Diese Arbeit wurde von der National Health and Medical Research Council, Australien (1008865), das Australian Research Council (LE110100125), das National Cancer Institute (CA138687-01), Erica Sloan wird von einem Early Career-Stipendium der National Breast Cancer abgestützt Foundation, Australien. Corina Kim-Fuchs wird durch ein Stipendium von der Krebsliga Schweiz und einem HDR-Stipendium von der Monash Institute of Pharmaceutical Sciences unterstützt. Eliane Angst wird durch einen Zuschuss aus dem Bern Krebsliga unterstützt.

Das Protokoll demonstriert wird unter der Leitung und Genehmigung des Autors Institution Tierpflege und Nutzung Ausschuss durchgeführt. Alle Versuche werden unter Einhaltung aller einschlägigen Richtlinien, Vorschriften und Aufsichtsbehörden ausgeführt. 1. Transduzierende Pankreaskarzinomzelllinien <ol><li> Wandlerprinzip Bauchspeicheldrüsenkrebszellen ausdrücken Luciferase wie zuvor beschrieben. 12,13 Panc-1 und Capan-1 Pankreaskarzinom Zelllinien transduziert mit Glühwürmchenluciferase kommen hier zum Einsatz. </li></ol> Hinweis: Renilla-Luciferase oder bakterielle Luciferase, können ebenfalls verwendet werden. 2. Bauchspeicheldrüsenkrebs Zellpräparation <ol><li> Kultur transduziert Bauchspeicheldrüsenkrebszellen bis 70% konfluent. </li><li> Heben Sie die Zellen der Bauchspeicheldrüse und die Rentabilität zu gewährleisten ist größer als 90%. </li><li> Resuspendieren in 2 x 10 7 Zellen / ml in einer 3:2-Mischung von gekühlten Matrigel: Phosphat gepufferte saline (PBS). </li><li> Halten Sie die Matrigel-Zellsuspension auf Eis vor der Injektion in der Bauchspeicheldrüse. </li></ol> Anmerkungen: Eine rasche Verfestigung Matrigel sicherzustellen, Verringerung der Volumen PBS zur Berücksichtigung des Volumens des Zellpellets. Griff Matrigel mit eiskaltem Instrumente und Spritzen zu allen Zeiten zu Erstarrung vor der Injektion zu verhindern. Die vorgeschlagene Anzahl von Zellen ist eine Führung und sollten empirisch für jede Zelllinie zu bestimmen. 3. Maus Vorbereitung <ol><li> Anesthetize die Maus mit eingeatmet 2-3% Isofluran. Bestimmen Narkosetiefe durch mangelnde Pedal Reflex zu einer sanften Prise Zehe. </li><li> Schmiermittel auf die Augen, um ein Austrocknen zu verhindern. </li><li> Positionieren Sie den Mauszeiger auf dem Rücken auf einem 37 ° C Heizkissen und sanft drehen Sie die Maus, um die linke Seite des Bauches zu erhöhen. </li><li> Bereiten Sie den Bauch mit einem 10% Povidoniod Lösung. </li></ol> Anmerkungen: Injectable Anästhesie kann anstelle von inhalativen Anästhesie verwendet werden. Präoperative Fasten ist nicht erforderlich. 