-Thermometer, um die Temperatur zu messen, bis die Maus vollständig wiederhergestellt wird. </li><li> Haus das Tier in einem Käfig vorzugsweise mit Papierbettwäsche, um Komfort zu gewährleisten. </li><li> Nach anfänglichen Erholung, überwachen das Tier Anzeichen von Unwohlsein, Blutungen, Schmerzen und Leiden. Geben Buprenorphin (0,05 -0,1 mg / kg), um Schmerzen zu lindern. Die Aktivität, Verhalten und Aussehen, sowie Wasser und der Nahrungsaufnahme für ca. 2 h genau zu überwachen. Wenn ambulante, bewegen Sie das Tier wieder in die Gegend für regelmäßige normale Gehäuse von Mäusen und weiterhin auf 6-12 h Intervalle für 24 Stunden nach der Operation zu überwachen. Führen Sie eine Gewichtskontrolle, falls gewünscht. </li><li> Allerdings, wenn das Tier ist mit Problemen erholt, geeignete unterstützende Behandlung Aktionen. Wenn Mäuse sind nicht in der Lage sich zu erholen, mit Menschlichkeit einschläfern Mäusen über CO 2 Ersticken und Genickbruch. Folgen Sie genehmigten Richtlinien des Instituts für die Euthanasie. </li><li> Nach der Operation, überprüfen Sie für die klinische Beweise für HCC in der Regel observab istle als signifikante Reduktion der Körperkondition. Anmerkung: An diesem Punkt wurde ein Primärtumor wahrscheinlich in der Leber entwickelt und metastatischen Ablagerungen wahrscheinlich in der Lunge entwickelt. </li><li> An dieser Stelle opfern alle Mäuse (Versuchs- und Kontroll) im Experiment durch humane Sterbehilfe beteiligt. Betreff die Mäuse zu Erstickung mit Kohlendioxid: sehr langsam setzen Kohlendioxid in die Kammer über einen Zeitraum von 5 bis 10 min. Dies führt dazu, Atemstillstand. </li></ol>

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The authors declare that they have no competing financial interests.

In diesem Artikel eine ausführliche Beschreibung des Verfahrens gegeben, dass kürzlich von Ogunwobi und Kollegen der erfolgreichen Etablierung eines orthotopen syngenen Mausmodell der HCC Metastase berichtet (Abbildung 2) 3. Die Entnahmerate von Tumoren, für dieses Verfahren ist im Allgemeinen hoch. Wir haben früher beobachteten eine Tumoraufnahmerate von 100% 3. Allerdings kann die Aufnahmerate variabel, abhängig von der Kompetenz der Prüfer sein. In einer aktuellen Experime...

Hepatocellular carcinoma (HCC) is one of the most lethal cancers, with poor prognosis and low life expectancy. Nearly all cancer-related deaths are due to the metastatic spread of the disease from the originating organ to additional distant organs2-6.HCC progression is a complex process. Therefore, being able to model the tumor microenvironment that is naturally found in metastatic HCC in animal models can prove to be a successful and useful way to reveal relevant mechanisms in humans.7Unfortunately, the mechanisms of cancer metastasis are still largely unclear. Therefore, there is a need to establish animal models that will enable us to elucidate the underlying molecular mechanisms of metastases of cancers such as HCC3,5.
Mouse model systems are a very useful approach for delineating mechanisms and evaluating novel strategies for treatment of metastatic human cancers5,8. The various mouse model systems that presently exist are a testament to efforts of researchers to correctly depict the complexity of the disease5,8,9.
Ogunwobi and colleagues recently used a survival surgical approach to demonstrate establishment of a novel orthotopic syngeneic mouse model for the study of metastasis in HCC3. Their work established a &#8220;patient-like&#8221; mouse model that recapitulates features of aggressive and metastatic HCCs3. They further demonstrated that this mouse model system can be used to study the biology of circulating tumor cells, and that this holds potential for gaining novel insights into the mechanisms of cancer metastasis3.
The aim of this paper is to describe in detail the methodology used in establishing this &#8220;patient-like&#8221; orthotopic syngeneic mouse model of HCC metastasis3. The methodology of how to implant BNL 1ME A.7R.1 mouse HCC cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c3 using survival surgery will be described. Unlike other mouse xenograft tumor models where human tumor cells are implanted into immune deficient mice, this system is syngeneic and is, therefore, suitable for studying the role of the immune system in tumor metastasis3,5,8. This approach will likely gain widespread use for studying the mechanisms of metastasis in solid organ cancers.

