Questo lavoro è sostenuto in parte dalla American Heart Association GRA Primavera 09 Postdoctoral Fellowship (09POST2250268) e il NIH R01 CA097465. Apprezziamo il dottor Wen Li e il dottor Vikas Gulani per la prova di protocollo MRI e la configurazione, e la signora Yvonne Parker per la sua assistenza su impianto del tumore.

Protocollo adattato da uno studio preliminare 11. 1. Coniugazione del Gd-DOTA per CLT1 Peptide <ol><li> Utilizzando lo standard di sintesi peptidica in fase solida, sintesi CLT1 peptide (CGLIIQKNEC) da amminoacidi Fmoc-protetti su una resina di cloruro di 2-chlorotrityl (1,0 mmol). </li><li> Dopo l&#39;aggiunta di amminoacidi finale, ciclizzare il peptide lineare in resina con tallio (III) trifluoroacetato (1.09 g, 2.0 mmol, 2 equivalenti) in DMF (20 ml) a 0 ° C per 2 h. </li><li> Successivamente, coniugato PEG e lisina sequenzialmente al N-terminale del peptide CLT1 (1,0 mmol) di resina facendo reagire in sequenza con Fmoc-NH-PEG-COOH (3.0 mmol), Fmoc-Lys (Fmoc)-OH (3.0 mmol), e un secondo lotto di Fmoc-Lys (Fmoc)-OH (6 mmoli). </li><li> Rimuovere Fmoc con piperidina. Aggiungere DOTA-tris (t-Bu) (4.58 g, 8.0 mmol, 2 equivalenti a ciascun gruppo amminico) per reagire con i quattro ammine libere dal dendrimero lisina sulla resina per 2 h. </li><li> Infine, fendere e deproteggere il prodottodalla resina mediante trattamento con un cocktail di acido trifluoroacetico, acqua, e triisobutylsilane (TIS) (20 ml, 95/2.5/2.5) per 8 ore a temperatura ambiente. Rimuovere la resina mediante filtrazione seguita da lavaggio con acido trifluoroacetico (TFA). Aggiungere filtrati associati a gocce alla etere etilico freddo (200 ml), centrifuga, e lavare con 4x etere etilico. Essiccare sotto vuoto a dare un solido incolore (1,99 g, 61% resa). </li><li> Purificare il prodotto grezzo con HPLC preparativa utilizzando un gradiente di 0-40% di solvente B (0.1% TFA in acetonitrile) in solvente A (0,1% soluzione acquosa TFA) per 20 min e 40-90% di solvente B in solvente A per 10 min . </li><li> Sciogliere CLT1-DL-(DOTA) 4 (250 mg, 0,077 mmol) in acqua deionizzata (15 ml) e portare il pH a 6 con 1 M NaOH. Aggiungere Gd (OAc) 3 .4 H 2 O (472 mg, 0,462 mmol, 1,5 equivalenti a DOTA monoammide) in porzioni alla soluzione, mantenendo il pH a 6 con 1 M NaOH. Mescolare la soluzione di reazione a temperatura ambiente per 48 ore. </li><li> Complesso il residuo Gd (III) con eacido thylenediaminetetraacetic (EDTA) (90 mg, 0,31 mmol), e purificare il prodotto grezzo con una colonna di esclusione dimensionale per dare il prodotto finale CLT1-DL-(Gd-DOTA) 4 (169 mg, 57%). </li></ol> 2. Caratterizzazione di mezzi di contrasto <ol><li> Misurare Gd (III) contenuto con la spettroscopia di emissione al plasma accoppiato induttivamente ottica. </li><li> Acquisire matrix-assisted laser desorbimento / ionizzazione spettri di massa a tempo di volo (MALDI-TOF) in modo lineare con l&#39;acido 2,5-diidrossibenzoico (2,5-DHB) come matrice. </li><li> Misurare tempi di rilassamento della soluzione acquosa dei mezzi di contrasto di CLT1-DL-(Gd-DOTA) 4 e un agente di controllo nontargeted sCLT1-DL-(Gd-DOTA) 4 con differenti concentrazioni a 60 MHz (1,5 T) con un rilassometro a 37 ° C. Utilizzare una sequenza di impulsi di inversione di recupero per misurare T 1 e una sequenza di Carr-Purcell-Meiboom-Gill con 500 echi per misurare T 2. Calcola T 1 e T 2 relassività di secoloe agenti alle pendici delle trame di 1 / T 1 e 1 / T 2 e le concentrazioni Gd. </li></ol> 3. Cella Cultura e Sviluppo di modello animale con tumore alla prostata PC3 ortotopico <ol><li> Cultura PC3 prostata cellule tumorali con l&#39;espressione costitutiva di proteina fluorescente verde (GFP) in mezzo RPMI supplementato con 5% di siero fetale bovino e penicillina / streptomicina / fungizone, raccolto mediante tripsinizzazione e risospendere alla densità di 2,5 x 10 4 cellule / l di PBS. </li><li> Mantenere maschi NIH topi nudi atimici (4-5 settimane) presso l&#39;impianto di atimico animale core, Case Western Reserve University (CWRU) secondo un protocollo animali approvato dalla Institutional Animal Care ed uso commissione CWRU. </li><li> Le superfici chirurgici sono stati spruzzati con soluzione di clorexidina 2% e poi coperte con teli assorbenti. Guanti chirurgici sterili sono stati indossati durante la procedura. Prima dell&#39;intervento degli animali, ip iniettare Avertin (250 mg / kg) per anesthetizposta i topi. In alternativa, ketamina / xilazina / Acepromazina può essere utilizzato ad una concentrazione di 50/5/1 mg / kg. Pomata oftalmica è stata applicata al mouse per mantenere un&#39;adeguata umidità durante l&#39;anestesia. </li><li> Avviare la chirurgia tagliando una piccola incisione attraverso la pelle e peritoneo lungo la linea mediana inferiore ad una lunghezza di circa 1 cm con un bisturi sterile. Un telo chirurgico sterile era coperto intorno al sito di incisione durante interventi chirurgici di sopravvivenza. </li><li> Esteriorizzare delicatamente e stabilizzare i lobi dorsali prostata per esporre la prostata. Iniettare la sospensione di cellule PC3-GFP in PBS (20 microlitri) nella prostata con