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>

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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><tbody><tr><td colspan="4">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&#039;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 colspan="4">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>

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 ...

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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

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

Ein Murine Orthotopische Blasentumormodell und Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

Forschung

Krebsforschung

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

Maus Blasenwand Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

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 ...

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 ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

Induktion von Invasive Transitional Handy Blasenkarzinom in Immune intakte menschliche MUC1 transgenen Mäusen: Ein Modell für Immuntherapie Entwicklung

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

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 ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established.
In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin.
This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

A high yield method for one-step negative purification of recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) overexpressed in Escherichia coli by using diethylaminoethyl resins in batch mode is described. HP-NAP purified by this method is beneficial for the development of vaccines, drugs, or diagnostics for H. pylori-associated diseases.

Ein-Schritt-Negative chromatographischer Reinigung von<em&gt; Helicobacter pylori</em&gt; Neutrophil-aktivierendes Protein überexprimiert in<em&gt; Escherichia coli</em&gt; Im Batch-Modus

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

Immunologie und Infektion

Establishing a Murine Superficial Bladder Cancer Model via an Intravesical Cell Administration Technique

This study presents an innovative method for establishing an orthotopic murine bladder tumor model with high efficiency and precise tumor localization. After anesthetizing female C57BL/6J mice in a supine position, a 24 G intravenous needle is inserted into the bladder to evacuate its contents. A 34 G dispensing needle is then introduced through the catheter, rotated five times to create a focal injury to the bladder dome mucosa, and subsequently removed. The MB49 cell suspension is aspirated and connected to a 30 G dispensing needle, which is inserted into the bladder via the catheter. Tumor cells are injected submucosally into the bladder under pressure. This technique results in minimal trauma to the mice, a high tumor take rate, and a fixed tumor location. It is characterized by simplicity and excellent reproducibility. This model provides an ideal experimental platform for developing intravesical therapies for bladder cancer, facilitating the advancement and optimization of treatment strategies for this malignancy.

This protocol describes a unique method for constructing an orthotopic model of superficial bladder cancer.

Etablierung eines Modells für oberflächliches Blasenkarzinom der Maus mittels intravesikaler Zellverabreichungstechnik

This study presents an innovative method for establishing an orthotopic murine bladder tumor model with high efficiency and precise tumor localization. ...

Evaluating the Anti-Tumor Efficacy of a Bacterial Immunomodulatory Protein in a Murine Model

Source: Codolo, G. et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/52743">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.</a>&#160;J. Vis. Exp. (2015)
This video demonstrates a technique to assess the anti-tumor activity of Helicobacter pylori neutrophil-activating protein, or HP-NAP, in a mouse bladder cancer model. HP-NAP interacts with tumor-cell neutrophils to produce pro-inflammatory cytokines, inducing the differentiation of na&#239;ve T cells into type 1 T helper (Th1) and type 1 cytotoxic (Tc1) T cell phenotypes. The Th1 and Tc1 cells secrete interferon-gamma, inhibiting tumor angiogenesis and promoting cytotoxic T cell-mediated tumor destruction, reducing tumor volume.

Bewertung der Anti-Tumor-Wirksamkeit eines bakteriellen immunmodulatorischen Proteins in einem Mausmodell

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Bewertung der Wirksamkeit der

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