Diese Arbeit wird teilweise von der American Heart Association GRA Frühling 09 Postdoctoral Fellowship (09POST2250268) und dem NIH R01 CA097465 unterstützt. Wir schätzen Dr. Wen Li und Dr. Vikas Gulani für MRI-Protokoll Test-und Setup, und Frau Yvonne Parker für ihre Unterstützung bei der Tumorimplantation.

Protokoll angepasst aus einer früheren Studie 11. 1. Konjugation von Gd-DOTA, um CLT1 Peptide <ol><li> Verwendung von Standard-Festphasenpeptidsynthese synthetisiert CLT1 Peptid (CGLIIQKNEC) von Fmoc-geschützten Aminosäuren an einer 2-Chlortritylchloridharz (1,0 mmol). </li><li> Nach Zugabe von endständigen Aminosäure, cyclisieren des linearen Peptids auf dem Harz mit Thallium (III) trifluoracetat (1,09 g, 2,0 mmol, 2 Äquivalente) in DMF (20 ml) bei 0 ° C für 2 Stunden. </li><li> Weiter, PEG-Konjugat und Lysin sequentiell an den N-Terminus des Peptids CLT1 (1,0 mmol) auf, das durch Umsetzung sequentiell mit Fmoc-NH-PEG-COOH (3,0 mmol) Fmoc-Lys (Fmoc)-OH (3,0 mmol), und eine zweite Charge von Fmoc-Lys (Fmoc)-OH (6 mmol). </li><li> Entfernen Fmoc mit Piperidin. Hinzufügen DOTA-Tris-(t-Bu) (4,58 g, 8,0 mmol, 2 Äquivalente zu jeder Aminogruppe), mit den vier freien Amine der Lysin Dendrimer auf das Harz für 2 Stunden reagieren. </li><li> Schließlich spalten und Entschützen das Produktvon dem Harz durch Behandlung mit einer Mischung aus Trifluoressigsäure, Wasser und Triisobutylsilan (TIS) (20 ml, 95/2.5/2.5) für 8 h bei RT. Entfernen des Harzes durch Filtration, gefolgt von Waschen mit Trifluoressigsäure (TFA). Fügen die vereinigten Filtrate tropfenweise zu kaltem Ethylether (200 ml), Zentrifuge, und Waschen mit Ethylether 4x. Trocknen unter Vakuum zu einem farblosen Feststoff (1,99 g, 61% Ausbeute). </li><li> Das Rohprodukt wird mit präparativer HPLC unter Verwendung eines Gradienten von 0-40% Lösungsmittel B (0,1% TFA in Acetonitril) in Lösungsmittel A (0,1% wässriger TFA-Lösung) für 20 min und 40-90% Lösungsmittel B in Lösungsmittel A für 10 min . </li><li> Auflösen CLT1-DL-(DOTA) 4 (250 mg, 0,077 mmol) in DI-Wasser (15 ml) und auf pH 6 unter Verwendung von 1 M NaOH. Hinzufügen Gd (OAc) 3 · 4H 2 O (472 mg, 0,462 mmol, 1,5 Äquivalente DOTA Monoamid) in Portionen zu der Lösung, während der pH bei 6 unter Verwendung von 1 M NaOH. Das Reaktionslösung bei Raumtemperatur für 48 Stunden. </li><li> Komplexe der Rest Gd (III) mit Ethylenediaminetetraacetic (EDTA) (90 mg, 0,31 mmol), und das rohe Produkt mit einer Größenausschlusssäule, um das Endprodukt zu erhalten CLT1-DL-(Gd-DOTA) 4 (169 mg, 57%). </li></ol> 2. Charakterisierung von Kontrastmittel <ol><li> Messen Gd (III)-Gehalt mit induktiv gekoppelten Plasma-optische Emissionsspektroskopie. </li><li> Erwerben Matrix-unterstützte Laserdesorption / Ionisation Time-of-Flight (MALDI-TOF)-Massenspektren im linearen Modus mit 2,5-Dihydroxybenzoesäure (2,5-DHB) als Matrix. </li><li> Messen Relaxationszeiten der wässrigen Lösung der Kontrastmittel von CLT1-DL-(Gd-DOTA) 4 und einem Steuermittel nontargeted sCLT1-DL-(Gd-DOTA) 4 mit verschiedenen Konzentrationen bei 60 MHz (1,5 T) mit einem Relaxometer bei 37 ° C. Verwenden Sie eine Inversion-Recovery-Pulssequenz zu T 1 und eine Carr-Purcell-Meiboom-Gill-Sequenz mit 500 Echos messen, um T 2 zu