Authors thank Jeffrey Sellers to assist orthotopic pancreatic cancer mouse modeling. This work was supported by Research Initiative Pilot Awards from the Department of Radiology at UAB and NIH grants 2P30CA013148 and P50CA101955.

Alle Verfahren wurden von der Institutional Animal Care und Verwenden Committee an der Universität von Alabama in Birmingham angenommen. 1. orthotopen Pankreastumormausling <ol><li> Kultur Standard menschlichen Pankreas-Krebszelllinien in Dulbeccos modifiziertem Eagle-Medium (DMEM) mit 10% fötalem Rinderserum. Behalten Sie alle Kulturen bei 37 ° C in feuchter Atmosphäre mit 5% CO 2. </li><li> Verwenden Sie 8-10 Wochen alte weibliche schwere kombinierte immundefizienten Mäusen. Platzieren Tierkäfigen bei 12 Stunden Licht und 12 Stunden Dunkelheit-Zyklus bei RT (21 ± 2 ° C) und 60% Luftfeuchtigkeit. </li><li> Anesthetize alle Tiere mit Belüftung mit 2% Isofluran mit Sauerstoff (2 l / min) im gesamten Operation gemischt. Bestätigen Sie die Narkosetiefe von toe Prise Reflex. Ort Tiere auf einem Heizkissen (37 ° C), um die Körpertemperatur aufrechtzuerhalten. Bewerben Veterinäraugensalbe auf die Augen bis zur Trockenheit während der Narkose zu verhindern. &amp;# 160; </li><li> Entfernen Sie die Haare in der linken oberen Quadranten des Bauches jeder Maus, und geben einen Analgetikum (carpofen, 5 mg / kg Körpergewicht subkutan) in der Umgebung. Tragen Sie eine Betadin-Lösung auf die freigelegte Haut. Bereiten autoklaviert chirurgische Instrumente. </li><li> Machen Sie eine 1 cm lange Inzision in der Haut und Bauchfell mit Iris gerade Schere. Entfernen Sie vorsichtig die Bauchspeicheldrüse aus dem Bauch mit chirurgischen Pinzette. </li><li> Einsatz 28 G Nadel einer 0,5 ml Insulinspritze in die Schwanzvene von Pankreas und dann langsam zu infundieren eine Lösung von 2,5 Millionen menschlichen Pankreaskarzinom-Zellen in 30 &amp; mgr; l DMEM. Bestätigen Sie, dass eine kleine Luftblase ist in der Spitze der Bauchspeicheldrüse durch die Lösung erstellt. </li><li> Sanft legen Sie die Bauchspeicheldrüse zurück in die Bauchhöhle mit chirurgischen Pinzette. Schließen Sie das Bauchfell und die Haut in 1 Schicht mit 2 unterbrochen 5-0 Prolene Nahtmaterial, und dann beenden Sie die Anästhesie. Sie ein Tier, das der Operation an die Firma von anderen Tieren unterzogen wurde, bis vollständig erholt nicht zurück.60; Nähte entfernen in 7 ~ 10 Tage nach der Operation. </li><li> Geben Sie eine weitere Dosis des Analgetikums (carpofen, 5 mg / kg Körpergewicht subkutan) bei 24 Stunden nach der Operation. </li><li> Überprüfen Tumorgröße durch Abtasten der OP-Bereich mit zwei Fingern. Tumoren in der Regel das Gefühl, dichter und holpriger als die umgebenden Gewebe und Organe. Normalerweise dauert es ca. 1 - 2 Wochen, um zu starten Gefühl ein Tumor. </li><li> Überwachen Tiere täglich auf Anzeichen von Krankheit. Wenn die Tiere krank erscheinen (fehlende normale Pflegen und Vermeidungsverhalten), beendet man sie mit Genickbruch während der Narkose. </li></ol> 2. Magnetic Resonance Imaging <ol><li> Anwenden MRI, wenn die Tumorgröße etwa 