<table><tbody><tr><td>Micro Dissecting Tweezer&amp;nbsp;</td><td>Roboz</td><td>(RS-5040)</td><td>Tip 0.20 x 0.12 mm</td></tr><tr><td>Graefe Micro Dissecting Forceps, serrated curved tip</td><td>Roboz</td><td>(RS-5111)</td><td>1 X 2 teeth, curved tip width 0.6 mm</td></tr><tr><td>Micro Dissecting Retractor-Agricola (3 by 3 prongs)</td><td>Roboz</td><td>(RS-6501)</td><td>Blunt 3 X 3 prongs, depth 4 mm, spread 25 mm</td></tr><tr><td>Micro Dissecting Retractor-Goldstein (3 by 3 prongs)</td><td>Roboz</td><td>(RS-6503)</td><td>Blunt, 3 X 3 prongs, depth 4 mm, spread 19 mm</td></tr><tr><td>Jameson Caliper (Measures tumors)</td><td>Roboz</td><td>(RS-6466)</td><td>80 mm/3 inch scale, chrome plated&amp;nbsp;</td></tr><tr><td>Micro Dissecting Scissors, large ring sissors, straight and sharp</td><td>Roboz</td><td>(RS-5852)</td><td>23 mm blades, length 4 inches, flat shanks and large rings</td></tr><tr><td>Scalpel with blades, for delicate dissecting procedures</td><td>Roboz</td><td>(RS-9861-36)</td><td></td></tr><tr><td>Scalpel handle&amp;nbsp;</td><td>Roboz</td><td>(RS-9884)</td><td>Solid</td></tr><tr><td>Littauer Stitch Sissors</td><td>Roboz</td><td>(RS-7074)</td><td>Length 4.5 inches</td></tr><tr><td>Brown needle holder, for easy suture tying</td><td>Roboz</td><td>(RS-7960)</td><td>Convex jaw, fine serrations</td></tr><tr><td>Reflex 7MM wound clips with reflex 7 clip applier</td><td>Roboz</td><td>(RS-9262)</td><td>safe, secure alternative method of wound closure</td></tr><tr><td>Instrument tray and lid</td><td>Roboz</td><td>RT-1350S</td><td></td></tr><tr><td>Mini-clipper with detachable blade</td><td>Roboz</td><td>(RS-5903)</td><td></td></tr><tr><td>Germinator 500 (the Germ Terminator) Dry Sterilizer</td><td>Roboz</td><td>Ds-400, Ds-401, DS-501</td><td>For fast decontamination of micro-dissecting instruments.&amp;nbsp;&amp;nbsp; Instruments decontaminate within 15 sec.&amp;nbsp;</td></tr><tr><td>Microinjection needles</td><td>VWR</td><td>BD305125</td><td>25G Needle</td></tr></tbody></table>

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.

Die Leber von Balb / c-Mäuse wurden mit 5 × 10 6 BNL 1ME A.7R.1 Maus HCC-Zellen implantiert. Klinische Beweise für HCC Entwicklung zu beobachten war 63 Tage nach der Operation. Demzufolge wurden die Mäuse auf humane Weise eingeschläfert. Autopsie wurde auf die eingeschläfert Mäusen durchgeführt. Die Lunge und Leber wurden reseziert und eine sorgfältige Brutto Prüfung durchgeführt, um makroskopischen Tumoren zu identifizieren. In einer Maus wurde ein oberflächlicher Tumor auf der Oberfläche der Le...

Watch this Scientific Journal Video about A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis at JoVE.com

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

Die Metastasierung von Krebs ist die Hauptursache der durch Krebs verursachten Todesfälle. Wir bieten eine eingehende Beschreibung der unser Überleben Chirurgie Methodik für die Festlegung eines &quot;Patienten-like&quot; orthotopen syngenen Mausmodellsystem zur Untersuchung der Mechanismen der Metastasierung bei soliden Organtumoren.

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 ...

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12.2K Aufrufe.  Hunter College of the City University of New York.  Die Metastasierung von Krebs ist die Hauptursache der durch Krebs verursachten Todesfälle. Wir bieten eine eingehende Beschreibung der unser Überleben Chirurgie Methodik für die Festlegung eines "Patienten-like" orthotopen syngenen Mausmodellsystem zur Untersuchung der Mechanismen der Metastasierung bei soliden Organtumoren. 

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

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

Erweiterte Tiermodell für Colorectal Metastase in der Leber: Imaging-Techniken und Eigenschaften des metastasierten Clones

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

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Krebsforschung

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Ein Portal Veneninjektion Modell zur Untersuchung der Leber Metastasierung von Brustkrebs

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

Ein Syngenetische Maus-Modell von metastasierendem Nierenzellkarzinom für Quantitative und Longitudinal Beurteilung der vorklinischen Therapien

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Orthotopen Mausmodell der Darmkrebs-

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

Biologie

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

Ein onkogenes Hepatozyten-induziertes orthotopisches Mausmodell von hepatozellulärem Krebs, der bei der Einstellung von Leberentzündungen und Fibrose entsteht

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

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 ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

Generierung von subkutanen und Intrahepatische Menschenleberzellkarzinom Xenografts in immundefizienten Mäusen

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Medizin

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

Nicht-invasive PET/MR-Bildgebung in einem orthotopen Mausmodell des hepatozellulären Karzinoms

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...

A Preclinical Murine Model of Hepatic Metastases

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.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Einer präklinischen Mausmodell der Lebermetastasen

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

Ein "Patient-Like" Orthotope Syngene Maus-Modell der Leberzellkarzinom Metastasen

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