4. Laparotomie <ol><li> Mit einer sterilen chirurgischen Instrumente einen 1,5 cm Schnitt in der Haut etwa 1 cm seitlich links von der Mittellinie. </li><li> Machen Sie eine 1,5 cm Einschnitt in der zugrunde liegenden Bauchmuskeln. </li><li> Suchen Sie die Milz mit der Zange und entfernen Sie vorsichtig die Milz aus der Bauchhöhle. Sichern Sie die Milz entlang einer sterilen Wattestäbchen, um die zugrunde liegenden Bauchspeicheldrüse aussetzen. </li><li> Finde den Schwanz der Bauchspeicheldrüse neben der Milz. </li><li> Mit einer 29 G 0,3 ml Insulinspritze, injizieren 20 ul der Matrigel-Zellsuspension in der Bauchspeicheldrüse. </li><li> Nach der Injektion, halten Sie die Spritze in der Bauchspeicheldrüse für 30-60 sec bis die Matrigel verfestigt hat. Dieser wichtige Schritt minimiert Zellleckstromcharakteristikfunktion. </li><li> Überprüfen Sie die Injektionsstelle, um sicherzustellen, keine Leckage aufgetreten. </li><li> Bringen Sie die Milz und Bauchspeicheldrüse in die Bauchhöhle. </li></ol> Hinweis: Achten Sie darauf, um zu vermeiden Punktion der Rückenseite der Bauchspeicheldrüse, die dünn sein kann. 5. Bauchdeckenverschluss <ol><li> Schließen Sie die Bauchmuskulatur der Maus mit einem resorbierbaren Nahtmaterial geflochten 4-0 mit einer runden Nadel mit einem kontinuierlichen Stich. </li><li> Schließen die äußere Haut mit einem nicht resorbierbaren monofilen Faden 6-0 mit einer Schneidnadel mit einem kontinuierlichen Abspielen. </li><li> Entfernen Sie die Maus vom Inhalationsanästhetikum und injiziert 0,05-0,1 mg / kg subkutan Buprenorphin. </li><li> Lassen Sie die Maus, um in seinem Käfig auf einem 37 ° C Heizkissen mit freiem Zugang zu Futter und Wasser gegeben zu erholen. Wenn Mäuse Anzeichen von Schmerzen wie gebeugt oder eingeschränkter Mobilität zu demonstrieren, kann Buprenorphin alle 12 h über einen 36 Stunden-Zeitraum angegeben werden. </li><li> Nach Wundheilung (7-10 Tage), betäuben die Maus und entfernen Sie die externe Nähte. </li></ol> 6. Bioluminescent-Tracking von Bauchspeicheldrüsenkrebs Cancer Progression <ol><li> Anesthetize die Maus mit Isofluran eingeatmet. </li><li> Injizieren 150 mg / kg D-Luciferin über die Schwanzvene. </li><li> Platzieren Sie die Maus in die Biolumineszenz-Imaging-System und erfassen Weißlicht-und Biolumineszenz Bilder wie zuvor beschrieben. 14,15 </li><li> Entfernen Sie die Maus vom Inhalationsanästhetikum und lassen Sie ihn in seinem eigenen Käfig zu erholen. </li></ol> Hinweis: Biolumineszenz-Bildgebung ist nicht-invasiv und kann periodisch durchgeführt werden, um Tumorwachstumskinetiken untersuchen. Um das Bild Tumor in der Bauchspeicheldrüse Schwanz ist es wichtig, mit der Maus auf die linke Seite legen, so dass die Tumor-Punkte in Richtung der Kamera. Wir abgebildet einmal pro Woche mit der Frequenz um bis zu dreimal pro Woche vor der experimentellen Endpunkt mit einem Lumina II Imaging-System (Perkin-Elmer, ehemals Caliper Life Sciences) läuft Wohnzimmer Imaging 4.3.1 Software mit Binning 4, FOV 12.5, F-Stop 1, Belichtung 1-60 sec (bestimmt durch die höchste exposure ohne Pixelsättigung).

