Questo lavoro è stato sostenuto dal National Health and Medical Research Council, Australia (1008865), l&#39;Australian Research Council (LE110100125), il National Cancer Institute (CA138687-01), Erica Sloan è supportato da una carriera Fellowship precoce dal National Breast Cancer Fondazione, in Australia. Corina Kim-Fuchs è sostenuto da una borsa di studio della Lega Tumori svizzera e una borsa di studio HDR da Monash Institute di Scienze Farmaceutiche. Eliane Angst è supportata da una sovvenzione da parte di Berna Cancer League.

Il protocollo viene mostrato è eseguita sotto la guida e l&#39;approvazione di cura degli animali dell&#39;istituzione dell&#39;autore e comitato utilizzo. Tutti gli esperimenti sono eseguiti in conformità a tutte le direttive, i regolamenti e le agenzie di regolamentazione. 1. Trasduzione del pancreas Cancer Cell Lines <ol><li> Cellule tumorali pancreatiche transduce di esprimere luciferasi come precedentemente descritto. 12,13 Panc-1 e Capan-1 linee cellulari tumorali pancreatiche trasdotte con lucciola luciferasi sono usati qui. </li></ol> Nota: Renilla luciferasi o luciferasi batterica possono anche essere utilizzati. 2. Pancreas Cancer Cell Preparazione <ol><li> Cultura trasdotte cellule del tumore pancreatico fino al 70% confluenti. </li><li> Sollevare le cellule pancreatiche e garantire la redditività è superiore al 90%. </li><li> Risospendere in 2 x 10 7 cellule / ml in una miscela 03:02 di Matrigel refrigerate: tampone fosfato saline (PBS). </li><li> Mantenere la sospensione Matrigel cellule in ghiaccio prima di iniezione nel pancreas. </li></ol> Note: Per assicurare la rapida solidificazione di Matrigel, ridurre il volume di PBS considerazione per il volume del pellet. Maneggiare Matrigel con strumenti di ghiaccio freddo e siringhe in ogni momento per evitare la solidificazione prima dell&#39;iniezione. Il numero di cellule suggerito è una guida e dovrebbe essere determinata empiricamente per ciascuna linea cellulare. 3. Topo Preparazione <ol><li> Anestetizzare il mouse utilizzando inalato 2-3% isoflurano. Determinare la profondità dell&#39;anestesia dalla mancanza di pedale riflesso di un dito del piede pizzico delicato. </li><li> Applicare lubrificante per gli occhi per evitare l&#39;essiccamento. </li><li> Posizionare il mouse sul dorso su una 37 ° C rilievo di riscaldamento e ruotare leggermente il mouse per aumentare il lato sinistro dell&#39;addome. </li><li> Preparare l&#39;addome con una soluzione di iodio povidone al 10%. </li></ol> Note: InjectabLe anestesia può essere usato al posto di anestesia inalatoria. Digiuno pre-operatorio non è necessario. 4. Laparotomia <ol><li> Utilizzando strumenti chirurgici sterili fare una incisione 1,5 cm in pelle di circa 1 cm a sinistra laterale dalla linea mediana. </li><li> Fare un cm incisione nel muscolo addominale sottostante 1.5. </li><li> Individuare la milza usando la pinza e rimuovere delicatamente la milza dalla cavità addominale. Fissare la milza lungo un batuffolo di cotone sterile per esporre il pancreas sottostante. </li><li> Individuare la coda del pancreas adiacente alla milza. </li><li> Utilizzando un 29 G 0,3 ml siringa da insulina, iniettare 20 microlitri della sospensione Matrigel-cellula nel pancreas. </li><li> Dopo l&#39;iniezione, tenere la siringa nel pancreas per 30-60 secondi fino a quando il Matrigel è solidificato. Questo importante passo minimizza perdita cellulare. </li><li> Ispezionare il sito di iniezione per garantire nessuna perdita si è verificata. </li><li> Restituire la milza e pancreas alla cavità addominale. </li></ol> Nota: Fare attenzione a non pungere la parte dorsale del pancreas che può essere sottile. 5. Addominale Chiusura muro <ol><li> Chiudere la muscolatura addominale del mouse con una sutura assorbibile intrecciata 4-0 con un ago rotondo con un punto continuo. </li><li> Chiudere la pelle esterna con un monofilamento non assorbibile 6-0 sutura con ago di taglio con un punto continuo. </li><li> Rimuovere il mouse dalla inalazione anestetico e iniettato 0,05-0,1 mg / kg per via sottocutanea buprenorfina. </li><li> Lasciare il mouse per recuperare nella sua gabbia posta su un 37 ° C rilievo di riscaldamento con libero accesso a cibo e acqua. Se i topi mostrano segni di dolore, come incurvando o mobilità ridotta, la buprenorfina può essere somministrata ogni 12 ore per un periodo di 36 ore. </li><li> Dopo la guarigione delle ferite (7-10 giorni), anestetizzare il mouse e rimuovere i punti di sutura esterni. </li></ol> 6. Monitoraggio bioluminescente di pancreas Cancer Progressione <ol><li> Anestetizzare il mouse utilizzando inalato isoflurano. </li><li> Iniettare 150 mg / kg di D-luciferina via coda vena. </li><li> Posizionare il mouse nel sistema di imaging bioluminescente e catturare immagini a luce bianca e bioluminescenza come descritto in precedenza. 14,15 </li><li> Rimuovere il mouse dalla inalazione anestetico e permettono di recuperare nella sua gabbia casa. </li></ol> Nota: Bioluminescenti imaging è non invasiva e può essere condotta periodicamente per indagare cinetica di crescita del tumore. Per tumore immagine nella coda pancreatica è importante mettere il mouse sul lato sinistro, quindi i punti tumorali verso la telecamera. Noi imaged una volta a settimana con la frequenza aumenta fino a tre volte alla settimana prima del punto finale sperimentale utilizzando un sistema di imaging Lumina II (Perkin-Elmer, ex Caliper Life Sciences) in esecuzione Living software 4.3.1 Imaging con binning 4, FOV 12,5, F-stop 1, esposizione 1 - 60 sec (determinata dalla più alta exposure senza pixel di saturazione).