messen. Berechnen T 1 und T 2-Relaxivität des thE Mittel von den Pisten der Grundstücke von 1 / T 1 und 1 / T 2 im Vergleich zu den Gd-Konzentrationen. </li></ol> 3. Zellkultur und Entwicklung von Tiermodell mit Orthotopische PC3 Prostata-Tumor <ol><li> Kultur PC3-Prostatakrebszellen mit konstitutiver Expression des grün fluoreszierenden Proteins (GFP) in RPMI-Medium mit 5% fötalem Rinderserum und Penicillin / Streptomycin / Fungizon Ernte durch Trypsinierung und resuspendieren supplementiert Dichte von 2,5 x 10 4 Zellen / &amp;mgr; l PBS. </li><li> Pflegen NIH männlichen Nacktmaus (4-5 Wochen alt) an der Athymische Tier Core Facility, Case Western Reserve University (CWRU) nach einem von der CWRU Institutional Animal Care und Verwenden Ausschuss genehmigt Tier-Protokoll. </li><li> Die chirurgischen Oberflächen wurden mit 2% Chlorhexidin-Lösung besprüht und dann mit saugenden Tüchern abgedeckt. Sterile OP-Handschuhe wurden während des Verfahrens tragen. Vor der Tierchirurgie, ip injizieren Avertin (250 mg / kg) zu anesthetize die Mäuse. Alternativ kann Ketamin / Xylazin / Acepromazin in einer Konzentration von 50/5/1 mg / kg verwendet werden. Augensalbe wurde in der Maus aufgetragen, um während der Narkose ausreichende Feuchtigkeit beizubehalten. </li><li> Startet die Operation, indem ein kleiner Einschnitt durch die Haut und Bauchfell entlang der unteren Mittellinie auf eine Länge von etwa 1 cm mit einem sterilen Skalpell. Eine sterile Operationstuch wurde um das Überleben während der Einschnittstelle Operationen abgedeckt. </li><li> Sanft exteriorisieren und stabilisieren die Prostata dorsalen Lappen, um die Prostata aus. Die Suspension wird von PC3-GFP-Zellen in PBS (20 ul) in die Prostata mit einer 30-Gauge-Nadel. Schließlich schließen Sie den Schnitt mit Wund Pusher 12. </li><li> Für post-Verfahren Überwachung, Überwachung Tiere zweimal pro Tag für 1 Woche. Zeichen der Krankheit gehören der Mangel an Essen, dunkler Urin Sammel um Harnröhre, und unsicherer Gang, die Infektion oder Verschluss der Blase in den Klammern geben könnte. </li><li> Nach 10 Tagen entfernen ter Heftklammern mit einer Heftklammer-Entferner. Überwachen Sie die Tiere täglich zu Tumorgröße und keine Anzeichen von Schmerzen wie Gewichtsverlust, Verhaltensabweichungen und Motor Mängel zu beurteilen. Euthanize die Tiere, die eine Tumorgröße über dem zulässigen Grenzwert ethisch oder Nachweis von Schmerzen beim Tumorwachstum zeigten. </li></ol> 4. Bestätigung der Tumorbindungsspezifität der Peptide mit Fluoreszenz-Imaging und Histologie <ol><li> Nach Inokulation der Tumorzellen ermöglichen ca. 4 Wochen nach Tumorwachstum vor Beginn der Bildgebung. Vor der Injektion der Peptid-Sonden erfassen GFP-Fluoreszenzbilder mit lebenden Mäusen auf einem Fluoreszenz-Imager, um die Gegenwart eines Tumors zu verifizieren. </li><li> Iv injizieren Texas Red markierten Peptide in den Tumor-tragenden Mäusen in einer Dosis von 10 nmol / Maus. </li><li> Opfere die Mäuse zwei Stunden später durch Genickbruch, sammeln den Tumor und wichtigsten Organe, und sofort Bild auf einem Fluoreszenz-Imager. Verwenden Sie grünes Licht Filter für GFP (Anregung: 444-490 nm, Emission: 515 nm Langpassfilter; Erwerb Einstellungen: 500 bis 720 in 10 nm-Schritten) und Rotlichtfilter für Texas Red (Anregung: 576-621 nm; Emission: 635 nm Langpassfilter; Erwerb Einstellungen: 630 bis 800 in 10 nm-Schritten). 