5-7 mm Durchmesser in der Regel 2 bis 4 Wochen nach der Zellimplantation. Verwenden Sie ein MR-Scanner, um die Kleintierbildgebung oder einem klinischen MR-Scanner mit einem spezialisierten Spule für die Kleintierbildgebung ausgestattet gewidmet. HINWEIS: Wir haben ein 9,4-T-MR Kleintierscanner mit einer Kombination aus einem 1 H-Volumen-Resonator/ Sender und eine Oberflächenspule Empfänger (30 mm im Durchmesser) (Bruker BioSpin Corp., Billerica, MA). Oberflächenspule eine bessere Signal-Rausch-Verhältnis (SNR) 14. </li><li> Vorbereiten einer gadoliniumhaltigen MRI-Kontrastmittel zu ~ 0,1 injizieren - 0,2 mmol / kg an jedes Tier in ~ 0.1 - 0.2 ml PBS (phosphatgepufferte Salzlösung). HINWEIS: Es wird verwendet, Gadoteridol und injiziert 0,2 mmol / kg in 0,15 ml PBS über einen Zeitraum von 15 sec (0,1 ml / sec). </li><li> Bereiten Sie eine Mikro-Polyethylen Rohr (Länge: 7,62 mm, Innendurchmesser: 0,28 mm, Außendurchmesser: 0,64 mm). Legen Sie eine 30 G-Nadel (12,7 mm Länge) in ein Ende des Rohres und einer 30 G stumpfe Spitze Nadel (9,5 mm Länge) in das andere Ende. Schließen Sie eine 1 ml Spritze mit MR-Kontrastmittels in die stumpfe Spitze Nadel und drücken Sie langsam die Spritze zu füllen Sie das gesamte Rohr mit dem MR-Kontrastmittels. </li><li> Anesthetize Tieren unter Belüftung mit ~ 1-2% von Isofluran mit Sauerstoff (2 l / min) gemischt gesamten Zubereitung und Bildgebung. Bestätigen Sie die Tiefe der anesthesia von toe Prise Reflex. Bewerben Veterinäraugensalbe auf die Augen bis zur Trockenheit während der Narkose zu verhindern. Erweitern sich die Schwanzvene mit einer Wärmelampe vor Nadeleinführung. Besorgen Sie sich die Mitte des 30 G-Nadel mit Kelly Zangen, und setzen Sie sie in die Schwanzvene. Band sowohl der Schwanz und Rohr auf ein Stück Kunststoff oder Karton Papier (10 mm Breite x 100 mm Länge), um den Schwanz gerade zu halten. </li><li> Legen Sie das Tier in Rückenlage in einem Tierbett mit zirkulierenden Warmwasser (oder warme Luft), die Körpertemperatur während der Bildgebung zu regulieren ausgestattet. Stellen Sie die Temperatur über dem Bett, um 37 ° C. Legen Sie eine rektale Temperatursonde, die Körpertemperatur während der Bildgebung zu überwachen. </li><li> Tragen Sie eine orthogonal gebogenen Kunststoffplatte in den Bauchbereich. Stellen Sie sicher, der Tumor hinter dem oberen Ende der Platine befindet, und ziehen Sie dann etwas nach unten das Board (~ 2 mm), um den Tumor zu gewährleisten wird vom Vorstand eingeholt. Kleben Sie die Platine zu Tierbett fest. </li><li> Kleben Sie einen Atem Pad Transducer (SA Instrument, Inc., Stony Brook, NY) im Brustbereich zu Tier Atmung während der Bildgebung zu überwachen. Legen Sie eine Oberflächenspule auf der Oberseite der Tumorregion, und kleben Sie sie auf Tierbett fest. Schieben des Tieres Bett in den MR-Scanner, um den Tumorbereich in der Mitte der Volumenspule platzieren (Innendurchmesser: 72 mm). </li><li> Führen Anpassung und Optimierung sowohl für die Sender und Empfänger durch Shimming gefolgt. </li><li> Beginnen Sie mit einer anatomischen MR-Sequenz, um