Diese Arbeit wurde von Associazione Italiana per la Ricerca sul Cancro, italienische Ministerium für Universität und Forschung, Projekte und Prin Progetti di Ricerca di Ateneo, gewähren N ° CPDA137871, Fondazione Cariplo unterstützt wird, gewähren N ° 2011-0485 zu MdB, und durch Finanziamento Giovani Studiosi, Universität Padua, um Gaia Codolo.

Alle Verfahren für die Behandlung der Tiere wurden von der italienischen Gesundheitsministerium (DM 204/2011-B) genehmigt worden. 1. Tiere <ol><li> Wachsen C57BL6 / J weiblichen Mäusen zu 8 Wochen in einzeln belüfteten Käfigen mit microisolation Filter. Hinweis: Frauen werden bevorzugt, da die anatomische Anpassung ihrer äußeren urogenitalen Gerät macht die Katheterisierung einfacher als bei Männern. </li></ol> 2. Zellkultur <ol><li> Aufrechterhaltung der Maus Urothelkarzinom Zelllinie MB49 in RPMI 1640 Medium mit 10% hitzeinaktiviertem fötalem Rinderserum und 50 ug / ml Gentamicin ergänzt bei 37 ° C in befeuchteter 5% CO 2. </li><li> Wachsen MB49-Zellen in T-75-Flaschen zu 80% Konfluenz. Trypsinieren und resuspendieren in 0,5 x 10 5 bis 0,5 × 10 6 Zellen pro 150 &amp; mgr; l PBS vor der Implantation in Mäuse. </li></ol> 3. Intravesikale Tumor Implant <ol><li> Betäuben Mäuse mit einer Mischung aus dem Narkose zoletil und xylor (33 mg / kg und 20 mg / kg) intraperitoneal injiziert. </li><li> Zeigen Tiere in Rückenlage und fixieren die Hinterbeine in die Unterlage mit Tesafilm. Pfoten haben ausreichend getrennt, um die Harnröhrenmündung gut sichtbar und für die Einführung des Katheters zugänglich machen werden. </li><li> Hinweis: Für die Katheterisierung, ist ein 24 G pädiatrischen Venenkatheter geeignet für 8 bis 10 Wochen alten Mäusen. Die Wahl des Katheters ist, um eine Leckage zwischen der Harnröhrenwand und dem Katheter Flüssigkeit zu vermeiden sehr wichtig. Größere Bohrung Katheter muss für größere oder älteren Mäusen verwendet werden. </li><li> Mit einem kleinen Zange, kneifen eine Kante des äußeren Teil der Harnröhre und drehen Sie sie ganz sanft in Richtung der &quot;Kopfende&quot; der Mäuse. Diese Aktion macht die Harnröhrenmündung. </li><li> Entfernen Sie den inneren Nadel des Katheters und legen Sie diese in die Harnröhre in einem Winkel von 45 °, leicht orientiertin Richtung der &quot;Schwanzende&quot; der Mäuse. Nicht mit Gewalt den Eingang des Katheters; im Falle von Widerstand, nur drehen Sie den Katheter sehr vorsichtig, bis es schlüpft in der Harnröhre. Wenn etwa 0,5-0,8 cm des Katheters in die Harnröhre, vorsichtig die Katheter parallel zum Schwanz der Mäuse und setzen Sie sie vollständig. </li><li> Verwendung einer 1 ml Spritze, deren Nadel in den Katheter eingeführt werden muß, zu waschen, die Blase von Restharn durch Injizieren 200-300 ul sterilem PBS, und saugen die gesamte Flüssigkeit aus der Blase. </li><li> Hinweis: Bei der Injektion von PBS, wird die Blase zu füllen und eine Schwellung zwischen den Hinterbeinen der Mäuse sichtbar sein; Dies bestätigt, dass die Katheter korrekt ist. Die meisten Katheter haben ein Totvolumen, die berücksichtigt werden müssen bei der Berechnung der Einspritzmenge. </li><li> Verwendung einer sterilen 1-ml-Spritze, Injizieren 200 ul 0,1 N HCl. Füllen Sie die Blase, um seine ganze Wand zu verbrennen. Nach 10 sec, entfernen Sie alle Säure und sofort Neutralisaze