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(56), e3175 (2011).</li><li>Lim, E., 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=Monitoring+tumor+metastases+and+osteolytic+lesions+with+bioluminescence+and+micro+CT+imaging.">Monitoring tumor metastases and osteolytic lesions with bioluminescence and micro CT imaging.</a> J. Vis. Exp. (52), e2775 (2011).</li><li>Burton, J. B., 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=Adenovirus-mediated+gene+expression+imaging+to+directly+detect+sentinel+lymph+node+metastasis+of+prostate+cancer.">Adenovirus-mediated gene expression imaging to directly detect sentinel lymph node metastasis of prostate cancer.</a> Nat Med. 14, 882-888 (2008).</li><li>Vezeridis, M. P., Doremus, C. M., Tibbetts, L. M., Tzanakakis, G., Jackson, B. 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Gli autori dichiarano di non avere interessi finanziari in competizione.

Qui si descrive un modello ortotopico per la valutazione longitudinale di sviluppo del tumore del pancreas e la progressione. Cinetica di crescita del tumore primario sono riproducibili (Figura 3) e possono essere non invasivo monitorato utilizzando bioluminescenza delle cellule luciferasi-tag, ad esempio, per le analisi di risposta del tumore alle nuove terapie anti-cancro del pancreas. Coerentemente con la malattia umana, il modello mostra invasione del pancreas locale (Figura 4A),</s...

Il tumore al pancreas è la quarta causa di morte per cancro, con un tasso di sopravvivenza a 5 anni del 4-6%. 1,2 Solo il 15% dei pazienti sono diagnosticati abbastanza presto nel corso della malattia per poter beneficiare di un intervento chirurgico, e tumori ricorrono in&gt; 80% dei pazienti. 3,4 La gemcitabina è usata per il trattamento di adenocarcinomi pancreatici, tuttavia, chemioresistenza è comune e spesso la droga ha poco impatto sulla sopravvivenza globale. 5 nuove strategie farmacologiche per curare il cancro al pancreas sono estremamente necessari. Il loro sviluppo dipende notevolmente migliorato la comprensione dei passaggi chiave della progressione di malattia che possono essere sensibili a un intervento terapeutico. Modelli ortotopico di cancro al pancreas emulare gli aspetti chiave della malattia umana, che li rende strumenti ideali per lo studio della biologia del cancro al pancreas. 6-9 In contrasto in vitro basati su cellule di pancreas comportamento delle cellule di cancro di und sottocutaneo in modelli in vivo di cancro al pancreas, modelli ortotopici consentire indagini su interazioni delle cellule tumorali con il microambiente del pancreas. La cinetica di progressione della malattia sono altamente riproducibili in modelli ortotopici e si verificano nel corso di un breve lasso di tempo (settimane), che li rende adatti per test pre-clinici di nuove terapie. Questo è in contrasto con i modelli transgenici in cui l&#39;esordio della malattia si verifica nel corso di un telaio più lungo e variabile tempo (mesi a 1 anno). Se utilizzato con 10 linee di cellule più aggressive, modelli ortotopico di cancro del pancreas hanno modelli di metastasi spontanee simili a quelle viste in pazienti. 8 Espressione di geni reporter bioluminescenti come la lucciola luciferasi facilita il monitoraggio longitudinale della crescita tumorale, la diffusione metastatica, la recidiva e la risposta alle terapie. 6,11 Qui si descrive un modello ortotopico di cancro al pancreas che utilizza Matrigel per la consegna cella localizzata e di imaging bioluminescenza in vivo per il monitoraggio non invasivo della progressione tumorale. Questo modello ortotopico di cancro al pancreas consente analisi non invasive di progressione della malattia e la risposta agli interventi terapeutici in modelli singenici o xenotrapianto.