den Tumor zu lokalisieren. Verwenden Sie einen T2-gewichteten (T2W) Turbo-Spin-Echo-Sequenz, um die axiale Bilder mit folgenden Erfassungsparameter zu erhalten. Wiederholungszeit (TR) / Echozeit (TE) = 3000/34 ms, 128 x 128-Matrix, 30 x 30 mm Sichtfeld, Anzahl der Mitte = 1, Echozuglänge = 4 und 20 zusammenhängende 1 mm dicke Scheiben in ein Interlaced-Modus, um die gesamte Tumorregion (1,6 min Gesamtscanzeit) zu decken. Hinweis: Da orthotopischen Tumoren der Bauchspeicheldrüse sind schwieriger als die subkutane diejenigen befinden, herkömmlichen localizer Bilder mit niedriger Auflösung kann nicht sinnvoll sein. </li><li> Erwerben Sie T1-gewichteten (T1W) Bilder mit verschiedenen Kippwinkel bis T1 Karte abzurufen. Zu diesem Zweck verwenden Sie ein Gradientenecho MultiFlip Winkel Ansatz mit den folgenden Parametern: Wiederholungszeit (TR) / Echozeit (TE) = 115/3 ms, 128 x 128-Matrix, eine 30 x 30 mm Sichtfeld, Anzahl der Mittelwerte = 4 ~ 5-7 zusammenhängenden 1 mm dicke Scheiben in einem Zeilensprung-Modus, um den Tumorbereich zu bedecken, und sieben Flipwinkel von 10, 20, 30, 40, 50, 60 und 70 (Gesamtscanzeit pro Flipwinkel: 1 min). HINWEIS: Es ist jedoch MultiFlip Winkel Ansatz effizient nur, wenn B1 Feldhomogenität ist hoch. Wenn nicht, können T1 Karten mit mehreren TR-Ansatz statt 15 erreicht werden. </li><li> Erwerben T1W Bilder vor und nach Gadolinium basiert MR Kontrastmittelinjektion. Verwenden Sie die gleichen Aufnahmeparameter und Geometrie für T1-Mapping, aber mit dem festen Kippwinkel von 30. Verwendung linearer Codierung stabilen Zustand zu gewährleisten, wenn das Zentrum des k-Raums erhalten wird, especially wenn eine kurze TR und ein niedriger Grad Kippwinkel verwendet. Erwerben 5 Basis-Images vor Kontrastmittelinjektion. So erwerben 40 Bilder nach Kontrastmittelinjektion (gesamte Scandauer: 45 min). Verwenden einer Spritzenpumpe Kontrastmittel mit einer konstanten Geschwindigkeit (0,01 ml / sec) zu injizieren. </li><li> Überwachen Tier Atmung kontinuierlich und passen Isofluran-Konzentration, um die Atemfrequenz auf 50 zu halten - 100 Atemzüge pro min. Überwachen Sie Tierkörpertemperatur im gesamten Imaging. </li><li> Nach Abschluss der DCE-MRI, nehmen Sie die Nadel und anderen Sonden, und legen Sie das Tier in einem leeren Käfig mit Papiertüchern gebettet. Massieren Sie sanft den unteren Bauchbereich. Der Käfig sollte die Hälfte unter einer Wärmelampe gelegt werden, dass das Tier in die und aus der Wärmegefälle zu bewegen, wie sie sich erholt. Sie ein Tier nicht unbeaufsichtigt lassen, bis er wieder zu sich kam, um ausreichende Brustlage zu halten. </li></ol> 3. Bildverarbeitung und -analyse <ol><li> Segment Tumor regi<html> on in T2W Bilder. In T2W Bildern ist die Signalintensität im Tumorbereich heller als die der umgebenden Gewebe, so dass die Tumorgrenze kann manuell abgegrenzt werden. HINWEIS: halbautomatische Segmentierungstechniken wie globale Schwellen oder aktive Konturierung verwendet werden 16,17, aber ungleichmäßige Hintergrundintensität korrigiert werden soll vor allem, wenn eine Oberflächenspule