durch Injizieren von 200 ul 0,1 N NaOH mit einer sterilen Spritze. Im Falle der Injektion von der höchsten Zahl der Zellen (0,5 × 10 6), wird die Ansäuerung Schritt nicht erforderlich. </li><li> Saugen Sie all die Restflüssigkeit und spülen den Hohlraum mit 300 ul sterilem PBS. </li><li> Leeren Sie die Blase durch Aspiration, und injizieren 150 ul PBS, das MB49-Zellen (Bereich von 0,5 x 10 5 bis 0,5 x 10 6) in die Blase. Lassen Sie die Spritze mit dem Katheter zusammengebaut, um das Austreten von Zellen zu vermeiden. Es ist besser, die Spritze durch Befestigung an der Unterlage mit Klebeband sichern. </li><li> Nach 1 Stunde sanft zu extrahieren den Katheter aus der Harnröhre, und platzieren Sie die Maus in den Käfig, unter einer Glühlampe bis sie wach: dies geschieht in der Regel zwischen 1 und 2 Stunden nach dem Ende der Behandlung. </li></ol> 4. Intravesikale Lieferung von HP-NAP <ol><li> Anmerkung: mindestens 3 Tage nach der Zell Instillation, ist es möglich, den BeginnBehandlung. </li><li> Catheterize wie in den Schritten 3.1 bis 3.4 beschrieben. Zu diesem Zeitpunkt ist es nicht notwendig, den Blasenwand mit HCl verbrennen. </li><li> Unter Verwendung einer 1 ml Spritze, waschen Sie die Blase von Restharn durch Einspritzen von 200-300 ul sterilem PBS und saugen Sie die gesamte Flüssigkeit aus der Blase. </li><li> Injizieren 50 ug HP-NAP pro 150 ul PBS in die Blase. Lassen Sie die Spritze an den Katheter angebracht ist, um eine Leckage von der Proteinlösung zu vermeiden. Befestigen Sie die Spritze durch Befestigung an der Unterlage mit Tesafilm. </li><li> Nach 1 Stunde sanft zu extrahieren den Katheter aus der Harnröhre und platzieren Sie die Maus in den Käfig, unter einer Glühlampe, bis sie wach. </li></ol> 5. Tumor Explantate und histologische Analyse <ol><li> Am Ende der Behandlung, die Tiere zu opfern, legen Sie sie in Rückenlage und fixieren die Hinterbeine in die Unterlage mit Tesafilm. </li><li> Mit Hilfe eines chirurgischen Skalpell einen vertikalen Schnitt etwa 1,52 cm lang durch die Haut und diePeritoneum, beginnend knapp über äußeren Genitalien auf die &quot;Kopfende&quot; der Mäuse. Um das Risiko einer Beschädigung der Blase zu minimieren, achten Sie besonders auf die Tiefe des Schnittes. </li><li> Die Blase wird in der Mitte-Links des Schnitts positioniert werden und der Tumor sichtbar als dunkle rosa / violett Masse sein. Nach seiner Exzision, legen Sie die gesamte Blase in einem histologischen Käfig und befestigen Sie in 10% gepuffertem Formalin vor dem Einbetten in Paraffin. </li><li> Zur histologischen und immunhistochemischen Analyse vorzubereiten Serienschnitte der Blase (46 &amp; mgr; m Dicke) mit einem Standard-Mikrotoms. </li><li> Färben die Schnitte mit Hämatoxylin / Eosin und fahren Sie mit der Berechnung der Tumorgröße (D xd 2) und Bestimmung des Prozentsatzes Nekrose pro Querschnittsfläche mit Hilfe eines computergestützten Bildanalysegerät. </li><li> Für Behälter Zählen, gehen durch Färben der Abschnitte für die endotheliale Marker CD31 / PECAM, mit einem Standard-Immunperoxidase-Technik. Scannen Sie die Abschnitte at niedriger Leistung (4 x), um den Bereich der höchsten Gefäßdichte zu identifizieren. Innerhalb dieser Region zählen einzelnen Mikrogefäße in fünf separate Zufallsfelder mit hoher Leistung (20 x). </li></ol>