un ago da 30 gauge. Infine chiudere l&#39;incisione con ferita AutoClip 12. </li><li> Per il monitoraggio post-procedura, di monitorare gli animali due volte al giorno per 1 settimana. I segni della malattia comprendono la mancanza di cibo, urina raccolta buio intorno all&#39;uretra, e andatura instabile che potrebbe indicare infezione o occlusione della vescica nei punti metallici. </li><li> Dopo 10 giorni, rimuovere tegli graffette con un fiocco di rimozione. Monitorare gli animali ogni giorno per valutare le dimensioni del tumore e le prove di dolore, come la perdita di peso, deviazioni comportamentali, e difetti del motore. Euthanize gli animali che hanno mostrato una dimensione del tumore superiore al limite eticamente consentito o evidenza di dolore durante la crescita del tumore. </li></ol> 4. Conferma della Tumore Legatura specificità dei peptidi con imaging di fluorescenza e Istologia <ol><li> Dopo l&#39;inoculazione delle cellule tumorali, permettere circa 4 settimane di crescita tumorale prima di iniziare l&#39;imaging. Prima dell&#39;iniezione delle sonde peptidiche, acquisire immagini di fluorescenza GFP con topi diretta su un imager fluorescenza per verificare la presenza di tumore. </li><li> Iv iniettare Texas Red etichettato peptidi al tumore cuscinetto topi alla dose di 10 nmol / mouse. </li><li> Sacrifica i topi 2 ore più tardi da dislocazione cervicale, raccogliere il tumore e gli organi più importanti, e l&#39;immagine immediatamente su un imager fluorescenza. Utilizzare i filtri di luce verde per GFP (eccitazione: 444-490 nm, emissione: 515 nm filtro passa lungo; impostazioni di acquisizione: 500-720 a passi di 10 nm) e filtri di luce rossa per Texas Red (eccitazione: 576-621 nm, emissione: 635 nm filtro passa lungo; acquisizione Impostazioni: 630-800 a passi di 10 nm). 10 msec tempo di esposizione per la GFP e 150 msec per Texas Red. </li><li> Subito dopo la misura di tutta la fluorescenza dei tessuti, raccoglie tessuti tumorali, fissare con la formalina, e cryosection a fette di 5 micron. </li><li> Dopo il risciacquo con PBS, montare con 1 goccia di soluzione di montaggio contenente 4 &#39;,6-diamidino-2-fenilindolo (DAPI), l&#39;immagine immediatamente su un microscopio confocale a scansione laser. </li></ol> 5. Magnetic Resonance Imaging (MRI) <ol><li> Circa 4 settimane dopo l&#39;inoculazione delle cellule tumorali, consentono ai tumori di crescere fino a 0,3-0,6 cm di diametro. Utilizzare uno scanner MRI 7 T con un volume di radio frequenza (RF) bobina per lo studio MRI. </li><li> Anestetizzare il mouse con una miscela ossigeno-isoflurano 2% in una camera di induzione isoflurano. Ophthalmic unguento è stato applicato al mouse per mantenere un&#39;adeguata umidità durante l&#39;anestesia. </li><li> Inserire un ago da 30 gauge in un 1,0 m di lunghezza tubo riempito con soluzione salina eparinizzata. Posizionare il catetere self-made nella vena della coda del mouse. </li><li> Posizionare il mouse nel magnete e tenere sotto anestesia inalatoria con il 1,5% isoflurano-ossigeno attraverso un cono di naso. </li><li> Posizionare un sensore respiratorio collegato ad un sistema di monitoraggio sull&#39;addome per monitorare la frequenza e la profondità della respirazione. Mantenere la temperatura corporea a 37 ° C mediante insufflazione di aria calda all&#39;interno del magnete attraverso un sistema di controllo in retroazione. </li><li> Iniziare con le immagini di sezione sagittale utilizzando una sequenza localizzante per identificare la posizione del tumore (TR / TE = 200/3.7 msec, FOV = 3,0 centimetri, spessore di strato = 2,2 millimetri, numero di slice = 16, media = 2, flip angle = 45 °, matrix = 128 x 128). </li><li> Utilizzare un gradiente sequenza di soppressione del grasso 2D T1-pesate per acquisire immagini assiali 2D per CE-MRI (TR / TE = 151.2/1.9 msec, FOV = 3,0 centimetri x 3,0 centimetri, sSpessore pidocchi = 1,2 mm, numero di slice = 12, media = 1, flip angle = 80 °, matrice = 128 x 128). </li><li> Dopo la pre-iniezione acquisizione delle immagini MR basale, iniziare a iniettare l&#39;agente mirata o agente di controllo ad una dose di 0,03 mmol / kg di Gd mediante flussaggio con 200 ml di soluzione salina. </li><li> Continuare ad acquisire immagini CE-MRI in diversi momenti fino a 30 min. </li></ol> 6. Image Processing and Analysis <ol><li> Utilizzare il software di imaging per l&#39;analisi delle immagini. </li><li> Disegnare le regioni di interesse (ROI) sull&#39;intero tumore ei reni nel piano dell&#39;immagine bidimensionale e misurare l&#39;intensità media del segnale. </li><li> Per quantificare i dati CE-MRI, misurare tumore o enhancement rene (ΔSNR) dal calcolo della maggiorazione in post-contrasto SNR SNR over pre-contrasto utilizzando la seguente equazione: ΔSNR = (S t / σ t) - (S 0 / σ 0), dove S 0 e S T indicano il segnale nel tumo o reni prima e dopo contrasto, e σ 0 e σ t sono la deviazione standard del rumore misurato dall&#39;aria sfondo prima e dopo contrasto. </li><li> Calcolare i valori di p utilizzando due code t-test dello studente, assumendo la significatività statistica p &lt;0.05. </li></ol>