10 ms Belichtungszeit für GFP und 150 ms für Texas-Rot. </li><li> Sofort nach der Messung von ganzen Gewebefluoreszenz, sammeln Tumorgewebe, fix mit Formalin und Kryoschnitt in 5 um Scheiben schneiden. </li><li> Nach dem Spülen mit PBS, montieren mit 1 Tropfen Eindeckmedium mit 4 &#39;,6-Diamino-2-phenylindol (DAPI), Bild sofort auf einem konfokalen Laser-Scanning-Mikroskop. </li></ol> 5. Magnetic Resonance Imaging (MRI) <ol><li> Etwa 4 Wochen nach Inokulation der Tumorzellen ermöglichen die Tumoren auf 0,3 bis 0,6 cm im Durchmesser wachsen. Verwenden Sie eine 7 T-MRT mit einem Volumen Radiofrequenz (RF)-Spule für die MRT-Studie. </li><li> Anesthetize die Maus mit einer 2% Isofluran-Sauerstoff-Gemisch in einer Isofluran Induktionskammer. Ophthalmic Salbe wurde auf die Maus aufgetragen, um während der Narkose ausreichend Feuchtigkeit zu halten. </li><li> Legen Sie eine 30-Gauge-Nadel in einem 1,0 m langen Schlauch mit Heparin-Kochsalzlösung gefüllt. Legen Sie die Self-made-Katheter in der Maus Schwanzvene. </li><li> Platzieren Sie die Maus in den Magneten und unter Inhalationsnarkose mit Isofluran 1,5%-Sauerstoff-halten über eine Nasenkegel. </li><li> Legen einen Atmungssensor mit einem Überwachungssystem auf dem Bauch, um Frequenz und Tiefe der Atmung überwachen. Aufrechterhaltung der Körpertemperatur bei 37 ° C durch Einblasen von Warmluft in den Magneten durch ein Rückkopplungssteuersystem. </li><li> Beginnen Sie mit Sagittalschnitt Bilder mit einem Lokalisierungssequenz, um die Tumorlokalisation (TR / TE = 200/3.7 ms, FOV = identifizieren 3,0 cm, Schichtdicke = 2,2 mm, Slice-Nummer = 16, Durchschnitts = 2, Flip-Winkel = 45 °, Matrix = 128 × 128). </li><li> Verwenden Sie ein 2D-T1-Gradienten Fettunterdrückung Sequenz 2D Axialbilder für die CE-MRI (TR / te = 151.2/1.9 ms FOV = 3,0 x 3,0 cm cm, s erwerbenLäuse Dicke = 1,2 mm, Slice-Nummer = 12, Durchschnitts = 1, Flip-Winkel = 80 °, Matrix = 128 x 128). </li><li> Nach Voreinspritzung Grundbild MR-Aufnahme, beginnen die zielorientierte Agens oder Kontrollmittel in einer Dosis von 0,03 mMol Gd / kg injiziert durch Spülen mit 200 ul Kochsalzlösung. </li><li> Weiterhin CE-MRI-Bilder zu verschiedenen Zeitpunkten bis zu 30 Minuten erhalten. </li></ol> 6. Bildverarbeitung und-analyse <ol><li> Verwenden Imaging-Software für die Bildanalyse. </li><li> Zeichnen Sie die Regionen von Interesse (ROI) über den gesamten Tumor und die Nieren in der zweidimensionalen Bildebene und messen durchschnittliche Signalintensität. </li><li> Um die CE-MRT-Daten zu quantifizieren, messen Tumor-oder Nierenkontrastverbesserung (ΔSNR) durch Berechnung der Erhöhung der Post-Kontrast-SNR über Vor-Kontrast-SNR nach der folgenden Gleichung: ΔSNR = (S t / σ t) - (S 0 / σ 0), wobei S 0 und S t bezeichnen das Signal in der TUModer oder die Nieren vor und nach dem Kontrast und σ 0 und σ t die Standardabweichung des Rauschens von der Hintergrundluft vor und nach dem Kontrast. </li><li> Berechnen Sie die p-Werte mit Hilfe der Schüler zweiseitigen t-Test, unter der Annahme, statistisch signifikant, p &lt;0,05. </li></ol>

