Ce travail est soutenu en partie par l&#39;American Heart Association GRA printemps 09 postdoctorale (09POST2250268) et le NIH R01 CA097465. Nous apprécions hautement le Dr Wen Li et le Dr Vikas Gulani pour le test de protocole IRM et la configuration, et Mme Yvonne Parker pour son aide lors de l&#39;implantation de la tumeur.

Protocole adapté d&#39;une étude préalable 11. Une. Conjugaison de Gd-DOTA à CLT1 Peptide <ol><li> En utilisant la synthèse classique de peptides en phase solide, synthèse de peptide CLT1 (CGLIIQKNEC) à partir des acides aminés protégés par Fmoc sur une résine de chlorure de 2-chlorotrityle (1,0 mmol). </li><li> Après l&#39;ajout d&#39;acides aminés finale, cycliser le peptide linéaire sur la résine avec du thallium (III) de trifluoroacétate (1,09 g, 2,0 mmol, 2 équivalents) dans du DMF (20 ml) à 0 ° C pendant 2 heures. </li><li> Ensuite, le conjugué PEG et la lysine en séquence à l&#39;extrémité N-terminale du peptide CLT1 (1,0 mmol) de résine en faisant réagir séquentiellement avec Fmoc-NH-PEG-COOH (3,0 mmol), Fmoc-Lys (Fmoc)-OH (3,0 mmol), et un second lot de Fmoc-Lys (Fmoc)-OH (6 mmol). </li><li> Retirer Fmoc avec de la pipéridine. Ajouter DOTA-tris (t-Bu) (4,58 g, 8,0 mmol, 2 équivalents par rapport à chaque groupe amino) à réagir avec les amines libres à partir de quatre dendrimère de la lysine sur la résine pendant 2 heures. </li><li> Enfin, cliver et déprotéger le produità partir de la résine par traitement avec un cocktail d&#39;acide trifluoroacétique, de l&#39;eau, et triisobutylsilane (TIS) (20 ml, 95/2.5/2.5) pendant 8 heures à température ambiante. Éliminer la résine par filtration suivie d&#39;un lavage avec de l&#39;acide trifluoroacétique (TFA). Ajouter les filtrats combinés goutte à goutte à de l&#39;éther d&#39;éthyle froid (200 ml), centrifugation et lavage à l&#39;éther éthylique 4x. Sécher sous vide pour donner un solide incolore (1,99 g, rendement de 61%). </li><li> On purifie le produit brut par HPLC preparative en utilisant un gradient de 0-40% de solvant B (TFA 0,1% dans de l&#39;acétonitrile) dans du solvant A (0,1% de solution aqueuse de TFA) pendant 20 min et de 40 à 90% de solvant B dans le solvant A pendant 10 min, . </li><li> Dissoudre CLT1-dl-(DOTA) 4 (250 mg, 0,077 mmol) dans de l&#39;eau DI (15 ml) et ajuster le pH à 6 en utilisant 1 M de NaOH. Ajouter Gd (OAc) 3 0,4 H 2 O (472 mg, 0,462 mmol, 1,5 équivalents à DOTA monoamide) en portions à la solution, tout en maintenant le pH à 6 avec 1 M NaOH. Agiter la solution de réaction à température ambiante pendant 48 h. </li><li> Complexe Gd résiduel (III) avec ethylenediaminetetraacetic acide acétique (EDTA) (90 mg, 0,31 mmol), et on purifie le produit brut avec une colonne d&#39;exclusion de taille pour donner le produit final CLT1-dl-(Gd-DOTA) 4 (169 mg, 57%). </li></ol> 2. Caractérisation des agents de contraste <ol><li> Mesurer Gd (III) contenu par spectroscopie d&#39;émission à plasma à couplage inductif-optique. </li><li> Acquérir désorption / ionisation laser à temps de vol (MALDI-TOF) des spectres de masse assistée par matrice en mode linéaire avec de l&#39;acide 2,5-dihydroxybenzoïque (2,5-DHB) comme matrice. </li><li> Mesurer les temps de relaxation de la solution aqueuse des agents de contraste de CLT1-dL (Gd-DOTA) 4 et un agent de contrôle non ciblée sCLT1-dL (Gd-DOTA) 4 avec différentes concentrations à 60 MHz (1,5 T) avec un relaxometer à 37 ° C. Utilisez une séquence d&#39;impulsions inversion-récupération de mesurer T 1 et une séquence Carr-Purcell-Meiboom-Gill avec 500 échos pour mesurer T 2. Calculer T 1 et T 2 relaxivités de eagents e des pentes des parcelles de 1 / T 1 