verwendet wird. </li><li> Neues T1 und Protonendichtekarten. In T1W Bilder mit einer Gradientenecho-Sequenz erfasst, vorausgesetzt, dass die Echozeit (TE) ist viel geringer als T2 * -Wert, der Pixelwert bestimmt wird durch <img alt="Gleichung 1" fo:content-width="3in" src="/files/ftp_upload/52641/52641eq1.jpg" /> wobei S 0 ist Protonendichte, T 1 T 1 Relaxationszeitkonstante, TR ist Wiederholzeit und θ ein Kippwinkel. Gleichung (1) kann umgeschrieben werden 641eq2.jpg &quot;/&gt; wenn S (θ) / sin &amp; theta; wird mit Y ersetzt ist, und S (θ) / tan &amp; theta; wird mit X ersetzt. Gleichung (2) ist eine lineare Gleichung, ihre Steigung und kann zum Abrufen von T1 und S &amp; sub0; -Werte betrugen. </li><li> Berechnen MR-Kontrastkonzentration im DCE-MR-Bilder. Wenn Gadolinium basierten MR-Kontrastmittel injiziert wird T 1 Relaxationszeitkonstante Laufe der Zeit verändert. So kann die Gleichung (1) neu geschrieben werden <img alt="Gleichung 3" fo:content-width="3in" src="/files/ftp_upload/52641/52641eq3.jpg" /> T 1 (t) ist mit MR-Kontrastkonzentration, C (t) bezogen, wie folgt, <img alt="Gleichung 4" fo:content-width="3in" src="/files/ftp_upload/52641/52641eq4.jpg" /> wobei r 1 longitudinalen Relaxivität von MR-Kontrastmittel. So kann durch Kombinieren der Gleichungen (3) und (4) wird MR Kontrastmittelkonzentration bestimmt <img alt = &quot;Gleichung 5&quot; fo: Content-width = &quot;3in&quot; src = &quot;/ files / ftp_upload / 52.641 / 52641eq5.jpg&quot; /&gt; </li><li> Quantifizieren der pharmakokinetischen Parameter der MR-Kontrastmittel. C p (t) stellt MR Kontrastmittelkonzentration im Blutplasma zur Zeit t nach Beginn Kontrastinjektion. C p (t) wird als arteriellen Inputfunktion (AIF). Wenn AIF zur Verfügung steht, können die pharmakokinetischen Parameter von MR-Kontrastmittels berechnet werden <img alt="Gleichung 6" fo:content-width="4in" src="/files/ftp_upload/52641/52641eq6.jpg" /> wobei C t (t) MR Kontrastmittelkonzentration in einem Zielgewebe, v p fraktionierten Blutplasmavolumen ist, v e fraktionierte extravasalen Extrazellulärvolumens und K trans Volumenübertragungskonstante. Flux Geschwindigkeitskonstante k ep, gleich K tran ists dividiert durch v e. Wenn AIF nicht verfügbar ist, dann wird der Referenzregion Modell kann statt 18,19 verwendet werden. Der Referenzbereich Modell basiert auf der strömungs beschränkt Kety Modell 20 und basieren nutzt Kontrastmittelkonzentration in einem Referenzbereich, um den Bedarf an AIF wie folgt zu entfernen, <img alt="Gleichung 7" fo:content-width="5in" src="/files/ftp_upload/52641/52641eq7.jpg" /> wobei C t, ROI (t), K trans, ROI und v e, sind ROI Kontrastmittelkonzentration, Volumenübertragungskonstante und fraktionierte extravaskulären und extrazellulärer Volumen, bzw. in der Region von Interesse (ROI), wobei C t, RR (t), K trans, RR und v e, RR sind die in der Referenzregion. Paravertebralen Muskeln wird oft als der Referenzbereich ausgewählt ist, und v e, RR in murine paravertebral Muskel wird angenommen bei 0,08 21 konstant zu sein. Wir nutzten die Referenzregion Modell. </li></ol>