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Kritische Schritte Die richtige Auswahl der Biomarker und auf kleine Peptide Um erfolgreich zu entwickeln eine gezielte Kontrastmittel mit geringen Abmessungen, müssen zwei wichtige Punkte beachtet werden. Zunächst ist es wichtig, die richtige molekulare Biomarker, die reichlich in erkrankten Geweben mit wenig Präsenz in normalen Geweben vorhanden sind, zu wählen. Unsere ausgewählten krebsbedingte Biomarker, geronnen Plasmaproteine, erfüllt di...

Effektive Bildgebung bei Krebserkrankungen molekulare Ziele ist von großer Bedeutung, um die Richtigkeit der früheren Erkennung und Diagnose von Krebs verbessern. Die Magnetresonanztomographie (MRT) ist ein leistungsfähiges klinischen Bildgebungsverfahren mit hoher räumlicher Auflösung und ohne ionisierende Strahlung ein. Allerdings ist keine gezielte Kontrastmittel für die MR klinischen Krebs molekulare Bildgebung zur Verfügung. Innovatives Design und Entwicklung von gezielten MRI-Kontrastmittel würde die Anwendung der MR-Bildgebung der molekularen Krebs stark voranzutreiben. Erhebliche Anstrengungen wurden unternommen, um gezielte Kontrastmittel für die MR-Bildgebung der Biomarker auf der Oberfläche von Krebszellen exprimiert zu entwickeln. Aufgrund der relativ geringen Sensitivität der MRT und niedriger Konzentration dieser Biomarker, ist es eine Herausforderung, genügend Kontrastverstärkung für eine effektive MR molekulare Bildgebung mit kleinen molekulare zielgerichtete Kontrastmittel 2,3 generieren. Um eine ausreichende Verstärkung zu erhalten, erfolg verschiedenen Delivery-Systemeh, wie Liposomen, Nanopartikel und Polymer-Konjugate mit einer hohen Nutzlast von paramagnetischen Gd (III)-Chelate sind vorbereitet, um lokale Konzentration von Kontrastmitteln an den Zielstellen 4,5 zu erhöhen. Obwohl diese Abgabesysteme konnten signifikante Tumorverstärkung in Tiermodellen zu erzeugen, in Folge ihrer großen Größen in langsame und unvollständige Ausscheidung aus dem Körper, was zu einer verlängerten Akkumulation toxischer Gd (III)-Ionen, die schwere Sicherheitsbedenken 6 verursachen kann. Kürzlich haben einige Studien gezeigt, dass die Grenzen der MRT für die molekulare Bildgebung kann durch Auswahl der richtigen molekularen Biomarkern mit hoher Orts Ausdruck in den Läsionen und mit kleinen molekularen Mittel, die leicht ausgeschieden werden können 7,8 überwunden werden. Das Schlüsselmerkmal dieser Mittel ist, dass sie molekulare Marker reichlich in den erkrankten Geweben mit geringer Anwesenheit in normalen Geweben vorhanden Ziel. Eine hohe Konzentration von Kontrastmittel können auf diese Ziele zu binden, was ausreiziente Kontrastverstärkung für eine effektive MR molekularen Bildgebung. Seit ihrer Größe kleiner als die renale Filtration Schwellenwert ist, kann ungebundenes Kontrastmittel leicht aus dem Körper mit reduziertem Hintergrundrauschen ausgeschieden werden. Wir haben eine universelle krebsbezogenen Biomarker geronnenen Plasma-Proteine, die in großen Mengen in Tumorstromas bestehen, ausgewählt, und werden selten in normalen Geweben 9 vorhanden. Wir synthetisierten eine gezielte Kontrastmittel, das eine kleine Zielpeptid CGLIIQKNEC (CLT1), die eine starke spezifische Bindung an das PC3 Prostata-Tumor-Modell 10, und vier Gd-DOTA Monoamid Chelate zeigten. Hier bieten wir eine Methodik für die MR-Bildgebung zur molekularen Krebstumoren in Mäusen zu erkennen.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>Fmoc protected amino acids</td>
			<td>EMD Chemicals Inc</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>DOTA-tris(t-Bu)</td>
			<td>TCI America</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>PyBOP, HOBt, HBTU</td>
			<td>Nova Biochem</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>DIPEA, Thallium(III) trifluoroacetate, TIS</td>
			<td>Sigma-Aldrich Corp.</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Texas Red, succinimidyl ester, single isomer</td>
			<td>Invitrogen</td>
			<td>T20175</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENTS</td>
		 </tr>
		 <tr>
			<td>Agilent 1100 HPLC system</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>ZORBAX 300SB-C18 PrepHT column</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>ICP-OES Optima 3100XL</td>
			<td>Perkin-Elmer</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>MALDI-TOF mass spectrometer</td>
			<td>Bruker</td>
			<td>AutoflexTM Speed</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Maestro FLEX In Vivo Imaging System</td>
			<td>Cambridge Research &amp; Instrumentation, Inc.</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Biospec 7T MRI scanner</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