et 1 / T 2 par rapport aux concentrations de Gd. </li></ol> 3. Culture cellulaire et le développement de modèle animal avec Orthotopique PC3 tumeur de la prostate <ol><li> Des cellules de cancer de la prostate Culture PC3 avec une expression constitutive de la protéine de fluorescence verte (GFP) dans du milieu RPMI supplémenté avec 5% de sérum bovin fœtal et de pénicilline / streptomycine / fungizone, récolte par traitement à la trypsine et remettre en suspension à une densité de 2,5 x 10 4 cellules / ul de PBS. </li><li> Maintenir NIH souris nude athymiques hommes (âgés de 4-5 semaines) à l&#39;installation athymique animale de base, Case Western Reserve University (CWRU) selon un protocole animale approuvé par le comité de protection et d&#39;utilisation des animaux CWRU. </li><li> Les surfaces chirurgicales ont été pulvérisées avec une solution de chlorhexidine à 2% et ensuite recouverts avec des rideaux absorbants. Gants chirurgicaux stériles ont été portés au cours de la procédure. Avant la chirurgie des animaux, ip injecter Avertin (250 mg / kg) à anesthetize les souris. En variante, la kétamine / xylazine / acépromazine peut être utilisé à une concentration de 50/5/1 mg / kg. Pommade ophtalmique a été appliquée à la souris pour maintenir une humidité suffisante au cours de l&#39;anesthésie. </li><li> Démarrer l&#39;opération de coupe par une petite incision à travers la peau et du péritoine le long de la ligne médiane inférieure à une longueur d&#39;environ 1 cm en utilisant un scalpel stérile. Un drap chirurgical stérile a été couvert autour du site d&#39;incision lors d&#39;interventions chirurgicales de survie. </li><li> Extérioriser doucement et stabiliser les lobes dorsaux de la prostate pour exposer la prostate. Injecter la suspension de cellules PC3-GFP dans du PBS (20 ul) dans la prostate avec une aiguille de calibre 30. Enfin fermer l&#39;incision avec la blessure autoclip 12. </li><li> Pour la surveillance post-procédure, de surveiller les animaux deux fois par jour pendant 1 semaine. Les signes de la maladie sont le manque de manger, urine foncée collecte autour de l&#39;urètre, et une démarche instable qui pourrait indiquer une infection ou à une occlusion de la vessie chez les agrafes. </li><li> Après 10 jours, retirez til l&#39;aide d&#39;agrafes d&#39;une agrafe remover. Surveiller les animaux tous les jours pour évaluer la taille de la tumeur et des preuves de la douleur comme la perte de poids, troubles du comportement, et des troubles moteurs. Euthanasier les animaux qui présentaient une taille de la tumeur ou de dépassement de la limite autorisée preuve éthique de la douleur au cours de la croissance tumorale. </li></ol> 4. Confirmation de la tumeur spécificité de liaison des peptides avec l&#39;imagerie de fluorescence et histologie <ol><li> Après l&#39;inoculation des cellules tumorales, permettre à environ 4 semaines de croissance de la tumeur avant de commencer avec la formation d&#39;image. Avant l&#39;injection des sondes peptidiques, acquérir des images de fluorescence de la GFP avec des souris vivantes sur un imageur de fluorescence pour vérifier la présence d&#39;une tumeur. </li><li> Iv injecter peptides marqués Texas Red à la tumeur chez des souris porteuses à une dose de 10 nmol / souris. </li><li> Sacrifier la souris 2 heures plus tard par dislocation cervicale, de recueillir la tumeur et les organes principaux, et immédiatement l&#39;image sur un imageur de fluorescence. Utilisez des filtres de lumière verte pour la GFP (excitation: 444-490 nm; émission: 515 nm filtre passe-haut; paramètres d&#39;acquisition: de 500 à 720 en 10 étapes nm) et des filtres de lumière rouge pour le rouge Texas (excitation: 576 à 621 nm; émission: 635 nm filtre passe-haut, l&#39;acquisition paramètres: 630-800 en 10 étapes nm). 