<ol>
	<li>American Cancer Society. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Facts and Figures. , (2013).</li><li>Hariharan, D., Saied, A., Kocher, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mortality+rates+for+pancreatic+cancer+across+the+world.">Analysis of mortality rates for pancreatic cancer across the world.</a> HPB (Oxford). 10, 58-62 (2008).</li><li>Li, D., Xie, K., Wolff, R., Abbruzzese, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+cancer.">Pancreatic cancer.</a> Lancet. 363, 1049-1057 (2004).</li><li>Oettle, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adjuvant+chemotherapy+with+gemcitabine+vs+observation+in+patients+undergoing+curative-intent+resection+of+pancreatic+cancer:+a+randomized+controlled+trial.">Adjuvant chemotherapy with gemcitabine vs observation in patients undergoing curative-intent resection of pancreatic cancer: a randomized controlled trial.</a> JAMA. 297, 267-277 (2007).</li><li>Andersson, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gemcitabine+chemoresistance+in+pancreatic+cancer:+molecular+mechanisms+and+potential+solutions.">Gemcitabine chemoresistance in pancreatic cancer: molecular mechanisms and potential solutions.</a> Scand. 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G., et al. <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+nude+mouse+model+for+evaluating+pathophysiology+and+therapy+of+pancreatic+cancer.">An orthotopic nude mouse model for evaluating pathophysiology and therapy of pancreatic cancer.</a> Pancreas. 26, 89-98 (2003).</li><li>Partecke, L. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+syngeneic+orthotopic+murine+model+of+pancreatic+adenocarcinoma+in+the+C57/BL6+mouse+using+the+Panc02+and+6606PDA+cell+lines.">A syngeneic orthotopic murine model of pancreatic adenocarcinoma in the C57/BL6 mouse using the Panc02 and 6606PDA cell lines.</a> Eur. Surg. Res. 47, 98-107 (2011).</li><li>Hingorani, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preinvasive+and+invasive+ductal+pancreatic+cancer+and+its+early+detection+in+the+mouse.">Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse.</a> Cancer Cell. 4, 437-450 (2003).</li><li>Sloan, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sympathetic+nervous+system+induces+a+metastatic+switch+in+primary+breast+cancer.">The sympathetic nervous system induces a metastatic switch in primary breast cancer.</a> Cancer Res. 70, 7042-7052 (2010).</li><li>Wang, X., McManus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+production.">Lentivirus production.</a> J. Vis. Exp. 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(56), e3175 (2011).</li><li>Lim, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+tumor+metastases+and+osteolytic+lesions+with+bioluminescence+and+micro+CT+imaging.">Monitoring tumor metastases and osteolytic lesions with bioluminescence and micro CT imaging.</a> J. Vis. Exp. (52), e2775 (2011).</li><li>Burton, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+gene+expression+imaging+to+directly+detect+sentinel+lymph+node+metastasis+of+prostate+cancer.">Adenovirus-mediated gene expression imaging to directly detect sentinel lymph node metastasis of prostate cancer.</a> Nat Med. 14, 882-888 (2008).</li><li>Vezeridis, M. P., Doremus, C. M., Tibbetts, L. M., Tzanakakis, G., Jackson, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+and+metastasis+following+orthotopic+transplantation+of+human+pancreatic+cancer+in+the+nude+mouse.">Invasion and metastasis following orthotopic transplantation of human pancreatic cancer in the nude mouse.</a> J. Surg. Oncol. 40, 261-265 (1989).</li><li>Fu, X., Guadagni, F., Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+metastatic+nude-mouse+model+of+human+pancreatic+cancer+constructed+orthotopically+with+histologically+intact+patient+specimens.">A metastatic nude-mouse model of human pancreatic cancer constructed orthotopically with histologically intact patient specimens.</a> Proc. Natl. Acad. Sci. U.S.A. 89, 5645-5649 (1992).</li><li>Reyes, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+xenografts+of+human+pancreatic+carcinomas+acquire+genetic+aberrations+during+dissemination+in+nude+mice.">Orthotopic xenografts of human pancreatic carcinomas acquire genetic aberrations during dissemination in nude mice.</a> Cancer Res. 56, 5713-5719 (1996).</li><li>Kim, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+orthotopic+and+heterotopic+human+pancreatic+cancer+xenografts+in+immunodeficient+mice.">Generation of orthotopic and heterotopic human pancreatic cancer xenografts in immunodeficient mice.</a> Nat Protoc. 4, 1670-1680 (2009).</li><li>Furukawa, T., Kubota, T., Watanabe, M., Kitajima, M., Hoffman, R. 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Die Autoren erklären, dass sie keine finanziellen Interessen haben.