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Authors do not have any conflicts of interest to declare.

Wir haben die detaillierten Methoden der orthotopen Pankreastumor Modellierung mit immundefizienten Mäusen, DCE-MRI von Bauch Tumoren in Mäusen, und die Quantifizierung ihrer kinetischen Parameter eingeführt. Im orthotopen Pankreastumor-Modellierung, müssen Sie beim Einsetzen einer Nadel in den Schwanz der Bauchspeicheldrüse entnommen werden. Wenn dies gelingt, werden Zellen auf den Kopf der Bauchspeicheldrüse Schaffung einer kleinen Blase übertragen werden. Bei der Anwendung eines orthogonal gebogenen Kunststoff...

Das übergeordnete Ziel dieser Methode ist es, dynamische kontrastverstärkten Magnetresonanztomographie (DCE-MRI) für orthotopen Pankreastumor-Xenografts bei Mäusen anzuwenden. DCE-MRI ist eine nicht-invasive Methode zur Mikrovaskulatur in einem Zielgewebe durch Überwachung der Änderung des MR Kontrast über einen bestimmten Zeitraum nach der Injektion beurteilt. DCE-MRI wurde verwendet, um bösartige Tumore zu diagnostizieren und Tumor-Antwort auf verschiedene Therapien 1-4 zu beurteilen. Quantitative DCE-MRI hat hohe Reproduzierbarkeit 5 vorgestellt. Pharmakokinetischen Parameter eines MR-Kontrastmittels in ein Zielgewebe quantifizieren, müssen alle zu unterschiedlichen Zeitpunkten und T1 Karte vor Kontrastmittelinjektion erhalten erworbenen DCE-MR-Bilder koregistriert 6 werden. Aufgrund der Atemwege und peristaltische Bewegungen im Bauchbereich, quantitative DCE-MRI hat begrenzte Anwendung für Magen-Darm-Tumoren. Wurden orthotopen Pankreastumormodelle verwendet werden, um zu bewertenPankreas-Tumor-Antwort folgenden biologischen Therapien und Chemotherapien 7,8. Orthotope Tumormodelle werden als überlegen gegenüber herkömmlichen subkutanen Modellen, da die Mikroumgebung in der ursprünglichen Tumorstelle reflektiert wird und dadurch die menschliche Tumor-Antwort auf die Therapie genauer vorhergesagt werden. Jedoch ist die Maus Bauchspeicheldrüse im linken oberen Quadranten des Abdomens angeordnet ist, so quantitative DCE-MRI des orthotopischen Pankreastumor-Xenotransplantaten in Mäusen nicht leicht implementiert. Wir haben ein Protokoll der DCE-MRI von Bauch Tumoren in Mäusen durch die Festsetzung der Tumoren mit einer orthogonal gebogenen Kunststoffplatte, um Bewegungsübertragung von der Brustbereich 9 zu verhindern etabliert. Die von diesem Platte aufgebracht Druck auf den Bauchbereich lokalisiert und nicht in Atemnot führte. Eine automatisierte Bild Koregistrierung Technik für DCE-MRI der Bauchorgane in einem Freiatemmodus validiert, aber es führt effectively nur dann, wenn die Zielregionen bewegen sich langsam und regelmäßig 10. Die Atemfrequenz von Tieren ist während der Bildvariablen werden so physische Zurückhaltung im Bauchbereich erforderlich sein, zuverlässig pharmakokinetischen Parameter in orthotopen Pankreastumormausmodelle abzurufen. Wir haben erfolgreich quantifiziert die pharmakokinetischen Parameter von einem MR-Kontrastmittel in orthotopen Pankreastumor-Xenotransplantaten mit dem orthogonal gebogenen Kunststoffplatte in DCE-MRI 11-13. Hier präsentieren wir die Einzelheiten des Verfahrens der orthotopen Pankreastumor-Modellierung, DCE-MRI der Tumorfremdtransplantaten in Mäusen, und die Quantifizierung der pharmakokinetischen Parameter.