<table border="1">
 <tbody>
 <tr>
 <td>Equipment</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Clean Bench coat</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Heating pad</td>
 <td></td>
 <td></td>
 <td>Set to 37 &deg;C </td>
 </tr>
 <tr>
 <td>Ivis Lumina ll Bioluminescent imager</td>
 <td>Caliper </td>
 <td></td>
 <td>Alternative bioluminescent imaging systems include In vivo F PRO (Carestream) and Photon Imager (Biospace Lab)</td>
 </tr>
 <tr>
 <td>Dissecting scissors</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Iris forceps (serrated)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Needle holder</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>27 G 0.3 ml insulin syringe</td>
 <td>Terumo</td>
 <td>T35525M2913</td>
 <td></td>
 </tr>
 </tbody>
</table>

bioluminescent orthotopic model of pancreatic cancer progression

<p class="jove_content"&gt; Il tumore al pancreas è estremamente povero tasso di sopravvivenza a cinque anni del 4-6%. Nuove opzioni terapeutiche sono criticamente necessari e dipendono da una migliore comprensione della biologia del cancro al pancreas. Per meglio comprendere l&#39;interazione delle cellule tumorali con il microambiente pancreatico, dimostriamo un modello ortotopico di cancro pancreatico che consente il monitoraggio non invasivo della progressione del cancro. Cellule tumorali pancreatiche luciferasi-tag sono risospesi in Matrigel e consegnati nella coda del pancreas durante la laparotomia. Matrigel solidifica a temperatura corporea per evitare perdite di cellule tumorali durante l&#39;iniezione. La crescita del tumore primario e metastasi in organi distanti sono monitorati dopo l&#39;iniezione del substrato luciferina luciferasi, utilizzando<em&gt; In vivo</em&gt; Imaging bioluminescenza emissione dalle cellule tumorali.<em&gt; In vivo</em&gt; Immagini può anche essere utilizzato per monitorare recidiva del tumore primario dopo la resezione. Questo modello ortotopico è adatto per entrambi i modelli singenici xenotrapianto e può essere utilizzato in studi pre-clinici per studiare l&#39;impatto di nuove terapie anti-tumorali sulla crescita del tumore pancreatico primario e delle metastasi.</p&gt;

Il metodo descrive un modello ortotopico di cancro al pancreas con intervento chirurgico, compresa l&#39;induzione dell&#39;anestesia, laparotomia, l&#39;iniezione di cellule tumorali in chiusura Matrigel e addominale (Figura 1A). Le cellule iniettate formano una bolla nella superficie del pancreas (Figura 1B). La progressione del cancro del pancreas può essere non invasivo monitorati utilizzando l&#39;imaging bioluminescenza in vivo per monitorare la proliferazione delle cell...

Watch this Scientific Journal Video about Bioluminescent Orthotopic Model of Pancreatic Cancer Progression at JoVE.com

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Migliore comprensione della biologia del cancro pancreatico è criticamente necessaria per consentire lo sviluppo di migliori opzioni terapeutiche per il trattamento di cancro al pancreas. Per rispondere a questa esigenza, abbiamo dimostrato un modello ortotopico di cancro al pancreas, che consente il monitoraggio non invasivo di progressione del cancro usando<em&gt; In vivo</em&gt; L&#39;imaging bioluminescenza.

Bioluminescente modello ortotopico di cancro al pancreas Progressione

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

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26.7K Views.  Monash University. Migliore comprensione della biologia del cancro pancreatico è criticamente necessaria per consentire lo sviluppo di migliori opzioni terapeutiche per il trattamento di cancro al pancreas. Per rispondere a questa esigenza, abbiamo dimostrato un modello ortotopico di cancro al pancreas, che consente il monitoraggio non invasivo di progressione del cancro usando In vivo L'imaging bioluminescenza.

Video: Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

Bioluminescent modello murino epatica Tumori

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

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Medicina

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

Un modello ortotopico di cancro alla vescica Murine

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-fotone Imaging di invasione delle cellule tumorali in un modello di mouse ortotopico di carcinoma orale a cellule squamose

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

In vivo doppio substrato Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

La radioterapia combinata in un modello di tumore ortotopico cervello di topo

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

Un modello ortotopico tumore della vescica e la valutazione del trattamento intravescicale sarna

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Procedure chirurgiche per un modello di Rat parziale trapianto di fegato con la ricostruzione arteriosa epatica

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

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

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

Un ortotopico cancro della vescica Modello di Gene Studi di consegna

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

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.