Hier beschreiben wir einen orthotopen Modell für Längs-Beurteilung von Pankreas-Tumor-Entstehung und Progression. Primäre Tumorwachstumskinetiken reproduzierbar sind (Abbildung 3) und kann nicht-invasiv überwacht mit Biolumineszenz-Bildgebung der Luciferase-markierten Zellen, für Analysen von Tumor-Reaktion auf neuartige Anti-Bauchspeicheldrüsenkrebs Therapeutika zB. Im Einklang mit der menschlichen Krankheit zeigt das Modell lokale Invasion Pankreas (4A), die Untersuchun...

Bauchspeicheldrüsenkrebs ist die vierthäufigste Ursache von Krebs-Todesfälle, mit einer 5-Jahres-Überlebensrate von 4-6%. 1,2 Nur 15% der Patienten sind früh genug in der Diagnose der Krankheit, um förderfähig zu sein für Chirurgie und Tumoren wiederkehren in&gt; 80% der Patienten. 3,4 Gemcitabin zur Behandlung von Pankreas-Adenokarzinomen verwendet wird, ist jedoch chemoresistance häufig und oft das Medikament hat wenig Einfluss auf das Gesamtüberleben. 5 Neue pharmakologische Strategien zur Behandlung von Bauchspeicheldrüsenkrebs dringend erforderlich. Ihre Entwicklung hängt wesentlich verbesserte Verständnis der wichtigsten Schritte des Fortschreitens der Krankheit, die empfindlich auf eine therapeutische Intervention kann. Orthotopen Modellen von Bauchspeicheldrüsenkrebs emulieren wichtigsten Aspekte der menschlichen Krankheit, so dass sie ein ideales Werkzeug für das Studium der Biologie von Bauchspeicheldrüsenkrebs. 6-9 Im Gegensatz zu in vitro-Zell-basierte Assays von Bauchspeicheldrüsenkrebs Zelle Verhalten einerd subkutan in-vivo-Modelle von Bauchspeicheldrüsenkrebs, erlauben orthotopen Modellen Untersuchung von Tumor-Zell-Interaktionen mit dem Pankreas Mikroumgebung. Die Kinetik des Fortschreitens der Krankheit sind in hohem Maße reproduzierbar in orthotopen Modellen auftreten und über einen kurzen Zeitraum (Wochen), die sie gut zu vorklinischen Erprobung neuartiger Therapeutika geeignet macht. Dies steht im Gegensatz zu transgenen Modellen, bei denen Ausbruch der Krankheit in ein längeres und variabler Zeitrahmen (Monate bis 1 Jahr). 10 Wenn mit aggressiveren Zelllinien verwendet werden, haben orthotopic Modelle von Bauchspeicheldrüsenkrebs Muster spontaner Metastase gleichen denen, die in Patienten. 8 Expression von Biolumineszenz Reportergene wie Glühwürmchen-Luciferase ermöglicht Längs Überwachung des Tumorwachstums, metastatischen Verbreitung, Wiederholung und Reaktion auf Therapeutika. 6,11 Hier beschreiben wir einen orthotopen Modell von Bauchspeicheldrüsenkrebs, die Matr nutztigel für lokalisierte Zelle Lieferung und in vivo Biolumineszenz-Bildgebung zur nicht-invasiven Überwachung der Tumorprogression. Diese orthotopen Modell des Pankreaskarzinoms ermöglicht nicht-invasive Analyse der Krankheitsverlauf und das Ansprechen auf therapeutische Interventionen in syngene oder Xenotransplantatmodellen.

<table border="1">
 <tbody>
 <tr>
 <td>Equipment</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Clean Bench coat</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Heating pad</td>
 <td></td>
 <td></td>
 <td>Set to 37 &deg;C </td>
 </tr>
 <tr>
 <td>Ivis Lumina ll Bioluminescent imager</td>
 <td>Caliper </td>
 <td></td>
 <td>Alternative bioluminescent imaging systems include In vivo F PRO (Carestream) and Photon Imager (Biospace Lab)</td>
 </tr>
 <tr>
 <td>Dissecting scissors</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Iris forceps (serrated)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Needle holder</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>27 G 0.3 ml insulin syringe</td>
 <td>Terumo</td>
 <td>T35525M2913</td>
 <td></td>
 </tr>
 </tbody>
</table>