10 ms temps d&#39;exposition pour la GFP et 150 ms pour le rouge Texas. </li><li> Immédiatement après la mesure de la fluorescence de tissus ensemble, collecter des tissus tumoraux, fixer avec du formol, et cryosection en tranches de 5 um. </li><li> Après rinçage avec du PBS, monter avec 1 goutte de milieu de montage contenant 4 &#39;,6-diamidino-2-phénylindole (DAPI), immédiatement l&#39;image sur un microscope confocal à balayage laser microscope. </li></ol> 5. Imagerie par résonance magnétique (IRM), <ol><li> Environ 4 semaines après l&#39;inoculation des cellules tumorales, permettent les tumeurs grandissent à 0,3-0,6 cm de diamètre. Utiliser un scanner IRM 7 T avec une fréquence radio de volume (RF) de bobines pour l&#39;IRM. </li><li> Anesthésier les souris avec un mélange d&#39;isoflurane et d&#39;oxygène de 2% dans une chambre d&#39;induction de l&#39;isoflurane. Ophthalmic pommade a été appliquée à la souris pour maintenir une humidité suffisante au cours de l&#39;anesthésie. </li><li> Insérer une aiguille de calibre 30 dans un long tube de 1,0 m rempli de sérum physiologique hépariné. Placer le cathéter de self-made dans la queue de la souris veine. </li><li> Placez la souris sur l&#39;aimant et garder sous anesthésie par inhalation avec 1,5% d&#39;isoflurane en oxygène via un cône de nez. </li><li> Placez un capteur respiratoire relié à un système de surveillance sur l&#39;abdomen pour surveiller le rythme et la profondeur de la respiration. Maintenir la température du corps à 37 ° C en soufflant de l&#39;air chaud dans l&#39;aimant grâce à un système de commande à rétroaction. </li><li> Commencez avec des images de coupe sagittale en utilisant une séquence de localisation pour identifier l&#39;emplacement de la tumeur (TR / TE = 200/3.7 de ms, FOV = 3,0 cm, épaisseur de coupe = 2,2 mm, le nombre de tranche = 16, moyenne = 2, l&#39;angle de bascule = 45 °, matrice = 128 x 128). </li><li> Utilisez une séquence de suppression des données 2D T1 gradient graisse d&#39;acquérir des images axiales 2D pour une IRM (TR / TE = 151.2/1.9 ms, FOV = 3,0 cm x 3,0 cm, sépaisseur de poux = 1,2 mm, le nombre de tranche = 12, moyenne = 1, l&#39;angle de bascule = 80 °, matrice = 128 x 128). </li><li> Après une pré-injection image MR référence acquisition, commencer à injecter l&#39;agent ciblé ou de l&#39;agent de contrôle à une dose de 0,03 mmol Gd / kg par rinçage avec 200 ul de solution saline. </li><li> Continuer d&#39;acquérir des images CE-IRM à différents points dans le temps pour un maximum de 30 minutes. </li></ol> 6. Traitement et analyse d&#39;images <ol><li> Utiliser un logiciel de formation d&#39;image pour l&#39;analyse d&#39;image. </li><li> Attirer les régions d&#39;intérêt (ROI) sur l&#39;ensemble de la tumeur et les reins dans le plan d&#39;imagerie en deux dimensions et à mesurer l&#39;intensité de signal moyenne. </li><li> Pour quantifier les données CE-IRM, de mesurer la tumeur ou des reins amélioration du contraste (ΔSNR) par le calcul de l&#39;augmentation de la post-contraste SNR sur pré-contraste SNR selon l&#39;équation suivante: ΔSNR = (S t / σ t) - (S 0 / σ 0), où S 0 et S t représentent le signal dans la tumou les reins ou avant et après contraste, et σ 0 et σ t sont l&#39;écart-type de bruit mesuré à partir de l&#39;air ambiant avant et après contraste. </li><li> Calculer les valeurs de p à l&#39;aide de deux test t bilatéral de l&#39;étudiant, en supposant que la signification statistique à p &lt;0,05. </li></ol>