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Nessun conflitto di interessi dichiarati.

Fasi critiche Selezione di Biomarker corretta e Targeting piccolo peptide Per sviluppare con successo un agente di contrasto mirato con dimensioni ridotte, due punti chiave devono essere considerati. In primo luogo, è importante selezionare marcatori molecolari appropriati che sono abbondantemente presenti in tessuti malati con scarsa presenza nei tessuti normali. Il nostro biomarcatore per cancro selezionata, proteine ​​del plasma coagulato, s...

Rappresentazione efficace di bersagli molecolari connessi con il cancro è di grande importanza per migliorare la precisione di rilevamento del cancro precoce e diagnosi. La risonanza magnetica (MRI) è una potente modalità di imaging clinico ad alta risoluzione spaziale e nessuna radiazione di ionizzazione 1. Tuttavia, nessun agente di contrasto mirata è disponibile per l&#39;imaging molecolare cancro MR clinica. Il design innovativo e lo sviluppo di agenti di contrasto per risonanza magnetica mirati sarebbero notevolmente progredire l&#39;applicazione di imaging molecolare cancro MR. Sono stati fatti notevoli sforzi per sviluppare agenti di contrasto mirate per RM dei biomarcatori espressi sulla superficie delle cellule tumorali. A causa della relativamente bassa sensibilità della RM e bassa concentrazione di questi biomarcatori, è una sfida di generare sufficiente aumento del contrasto per l&#39;imaging molecolare RM efficace con piccoli mezzi di contrasto a bersaglio molecolare 2,3. Al fine di ottenere il miglioramento sufficiente, vari sistemi di erogazione such come liposomi, nanoparticelle e polimeri coniugati con un alto carico utile di paramagnetici di Gd (III) chelati sono stati predisposti per aumentare la concentrazione locale di agenti di contrasto ai siti bersaglio 4,5. Sebbene questi sistemi di consegna sono stati in grado di generare un miglioramento significativo del tumore nei modelli animali, loro grandi dimensioni comportato l&#39;eliminazione lenta e incompleta dal corpo, con conseguente accumulo prolungato di Gd (III) ioni tossici, che possono provocare gravi problemi di sicurezza 6. Recentemente, alcuni studi hanno dimostrato che i limiti di MRI per l&#39;imaging molecolare possono essere superati selezionando marcatori molecolari adeguate con elevata espressione locale nelle lesioni e utilizzando piccoli agenti molecolari che possono essere facilmente escreti 7,8. La caratteristica fondamentale di questi agenti è quanto si rivolgono marcatori molecolari abbondantemente presenti nei tessuti malati con scarsa presenza nei tessuti normali. Un&#39;elevata concentrazione di agenti di contrasto in grado di legarsi a questi obiettivi, con conseguente sufficiente miglioramento del contrasto per l&#39;imaging molecolare efficace MR. Poiché la loro dimensione è inferiore alla soglia di filtrazione renale, agenti di contrasto legate possono facilmente essere espulso dal corpo con rumore di fondo ridotto. Abbiamo selezionato un biomarcatore per cancro universale, proteine ​​del plasma coagulato, che esistono in abbondanza nella stroma tumorale, e sono raramente presenti nei tessuti normali 9. Abbiamo sintetizzato un agente di contrasto mirato con un piccolo peptide di targeting CGLIIQKNEC (CLT1), che ha mostrato forte legame al modello di tumore della prostata PC3 10 specifica, e quattro chelati monoammide Gd-DOTA. Qui, forniamo una metodologia di imaging molecolare del cancro MR per rilevare i tumori nei topi.