bioluminescent orthotopic model of pancreatic cancer progression

<p class="jove_content"&gt; Bauchspeicheldrüsenkrebs hat eine extrem schlechte Fünf-Jahres-Überlebensrate von 4-6%. Neue therapeutische Optionen dringend erforderlich und hängen von besseren Verständnis von Bauchspeicheldrüsenkrebs Biologie. Zum besseren Verständnis der Wechselwirkung von Krebszellen mit dem Pankreas Mikroumgebung zeigen wir eine orthotopic Modell von Bauchspeicheldrüsenkrebs, die nicht-invasive Überwachung der Tumorprogression ermöglicht. Luciferase-tagged Bauchspeicheldrüsenkrebs Zellen in Matrigel resuspendiert und lieferte in den Pankreasschwanz während Laparotomie. Matrigel erstarrt bei Körpertemperatur, um ein Auslaufen von Krebszellen bei der Injektion. Primäre Tumorwachstum und Metastasierung in andere Organe werden nach der Injektion des Luciferase-Luciferin-Substrat überwacht, wobei<em&gt; In vivo</em&gt; Abbildung Biolumineszenz-Emission aus den Krebszellen.<em&gt; In vivo</em&gt; Bildgebung kann auch verwendet werden, um primäre Rezidiv nach Resektion zu verfolgen. Diese orthotopen Modell ist mit beiden syngene und Xenotransplantatmodellen geeignet und kann in prä-klinischen Studien verwendet werden, um die Auswirkungen von neuartigen Anti-Krebs-Therapeutika auf das Wachstum der primären Pankreastumorzellen und Metastasierung zu untersuchen.</p&gt;

Diese Methode beschreibt ein orthotopen Modell von Bauchspeicheldrüsenkrebs mit chirurgischen Eingriffen, einschließlich der Einleitung der Narkose, Laparotomie, Injektion von Krebszellen in Matrigel und Bauchdeckenverschluss (Abbildung 1A). Die injizierten Zellen bilden eine Blase in der Oberfläche der Bauchspeicheldrüse (1B). Bauchspeicheldrüsenkrebs Progression kann nicht-invasiv überwacht mit in vivo Biolumineszenz-Bildgebung zu Krebszellproliferation und Verbreitung ...

Watch this Scientific Journal Video about Bioluminescent Orthotopic Model of Pancreatic Cancer Progression at JoVE.com

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Verbessertes Verständnis von Bauchspeicheldrüsenkrebs Biologie ist dringend erforderlich, um die Entwicklung von besseren therapeutischen Optionen zur Behandlung von Bauchspeicheldrüsenkrebs zu ermöglichen. Um diesen Bedarf zu demonstrieren wir eine orthotopic Modell von Bauchspeicheldrüsenkrebs, die nicht-invasive Überwachung der Tumorprogression ermöglicht mit<em&gt; In vivo</em&gt; Biolumineszenz-Bildgebung.

Bioluminescent orthotopen Modell von Bauchspeicheldrüsenkrebs Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

bioluminescent-orthotopic-model-of-pancreatic-cancer-progression

Research

JoVE Journal

Medicine

26.8K Aufrufe.  Monash University. Verbessertes Verständnis von Bauchspeicheldrüsenkrebs Biologie ist dringend erforderlich, um die Entwicklung von besseren therapeutischen Optionen zur Behandlung von Bauchspeicheldrüsenkrebs zu ermöglichen. Um diesen Bedarf zu demonstrieren wir eine orthotopic Modell von Bauchspeicheldrüsenkrebs, die nicht-invasive Überwachung der Tumorprogression ermöglicht mit In vivo Biolumineszenz-Bildgebung.

Serie: Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

Murine Bioluminescent Leber Tumor-Modell

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

Forschung

Medizin

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 ...

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 ...

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

In vivo Dual-Substrat Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

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 Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

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 ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Chirurgische Verfahren für eine Ratte Modell Teilweise Lebertransplantation mit arteriellen Rekonstruktion

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

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  ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.