<ol>
	<li>Brown, M. A., Semelka, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> MRI Basic Principles and Applications. , (2003).</li><li>Artemov, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+magnetic+resonance+imaging+with+targeted+contrast+agents.">Molecular magnetic resonance imaging with targeted contrast agents.</a> J. Cell. Biochem. 90, 518-524 (2003).</li><li>Kalber, T. L., Kamaly, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low+molecular+weight+folate+receptor+targeted+contrast+agent+for+magnetic+resonance+tumor+imaging.">A low molecular weight folate receptor targeted contrast agent for magnetic resonance tumor imaging.</a> Mol. Imaging Biol. 13, 653-662 (2011).</li><li>Schmieder, A. H., Winter, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+MR+imaging+of+melanoma+angiogenesis+with+alphanubeta3-targeted+paramagnetic+nanoparticles.">Molecular MR imaging of melanoma angiogenesis with alphanubeta3-targeted paramagnetic nanoparticles.</a> Magn. Reson. Med. 53, 621-627 (2005).</li><li>Mulder, W. J., Strijkers, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR+molecular+imaging+and+fluorescence+microscopy+for+identification+of+activated+tumor+endothelium+using+a+bimodal+lipidic+nanoparticle.">MR molecular imaging and fluorescence microscopy for identification of activated tumor endothelium using a bimodal lipidic nanoparticle.</a> FASEB J. 19, 2008-2010 (2005).</li><li>Wang, S. J., Brechbiel, M., Wiener, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+a+new+MRI+contrast+agent+prepared+from+polypropyleneimine+dendrimers,+generation+2.">Characteristics of a new MRI contrast agent prepared from polypropyleneimine dendrimers, generation 2.</a> Invest. Radiol. 38, 662-668 (2003).</li><li>Amirbekian, V., Aguinaldo, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerosis+and+matrix+metalloproteinases:+experimental+molecular+MR+imaging+in+vivo.">Atherosclerosis and matrix metalloproteinases: experimental molecular MR imaging in vivo.</a> Radiology. 251, 429-438 (2009).</li><li>Overoye-Chan, K., Koerner, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EP-2104R:+a+fibrin-specific+gadolinium-Based+MRI+contrast+agent+for+detection+of+thrombus.">EP-2104R: a fibrin-specific gadolinium-Based MRI contrast agent for detection of thrombus.</a> J. Am. Chem. Soc. 130, 6025-6039 (2008).</li><li>Pilch, J., Brown, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptides+selected+for+binding+to+clotted+plasma+accumulate+in+tumor+stroma+and+wounds.">Peptides selected for binding to clotted plasma accumulate in tumor stroma and wounds.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 2800-2804 (2006).</li><li>Rohrer, M., Bauer, H., Mintorovitch, J., Requardt, M., Weinmann, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+magnetic+properties+of+MRI+contrast+media+solutions+at+different+magnetic+field+strengths.">Comparison of magnetic properties of MRI contrast media solutions at different magnetic field strengths.</a> Invest. Radiol. 40, 715-724 (2005).</li><li>Wu, X., Burden-Gulley, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+evaluation+of+a+peptide+targeted+small+molecular+Gd-DOTA+monoamide+conjugate+for+MR+molecular+imaging+of+prostate+cancer.">Synthesis and evaluation of a peptide targeted small molecular Gd-DOTA monoamide conjugate for MR molecular imaging of prostate cancer.</a> Bioconjugate Chem. 23, 1548-1556 (2012).</li><li>Burden-Gulley, S. M., Gates, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+molecular+diagnostic+of+glioblastomas:+detection+of+an+extracellular+fragment+of+protein+tyrosine+phosphatase+mu.">A novel molecular diagnostic of glioblastomas: detection of an extracellular fragment of protein tyrosine phosphatase mu.</a> Neoplasia. 12, 305-316 (2010).</li><li>McBride, J. 	. D., Birgit, H., Leatherbarrow, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resin-coupled+cyclic+peptides+as+proteinase+inhibitors.">Resin-coupled cyclic peptides as proteinase inhibitors.</a> Protein and Peptide. 3 (3), 193-198 (1996).</li><li>Cline, D. J., Thorpe, C., Schneider, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+method+for+facile+intramolecular+disulfide+formation+in+synthetic+peptides.">General method for facile intramolecular disulfide formation in synthetic peptides.</a> Anal. Biochem. 335 (1), 168-170 (2004).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Étapes critiques Sélection de biomarqueurs appropriés et ciblage petit peptide Pour développer avec succès un agent de contraste ciblées avec une petite taille, deux points essentiels doivent être pris en considération. Tout d&#39;abord, il est important de sélectionner des biomarqueurs moléculaires appropriés qui sont présents en abondance dans les tissus malades, où la présence dans les tissus normaux. Notre biomarqueur lié au cance...