<ol>
	<li>Siegel, R., Ma, J., Zou, Z., Cancerstatistics Jemal, 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> CA Cancer J Clin. 64, 9-29 (2014).</li><li>Saint, F., 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=Prognostic+value+of+a+T+helper+1+urinary+cytokine+response+after+intravesical+bacillus+Calmette-Guerin+treatment+for+superficial+bladder+cancer.">Prognostic value of a T helper 1 urinary cytokine response after intravesical bacillus Calmette-Guerin treatment for superficial bladder cancer.</a> J Urol. 167, 364-367 (2002).</li><li>Shahin, O., Thalmann, G. N., Rentsch, C., Mazzucchelli, L., Studer, U. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+retrospective+analysis+of+153+patients+treated+with+or+without+intravesical+bacillus+Calmette-Guerin+for+primary+stage+T1+grade+3+bladder+cancer:+recurrence,+progression+and+survival.">A retrospective analysis of 153 patients treated with or without intravesical bacillus Calmette-Guerin for primary stage T1 grade 3 bladder cancer: recurrence, progression and survival.</a> J Urol. 169, 96-100 (2003).</li><li>Lamm, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complications+of+bacillus+Calmette-Guerin+immunotherapy.">Complications of bacillus Calmette-Guerin immunotherapy.</a> Urol Clin North Am. 19, 565-572 (1992).</li><li>De Jager, 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=Long-term+complete+remission+in+bladder+carcinoma+in+situ+with+intravesical+TICE+bacillus+Calmette+Guerin.+Overview+analysis+of+six+phase+II+clinical+trials.">Long-term complete remission in bladder carcinoma in situ with intravesical TICE bacillus Calmette Guerin. Overview analysis of six phase II clinical trials.</a> Urology. 38, 507-513 (1991).</li><li>Dovedi, S. J., Davies, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+targeted+therapies+for+bladder+cancer:+a+disease+waiting+for+a+drug.">Emerging targeted therapies for bladder cancer: a disease waiting for a drug.</a> Cancer Metastasis Rev. 28, 355-367 (2009).</li><li>Evans, D. J., 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=Characterization+of+a+Helicobacter+pylori+neutrophil-activating+protein.">Characterization of a Helicobacter pylori neutrophil-activating protein.</a> Infect Immun. 63, 2213-2220 (1995).</li><li>Grant, R. A., Filman, D. J., Finkel, S. E., Kolter, R., Hogle, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crystal+structure+of+Dps,+a+ferritin+homolog+that+binds+and+protects+DNA.">The crystal structure of Dps, a ferritin homolog that binds and protects DNA.</a> Nat Struct Biol. 5, 294-303 (1998).</li><li>Amedei, A., 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+neutrophil-activating+protein+of+Helicobacter+pylori+promotes+Th1+immune+responses.">The neutrophil-activating protein of Helicobacter pylori promotes Th1 immune responses.</a> J Clin Invest. 116, 1092-1101 (2006).</li><li>Bernard, M., D'Elios, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+modulating+activity+of+the+Helicobacter+pylori+HP-NAP:+Friend+or+foe.">The immune modulating activity of the Helicobacter pylori HP-NAP: Friend or foe.</a> Toxicon. 56, 1186-1192 (2010).</li><li>Codolo, 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=The+neutrophil-activating+protein+of+Helicobacter+pylori+down-modulates+Th2+inflammation+in+ovalbumin-induced+allergic+asthma.">The neutrophil-activating protein of Helicobacter pylori down-modulates Th2 inflammation in ovalbumin-induced allergic asthma.</a> Cell Microbiol. 10, 2355-2363 (2008).</li><li>Gunther, J. 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=Optimizing+syngeneic+orthotopic+murine+bladder+cancer+(MB49).">Optimizing syngeneic orthotopic murine bladder cancer (MB49).</a> Cancer Res. 59, 2834-2837 (1999).</li><li>Codolo, 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=HP-NAP+inhibits+the+growth+of+bladder+cancer+in+mice+by+activating+a+cytotoxic+Th1+response.">HP-NAP inhibits the growth of bladder cancer in mice by activating a cytotoxic Th1 response.</a> Cancer Immunol Immunother. 61, 31-40 (2012).</li><li>Chan, E., Patel, A., Heston, W., Larchian, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+orthotopic+models+for+bladder+cancer+research.">Mouse orthotopic models for bladder cancer research.</a> BJU Int. 104, 1286-1291 (2009).</li><li>Kubota, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+models+of+human+cancer+xenografted+in+the+nude+mouse:+the+importance+of+orthotopic+transplantation.">Metastatic models of human cancer xenografted in the nude mouse: the importance of orthotopic transplantation.</a> J Cell Biochem. 56, 4-8 (1994).</li><li>Loi, M., 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+use+of+the+orthotopic+model+to+validate+antivascular+therapies+for+cancer.">The use of the orthotopic model to validate antivascular therapies for cancer.</a> Int J Dev Biol. 55, 547-555 (2011).</li><li>Miyazaki, 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=Preconditioning+methods+influence+tumor+property+in+an+orthotopic+bladder+urothelial+carcinoma+rat+model.">Preconditioning methods influence tumor property in an orthotopic bladder urothelial carcinoma rat model.</a> Mol Clin Oncol. 2, 65-70 (2014).</li><li>Horiguchi, Y., 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=Establishment+of+orthotopic+mouse+superficial+bladder+tumor+model+for+studies+on+intravesical+treatments.">Establishment of orthotopic mouse superficial bladder tumor model for studies on intravesical treatments.</a> Hum Cell. 21, 57-63 (2008).</li></ol>