<table border="0" cellpadding="0" cellspacing="0" width="1275">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Name of Material/ Equipment</td>
			<td style="width:199px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:712px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Invitrogen</td>
			<td>11965-118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td>Harlan Laboratories</td>
			<td>BT-9501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine</td>
			<td style="width:199px;">Purdue products</td>
			<td style="width:119px;">67618-153-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-0 Prolene sutures</td>
			<td style="width:199px;">Ethicon</td>
			<td style="width:119px;">8720H</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">9.4T MR scanner</td>
			<td>Bruker Biospin Corporation</td>
			<td style="width:119px;">BioSpec 94/20 USR</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Gadoteridol</td>
			<td>Bracco Diagnostics Inc</td>
			<td style="width:119px;">NDC 0270-1111-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-polyethelene tube</td>
			<td style="width:199px;">Strategic Applications, Inc</td>
			<td style="width:119px;">#PE-10-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30G blunt tip needle</td>
			<td style="width:199px;">Strategic Applications, Inc</td>
			<td style="width:119px;">89134-194</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monitoring and gating system</td>
			<td style="width:199px;">SA instruments, Inc</td>
			<td style="width:119px;">Model 1030</td>
			<td>This is an MR compatiable system to measure resiratory rating and body temperature of small animals at the same time.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe pump</td>
			<td>New Era Pump Systems, Inc.</td>
			<td>NE-1600</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Menschliche Pankreastumorzellen erfolgreich wachsen in Mauspankreas Schaffung eines soliden Tumors. 1 zeigt Fotografien von (a) einem normalen Pankreas wo Tumorzelllösung eingespritzt wird, und (B) ein Vertreter der Maus trägt eine orthotope Pankreas Tumorxenotransplantates (MIA PaCa-2 ). Tumor im linken oberen Quadranten des Abdomens, neben der Milz. Es dauert normalerweise 2-4 Wochen, die Tumoren zu 5 aufwachsen - 7 mm Durchmesser nach Zellimplantation. Bewegung orthotop...

Watch this Scientific Journal Video about Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model at JoVE.com

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

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.

Dynamic Contrast Verbesserte Spintomographie von einem orthotopen Mausmodell Bauchspeicheldrüsenkrebs

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

dynamic-contrast-enhanced-magnetic-resonance-imaging-an-orthotopic

Research

JoVE Journal

Medicine

15.0K Aufrufe.  University of Alabama at Birmingham. 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. 

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

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 ...

Forschung

Medizin

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multimodale Bildgebung von Angiogenese in einem Nude Rattenmodell der Brustkrebs Knochenmetastasen mittels Magnetresonanztomographie, volumetrische Computertomographie und Ultraschall

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

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 Mouse Model of Anaplastic Thyroid Carcinoma

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

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

Ein orthotopen Mausmodell des Anaplastic Schilddrüsenkarzinom

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

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

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

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

Ein orthotopen Blasenkrebs-Modell für Gene Delivery Studies

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

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

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 ...

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  ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Ein Orthotopische Glioblastoma Maus-Modell Pflege Gehirn Parenchymale Körperliche Einschränkungen und geeignet für Zwei-Photonen-Intravitalmikroskopie

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Tracking the Mammary Architectural Features and Detecting Breast Cancer with Magnetic Resonance Diffusion Tensor Imaging

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of life and survival of breast cancer patients. In this paper a new approach for the detection of breast cancer is described, based on tracking the mammary architectural elements using diffusion tensor imaging (DTI). 
The paper focuses on the scanning protocols and image processing algorithms and software that were designed to fit the diffusion properties of the mammary fibroglandular tissue and its changes during malignant transformation. The final output yields pixel by pixel vector maps that track the architecture of the entire mammary ductal glandular trees and parametric maps of the diffusion tensor coefficients and anisotropy indices. 
The efficiency of the method to detect breast cancer was tested by scanning women volunteers including 68 patients with breast cancer confirmed by histopathology findings. Regions with cancer cells exhibited a marked reduction in the diffusion coefficients and in the maximal anisotropy index as compared to the normal breast tissue, providing an intrinsic contrast for delineating the boundaries of malignant growth. Overall, the sensitivity of the DTI parameters to detect breast cancer was found to be high, particularly in dense breasts, and comparable to the current standard breast MRI method that requires injection of a contrast agent. Thus, this method offers a completely non-invasive, safe and sensitive tool for breast cancer detection.

We describe how to obtain parametric and vector maps of the diffusion tensor of the breast using magnetic resonance imaging. The protocol and final output following imaging processing are tailored for tracking breast architectural features and detecting breast malignancy.

Tracking der Brust architektonische Details und Erkennen von Brustkrebs mit Magnet-Resonanz-Diffusion Tensor Imaging

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Gleichzeitige PET / MRT-Bildgebung während Maus zerebrale Hypoxie-Ischämie

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...