Imagerie efficace de cibles moléculaires liés au cancer est d&#39;une grande importance pour améliorer la précision de la détection du cancer plus tôt et le diagnostic. Imagerie par résonance magnétique (IRM) est un puissant modalité d&#39;imagerie clinique à haute résolution spatiale et aucune ionisation rayonnement 1. Toutefois, aucun agent de contraste cible est disponible pour l&#39;imagerie moléculaire du cancer du MR clinique. Conception et développement de l&#39;innovation d&#39;agents de contraste IRM ciblées seraient grandement progresser l&#39;application de l&#39;imagerie moléculaire du cancer MR. D&#39;importants efforts ont été faits pour développer des agents de contraste pour l&#39;IRM ciblées des biomarqueurs exprimés à la surface des cellules cancéreuses. En raison de relativement faible sensibilité de l&#39;IRM et à faible concentration de ces biomarqueurs, c&#39;est un défi de générer amélioration du contraste suffisant pour l&#39;imagerie moléculaire de M. efficace en utilisant de petits moléculaire des agents de contraste ciblées 2,3. Afin d&#39;obtenir l&#39;amélioration suffisante, divers systèmes de transmission SUCh que des liposomes, des nanoparticules et des polymères conjugués avec une charge utile élevée paramagnétique Gd chélates (III) ont été préparés pour augmenter la concentration locale d&#39;agents de contraste au niveau des sites cibles 4,5. Bien que ces systèmes de délivrance ont été capables de générer de l&#39;amélioration significative de la tumeur dans les modèles animaux, leurs grandes dimensions ont donné lieu à l&#39;élimination lente et incomplète du corps, résultant en une accumulation prolongée de Gd (III) des ions toxiques, qui peuvent provoquer des graves problèmes de sécurité 6. Récemment, certaines études ont montré que les limites de l&#39;IRM pour l&#39;imagerie moléculaire peuvent être surmontés par la sélection des biomarqueurs moléculaires appropriés à haute expression locale dans les lésions et l&#39;utilisation de petits agents moléculaires qui peuvent être facilement excrétés 7,8. La principale caractéristique de ces agents est qu&#39;ils ciblent des marqueurs moléculaires présents abondamment dans les tissus malades avec peu de présence dans les tissus normaux. Une forte concentration d&#39;agents de contraste peut se lier à ces objectifs, entraînant sufficace amélioration du contraste pour l&#39;imagerie moléculaire de M. efficace. Etant donné que leur taille est plus petite que le seuil de filtration rénale, les agents de contraste non liés peuvent être aisément excrétés par le corps avec un bruit de fond réduit. Nous avons sélectionné un biomarqueur universel sur le cancer, les protéines de plasma coagulé, qui existent en abondance dans le stroma de la tumeur, et sont rarement présents dans les tissus normaux 9. Nous avons synthétisé un agent de contraste contenant ciblée un petit peptide de ciblage CGLIIQKNEC (CLT1), qui a montré une forte liaison spécifique pour le modèle de tumeur de la prostate PC3 10, et quatre chélates de monoamides Gd-DOTA. Ici, nous fournissons une méthode pour l&#39;imagerie moléculaire du cancer M. de détecter des tumeurs chez la souris.