Die Autoren erklären, dass sie keine konkurrierenden finanziellen Interessen.

Der Großteil der Fortschritte in der Krebstherapie erfordern Tests in Tiermodellen, bevor klinische Studien. Die Möglichkeit, das Studium der Tumorbiologie in vivo, unter Ausnutzung der Tier-Modelle, ist ein entscheidender Werkzeug für Forscher untersuchen Krebs Pathogenese, so dass die Bewertung der unterschiedlichen Therapieansätze. Orthotopen Modellen bleibt der Goldstandard 14-15, sowohl wegen der riesigen Menge an Zelllinien, die Einrichtung eines Tumor-Modell, und weil sie die Umgebung des ...

Blasenkrebs ist eine der häufigsten Krebserkrankungen des Urogenitaltrakts, mit fast 75.000 neue Fälle jedes Jahr in den USA 1. Hohe Rezidiv erfordern lebenslanges Follow-up, die Blasenkrebs eine der teuersten Krebsarten behandelbar machen. Der Goldstandard für die Behandlung hochgradigen NMIBC ist transurethralen Resektion, gefolgt von intravesikale Immuntherapie mit BCG. Obwohl der genaue Mechanismus der Antitumoraktivität von BCG, muss noch vollständig aufgeklärt werden, wird akzeptiert, dass die Aktivierung der zellvermittelten Immunantwort der T-Helfer (Th) 1 und zytotoxische T (Tc) 1-Zellen angereichert ist für Der Erfolg der Therapie 2. Trotz der Tatsache, dass BCG bleibt die Behandlung der Wahl für NMIBC ein hoher Anteil der Patienten nicht auf die Therapie zu antworten; Darüber hinaus kann sie eine Reihe von Nebenwirkungen haben: ca. 70% der behandelten Tumore nach einer gewissen Zeit wieder auftreten und ~ 15% Fortschritt auf die muskelinvasiven Form desKrankheit. Andere Nebenwirkungen, die mit BCG-Behandlung verbunden sind Dysurie, Zystitis und Nierenentzündung 3-6. Die Entwicklung neuer Therapiestrategien zu beachten die Verwendung von präklinischen Modellen zu nehmen, nach der ersten in-vitro-Bewertung; Dies ist besonders wichtig bei Tumoren, deren Mikromilieu deutlich ihre Entwicklung und Ansprechbarkeit auf die Behandlung beeinflussen. Im Laufe des letzten Jahrzehnts haben verschiedene Aspekte der Immunmodulationsaktivität von HP-NAP, einer durch das Bakterium Helicobacter pylori hergestellte Protein gerichtet, zunächst als fördern kann endothelialen Adhäsion von polymorphkernigen Zellen (PMNs) 7 identifiziert. Strukturell gehört HP-NAP an die DNA-Schutz-Protein unter Bedingungen ausgehungert (DPS) Familie 8 und besteht aus 12 in einer dodecameren Schale angeordneten identischen Untereinheiten. Wir haben gezeigt, dass HP-NAP isteinen Toll-like Rezeptor (TLR) 2-Agonisten mit einer starken immunmodulierende Aktivität zum Antreiben der Differenzierung von T-Lymphozyten verantwortlich Richtung des Th1-Phänotyp in vitro und in vivo 9-10. Im Rahmen dieser Tätigkeit ist HP-NAP in der Lage, die Th2-Immunantwort in die vorteil Th1-Antwort in einem Mausmodell von allergischem Asthma 11 umzuleiten. Um die Th1-abhängige Anti-Tumor-Potenzial von HP-NAP bewerten, nutzten wir einem Mausmodell von Blasenkrebs durch O&#39;Donnel und Kollegen 12 zur Bewertung der Auswirkungen der BCG Verwaltung vor einigen Jahren entwickelt. Bei diesem Protokoll, zeigen wir, dass HP-NAP hat eine starke Antitumor-Potential gegen Blasenkrebs und dass die Wirksamkeit von HP-NAP Verabreichung Parallelen der signifikante Akkumulation im Tumor und regionale Lymphknoten, sowohl Th1- und Tc1 Lymphozyten produzieren Interferon ( IFN) -γ 13 </sup&gt;. Tumore von HP-NAP behandelten Mäusen isoliert zeigte mehr Nekrose und weniger Vaskularisation als die unbehandelte Gegenstück. Der vorliegende Bericht liefert eine stepbystep Protokoll Detaillierung der Vorbereitung der Tiere, deren Harnröhrenkatheter, der chemischen Verbrennung für die