mr molecular imaging of prostate cancer with a small molecular clt1 peptide targeted contrast agent

Tumorextrazellulären Matrix hat Fülle von Krebs-verwandte Proteine, die als Biomarker für Krebs molekularen Bildgebung verwendet werden können. In dieser Arbeit haben wir gezeigt, effektive MR Krebs molekularen Bildgebung mit einer kleinen molekulares Peptid gezielt Gd-DOTA monoamid komplex wie eine gezielte MRT-Kontrastmittel spezifischen geronnen Plasmaproteinen in Tumor Stroma. Wir führten den Versuch, die Wirksamkeit des Mittels zum nicht-invasiven Nachweis von Prostata-Tumor in einer Maus MRI orthotopen PC-3-Prostatakrebs-Modell. Die gezielte Kontrastmittel wirksam war, um signifikante Tumorkontrastverstärkung bei einer niedrigen Dosis von 0,03 mmol Gd / kg zu produzieren. Das Peptid gezielte MRT-Kontrastmittel ist vielversprechend für MR molekularen Bildgebung der Prostata-Tumor.

Abbildung 1 zeigt die Synthese des gezielten Kontrastmittel CLT1-DL-(Gd-DOTA) 4 und dem Gesamtkonzept des Experiments. CLT1-DL-(Gd-DOTA) 4 zeigt deutlich höhere Relaxivität als klinische Gd-DOTA (Tabelle 1). 1,5 T, ist die T 1-Relaxivität des Gadolinium pro CLT1-DL-(Gd-DOTA) 4 in PBS (pH 7,4) etwa 3-mal höher als die von Gd-DOTA 10. Maestro Abbildungs ​​bestätigt die starke spezifische Bindung von Texas Red markierten CLT1 (CLT1-TR), Tumor ...