<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

Tumeur matrice extracellulaire a l&#39;abondance des protéines liées au cancer qui peuvent être utilisés en tant que biomarqueurs pour l&#39;imagerie moléculaire du cancer. Dans ce travail, nous avons démontré l&#39;imagerie moléculaire du cancer de M. efficace avec un petit peptide moléculaire complexe monoamide Gd-DOTA ciblée comme un agent de contraste IRM spécifique ciblé aux protéines plasmatiques coagulé dans le stroma de la tumeur. Nous avons effectué l&#39;expérience de l&#39;évaluation de l&#39;efficacité de l&#39;agent pour la détection non-invasive de tumeur de la prostate par IRM dans un modèle orthotopique de cancer de la prostate PC-3 souris. L&#39;agent de contraste ciblées était efficace pour produire importante amélioration du contraste de la tumeur à une dose faible de 0,03 mmol Gd / kg. Le peptide agent de contraste IRM ciblée est prometteur pour l&#39;imagerie moléculaire M. de tumeur de la prostate.

La figure 1 illustre la synthèse de l&#39;agent de contraste ciblées CLT1-dL (Gd-DOTA) 4 et le schéma global de l&#39;expérience. CLT1-dL (Gd-DOTA) 4 spectacles de relaxation beaucoup plus élevé que clinique Gd-DOTA (tableau 1). À 1,5 T, T 1 de relaxation par le gadolinium de CLT1-dL (Gd-DOTA) 4 dans du PBS (pH 7,4) est environ 3 fois plus élevée que celle de Gd-DOTA 10. imagerie de Maestro confirme la forte liaison spécifique de Texas Red...

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

Pour démontrer l&#39;imagerie moléculaire du cancer MR avec un petit peptide agent de contraste IRM spécifique aux protéines de plasma coagulé dans le stroma de la tumeur dans un modèle de cancer de la prostate de la souris ciblé.

M. imagerie moléculaire du cancer de la prostate avec un agent de contraste ciblé Petit moléculaire CLT1 Peptide

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

mr-molecular-imaging-prostate-cancer-with-small-molecular-clt1

Research

JoVE Journal

Medicine

11.1K vues.  Case Western Reserve University.  Pour démontrer l'imagerie moléculaire du cancer MR avec un petit peptide agent de contraste IRM spécifique aux protéines de plasma coagulé dans le stroma de la tumeur dans un modèle de cancer de la prostate de la souris ciblé. 

Vidéo: 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 par fluorescence dans le proche infrarouge (NIRF) intravasculaire d&#39;imagerie moléculaire de la plaque inflammatoire, une approche multimodale d&#39;imagerie de l&#39;athérosclérose

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

Recherche

Médecine

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).

Imagerie navire Contraste rehaussé utilisant 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 de la fixation des nanoparticules dans les tumeurs en temps réel par imagerie intravitale

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.

L&#39;imagerie moléculaire à la cible transplanté des cellules progénitrices musculaires

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 des macrophages imagerie utilisant MR ciblée agent de contraste pour l&#39;évaluation longitudinale d&#39;arthrite septique

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.

Un modèle murin orthotopique de cancer de la prostate humain métastases

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.

Isolement de cellules souches du cancer à partir d&#39;échantillons cancer de la prostate humaine

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.

Contraste dynamique amélioré imagerie par résonance magnétique d&#39;un modèle orthotopique cancer du pancréas de souris

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.

Génération de cancer de la prostate des patients Dérivé modèles de xénogreffe de cellules tumorales circulantes

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;

Une tumeur au cerveau / organotypiques Slice système de co-culture pour l&#39;étude des tumeurs Microenvironnement et ciblée Thérapies de drogue

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