Befestigung der Zellen an die Blasenschleimhaut erforderlich ist und die Injektion der Tumorzellen. Wir beschreiben auch die topische Verabreichung von HP-NAP, die als Prototyp einer für die Behandlung von Blasenkrebs entwickelt therapeutische Wirkstoffe betrachtet werden kann. Die durch den Vergleich Tumoren von Steuerung und HP-NAP behandelten Tieren isoliert erlangten Beweismittel betont nicht nur die Tatsache, dass HP-NAP könnte ein guter Kandidat für Blasenkrebs Immuntherapie, sondern auch die allgemeine Wirksamkeit der Versuchsanordnung ist.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>C57BL/6J female mice</td>
			<td>Harlan Italy (Udine, Italy)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MB49 Cells</td>
			<td></td>
			<td></td>
			<td>Obtained from Prof. O&#39;Donnel, University of Iowa Carver College of Medicine, Iowa, USA</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Sigma-Aldrich (St. Louis, Missouri, USA)</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D1408</td>
			<td>10X, to be diluted in apyrogenic water</td>
		</tr>
		<tr>
			<td>Flask</td>
			<td>Becton Dickinson (Franklin Lakes, New Jersey, USA)</td>
			<td>353135</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1ml</td>
			<td>Becton Dickinson</td>
			<td>301358</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Life Technologies (Waltham, Massachusetts, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Life Technologies</td>
			<td>15710-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Xilor</td>
			<td>Bio 98 s.r.l. (Milano, Italy)</td>
			<td></td>
			<td>2% Xylazin</td>
		</tr>
		<tr>
			<td>Zoletil</td>
			<td>Virbac (Carros, France)</td>
			<td>359713301992</td>
			<td>5% Zolazepam + 5% Tiletamine</td>
		</tr>
		<tr>
			<td>24G Catheter</td>
			<td>Terumo (Rome, Italy)</td>
			<td>SR+DM2419PX</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Carlo Erba Reagents (Milano, Italy)</td>
			<td>403871</td>
			<td>Liquid</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>JT Baker (Center Valley, Pennsylvania, USA)</td>
			<td>10095011</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Surgical Scalpel</td>
			<td>Albion Surgical Limited (Sheffield, England)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica Microsystem ( Wetzlar, Germany)</td>
			<td>RM2235</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>VWR International (Radnor, Pennsylvania, USA)</td>
			<td>631-0108</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Analyzer</td>
			<td>Zeiss (Jena, Germany)</td>
			<td>Cyres System</td>
			<td></td>
		</tr>
	</tbody>
</table>
Materials C57BL/6J female mice Harlan Italy (Udine, Italy) MB49 Cells Obtained from Prof. O&#39;Donnel, University of Iowa Carver College of Medicine, Iowa, USA RPMI Sigma-Aldrich (St. Louis, Missouri, USA) R8758 FBS Sigma-Aldrich F7524 PBS Sigma-Aldrich D1408 10X, to be diluted in apyrogenic water Flask Becton Dickinson (Franklin Lakes, New Jersey, USA) 353135 Syringe 1ml Becton Dickinson 301358 Trypsin Life Technologies (Waltham, Massachusetts, USA) Gentamycin Life Technologies 15710-049 Xilor Bio 98 s.r.l. (Milano, Italy) 2% Xylazin Zoletil Virbac (Carros, France) 359713301992 5% Zolazepam + 5% Tiletamine 24G Catheter Terumo (Rome, Italy) SR+DM2419PX HCl Carlo Erba Reagents (Milano, Italy) 403871 Liquid NaOH JT Baker (Center Valley, Pennsylvania, USA) 10095011 Powder Equipment Surgical Scalpel Albion Surgical Limited (Sheffield, England) Microtome Leica Microsystem ( Wetzlar, Germany) RM2235 Microscope Slides VWR International (Radnor, Pennsylvania, USA) 631-0108 Image Analyzer Zeiss (Jena, Germany) Cyres System