Watch this Scientific Journal Video about MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent at JoVE.com

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Um MR Krebs molekulare Bildgebung mit einem kleinen Peptid gezielte MRT-Kontrastmittel spezifischen geronnen Plasmaproteine ​​in Tumorstroma in einem Mausmodell zeigen, Prostata-Krebs.

MR Molekulare Bildgebung von Prostatakrebs mit einem kleinen Molecular Targeted CLT1 Peptid-Kontrastmittel

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

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JoVE Journal

Medicine

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Serie: MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

In vivo Near Infrared Fluorescence (NIRF) intravaskuläre Molecular Imaging von entzündlichen Plaque, einen multimodalen Ansatz, um Imaging der Atherosklerose

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

Forschung

Medizin

Contrast Enhanced Vessel Imaging using MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between different soft tissues and body fluids is inherently poor in micro-CT images 1. Under these circumstances, visualization of blood vessels becomes a nearly impossible task. To overcome this and to improve the visualization of blood vessels exogenous contrast agents can be used. Herein, we present a methodology for visualizing the vascular network in a rodent model. By using a long-acting aqueous colloidal polydisperse iodinated blood-pool contrast agent, eXIA 160XL, we optimized image acquisition parameters and volume-rendering techniques for finding blood vessels in live animals. Our findings suggest that, to achieve a superior contrast between bone and soft tissue from vessel, multiple-frames (at least 5-8/ frames per view), and 360-720 views (for a full 360&deg; rotation) acquisitions were mandatory. We have also demonstrated the use of a two-dimensional transfer function (where voxel color and opacity was assigned in proportion to CT value and gradient magnitude), in visualizing the anatomy and highlighting the structure of interest, the blood vessel network. This promising work lays a foundation for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic or xenograft tumor-bearing mice.

Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).

Kontrastverstärkten Vessel Imaging mit MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between ...

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Evaluation von Nanopartikel-Aufnahme in Tumoren in Echtzeit über Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Molecular Imaging Transplantierte Muscle Progenitor Zielzellen

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are intensively and increasingly recruited in septic joints from the early phases of infection and the infiltration is supposed to regress once efficient removal of the pathogens is obtained. The ability to identify in vivo macrophage activity in an infected joint can therefore provide two main applications: early detection of acute synovitis and monitoring of therapy.
In vivo noninvasive detection of macrophages can be performed with magnetic resonance imaging using iron nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO). After intravascular or intraarticular administration, USPIO are specifically phagocytized by activated macrophages, and, due to their magnetic properties, induce signal changes in tissues presenting macrophage infiltration. A quantitative evaluation of the infiltrate is feasible, as the area with signal loss (number of dark pixels) observed on gradient echo MR images after particles injection is correlated with the amount of iron within the tissue and therefore reflects the number of USPIO-loaded cells.
We present here a protocol to perform macrophage imaging using USPIO-enhanced MR imaging in an animal model of septic arthritis, allowing an initial and longitudinal in vivo noninvasive evaluation of macrophages infiltration and an assessment of therapy action.

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

We demonstrate how to perform macrophage MR imaging using ultrasmall superparamagnetic contrast agent (USPIO) in septic arthritis, allowing an initial and longitudinal in vivo non-invasive evaluation of macrophages infiltration and an assessment of therapy efficacy.

In-vivo-Bildgebung mit MR Makrophagen gezielte Kontrastmittel für die Längs-Auswertung der septische Arthritis

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages 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  ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolierung von Krebs Stammzellen aus menschlichen Prostatakrebs-Proben

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

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

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

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

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 ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Generation von Prostatakrebs-Patienten gewonnen Xenograft-Modelle von zirkulierenden Tumorzellen

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Ein Hirntumor / Organotypischen Co-Kultursystem für die Untersuchung Tumor-Microenvironment und gezielte Arzneimitteltherapie

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...