<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

Matrice extracellulare tumore ha abbondanza di proteine ​​del cancro correlati che possono essere utilizzate come marcatori per l&#39;imaging molecolare cancro. In questo lavoro, abbiamo dimostrato efficace imaging molecolare del cancro RM con un piccolo peptide molecolare mirata complesso monoammide Gd-DOTA come agente di contrasto per risonanza magnetica mirato specifico alle proteine ​​plasmatiche coagulato in stroma tumorale. Abbiamo eseguito l&#39;esperimento di valutare l&#39;efficacia dell&#39;agente per il rilevamento non invasivo di tumore della prostata con MRI in un ortotopico PC-3 modello di cancro della prostata mouse. Il mezzo di contrasto mirate si è dimostrato efficace per produrre miglioramento significativo contrasto del tumore con una dose bassa di 0,03 mmol Gd / kg. Il peptide mirato agente di contrasto per risonanza magnetica è promettente per l&#39;imaging molecolare RM del tumore alla prostata.

La figura 1 illustra la sintesi del mezzo di contrasto mirato CLT1-DL-(Gd-DOTA) 4 e lo schema complessivo dell&#39;esperimento. CLT1-DL-(Gd-DOTA) 4 mostra relassività molto superiore clinica Gd-DOTA (Tabella 1). A 1,5 T, T 1 relassività per gadolinio di CLT1-DL-(Gd-DOTA) 4 in PBS (pH 7,4) è circa 3 volte superiore a quella di Gd-DOTA 10. Imaging Maestro conferma la forte legame di Texas Red etichettato CLT1 (CLT1-TR) per tumore con un minimo lega...

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

Per dimostrare imaging molecolare del cancro RM con un piccolo peptide mirato MRI mezzo di contrasto specifico alle proteine ​​plasmatiche coagulato in stroma tumorale in un modello di cancro alla prostata del mouse.

MR Imaging Molecolare del cancro alla prostata con un piccolo Molecular CLT1 Peptide mirato di contrasto agente

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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11.1K Views.  Case Western Reserve University.  Per dimostrare imaging molecolare del cancro RM con un piccolo peptide mirato MRI mezzo di contrasto specifico alle proteine ​​plasmatiche coagulato in stroma tumorale in un modello di cancro alla prostata del mouse. 

Video: 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 fluorescenza (NIRF) intravascolare Imaging Molecolare della placca infiammatoria, un approccio multimodale per Imaging di aterosclerosi

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

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Medicina

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).

Contrast Imaging nave migliorata utilizzando 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.

Valutazione della captazione nanoparticelle nei tumori in tempo reale utilizzando intravitale 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.

Imaging molecolare a Target trapiantate cellule progenitrici muscolari

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 Macrofago Imaging Utilizzando MR mirata di contrasto agente per la valutazione longitudinale di artrite settica

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 ortotopico Modello murino di Human Prostate Cancer Metastasi

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.

Isolamento di cancro cellule staminali da campioni umani di cancro alla prostata

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.

Contrasto dinamico migliorato risonanza magnetica di un modello ortotopico cancro del pancreas mouse

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.

Generazione di cancro alla prostata del paziente Derivato modelli di xenotrapianto di cellule tumorali circolanti

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;

Un tumore cerebrale / organotipica fetta sistema di co-coltura per lo Studio microambiente tumorale e mirata terapie farmacologiche

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