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.

Abbildung 1 zeigt die Kathetertechnik; die Maus katheterisiert und mit 0,5 x 10 6 MB49-Zellen eingeimpft. 3 Tage nach der Injektion von Tumorzellen wird eine Behandlung mit HP-NAP gestartet, um ihnen zu ermöglichen, an der Blasenwand zu befestigen und zu vermehren. Alle Tiere in der Kontrollgruppe gehörenden Entwicklung des Tumors; einige von ihnen vor dem Ende des 13-Tage-Zeitraum auf Grund der Verstopfung der Harnröhre sterben. Nach der ersten Injektion mit H...

Watch this Scientific Journal Video about 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 at JoVE.com

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 ...

evaluation-efficacy-h-pylori-protein-hp-nap-as-therapeutic-tool-for

Research

JoVE Journal

Medicine

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

7.2K Aufrufe.  University of Padua. 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.

Serie: 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

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 ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarker in einem Tiermodell für die Aufdeckung Neural, hämatologischen und Behavioral Korrelate der PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

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 ...

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 ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Exzitotoxische Stimulation der Hirn Microslices als<em&gt; In-vitro-</em&gt; Modell der Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

Präklinische Orthotopische Mausmodell der menschlichen Prostata-Krebs

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

Zweite harmonische Generation Signale in Kaninchen Sklera als Instrument für die Bewertung des therapeutischen Gewebes Vernetzung (TXL) für Kurzsichtigkeit

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...