Este trabalho foi financiado pelo National Health and Medical Research Council, na Austrália (1008865), o Australian Research Council (LE110100125), o Instituto Nacional do Câncer (CA138687-01), Erica Sloan é apoiado por uma bolsa de carreira precoce do cancro da mama Nacional Fundação, na Austrália. Corina Kim-Fuchs é apoiado por uma bolsa da Liga Cancer suíço e uma bolsa HDR a partir de Monash Instituto de Ciências Farmacêuticas. Eliane Angst é apoiado por uma bolsa da Bern Cancer League.

O protocolo que está sendo demonstrado é realizada sob a orientação e aprovação de cuidados com os animais da instituição do autor e comitê uso. Todos os experimentos são executados em conformidade com todas as diretrizes, regulamentos e agências reguladoras. 1. Transdução pancreáticas linhas celulares de cancro <ol><li> Transduzir as células de cancro do pâncreas para expressar luciferase, tal como descrito anteriormente. 12,13 Panc-1 e Capan-1 linhas de células de cancro pancreático transduzidas com luciferase do pirilampo são usados ​​aqui. </li></ol> Nota: a luciferase de Renilla, ou luciferase bacteriana podem também ser usados. 2. Pancreatic Cancer celular Preparação <ol><li> Cultura transduzidas células de câncer pancreático até 70% confluentes. </li><li> Levante as células pancreáticas e assegurar a viabilidade é superior a 90%. </li><li> Ressuspender a 2 x 10 7 células / ml numa mistura 03:02 de Matrigel refrigerados sal fosfato tamponadoine (PBS). </li><li> Manter a suspensão de células de Matrigel em gelo antes da injecção no pâncreas. </li></ol> Notas: Para assegurar uma rápida solidificação de Matrigel, reduzir o volume de PBS para ter em conta o volume do sedimento celular. Pega Matrigel utilizando instrumentos gelados e seringas em todos os momentos para evitar a solidificação, antes da injecção. O número de células é sugerido um guia e deve ser determinada empiricamente para cada linha celular. 3. Rato Preparação <ol><li> Anestesiar o mouse usando inalado 2-3% de isoflurano. Determinar a profundidade da anestesia por falta de pedal reflexo de uma pitada dedo gentil. </li><li> Aplique lubrificante para os olhos para evitar a dessecação. </li><li> Posicione o mouse sobre a sua volta em uma almofada de aquecimento ° C 37 e vire suavemente o mouse para levantar o lado esquerdo do abdômen. </li><li> Preparar o abdómen com uma solução a 10% de iodo povidona. </li></ol> Notas: Injectable anestesia pode ser usado em vez da anestesia por inalação. Jejum pré-operatório, não é necessário. 4. Laparotomia <ol><li> Usando instrumentos cirúrgicos estéreis fazer uma incisão de 1,5 cm na pele de aproximadamente 1 cm lateral esquerdo da linha média. </li><li> Faça uma incisão de 1,5 cm no músculo abdominal subjacente. </li><li> Mostrar o baço utilizando a pinça e retirar cuidadosamente o baço a partir da cavidade abdominal. Fixe o baço ao longo de um cotonete estéril para expor o pâncreas subjacente. </li><li> Localizar a cauda do pâncreas adjacente ao baço. </li><li> Utilizando uma seringa de 0,3 ml de L insulina 29, injecta-se 20 ul da suspensão de células no pâncreas Matrigel. </li><li> Após a injecção, segure a seringa no pâncreas por 30-60 segundos até que o Matrigel solidificou. Este passo importante minimiza fugas célula. </li><li> Inspecione o local da injeção para garantir que não haja vazamento ocorreu. </li><li> Devolver o baço e pâncreas, na cavidade abdominal. </li></ol> Nota: Tome cuidado para evitar a perfuração do lado dorsal do pâncreas que pode ser fino. 5. Fechamento da parede abdominal <ol><li> Feche a musculatura abdominal do rato com uma sutura absorvível 4-0 trançada com uma agulha rodada meio de sutura contínua. </li><li> Feche a pele externa com um monofilamento não absorvível 6-0 com agulha cortante meio de sutura contínua. </li><li> Remova o mouse do anestésico inalatório e injetado 0,05-0,1 mg / kg de buprenorfina subcutânea. </li><li> Permitir que o rato se recuperar a sua gaiola colocada sobre uma almofada de aquecimento ° C, 37 º C com acesso livre a comida e água. Se os ratos demonstrar sinais de dor como hunching ou com mobilidade reduzida, a buprenorfina pode ser dada a cada 12 horas durante um período de 36 horas. </li><li> Após a cicatrização de feridas (7-10 dias), anestesiar o mouse e remover as suturas externas. </li></ol> 6. Rastreamento bioluminescente do Canc pâncreaser Progressão <ol><li> Anestesiar o mouse usando isoflurano inalado. </li><li> Injectar 150 mg / kg de D-luciferina, através da veia da cauda. </li><li> Posicione o mouse no sistema de imagem de bioluminescência e capturar imagens de luz branca e bioluminescência como descrito anteriormente. 14,15 </li><li> Remova o mouse do anestésico inalatório e permitir que ele se recuperar em sua gaiola. </li></ol> Nota: a imagem de bioluminescência é não-invasiva e pode ser realizada periodicamente para investigar a cinética de crescimento do tumor. Para tumor imagem na cauda do pâncreas é importante colocar o mouse sobre o seu lado esquerdo, de modo que os pontos do tumor para a câmera. Nós fotografada uma vez por semana com a freqüência aumentada até três vezes por semana, antes do ponto final experimental utilizando um sistema de imagem II Lumina (Perkin-Elmer, ex-Caliper Life Sciences) rodando software 4.3.1 Imagem de estar com binning 4, FOV 12.5, F-stop 1, exposição de 1 - 60 seg (determinada pela maior exposure sem saturação de pixels).

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Os autores declaram que não têm interesses financeiros concorrentes.

Aqui nós descrevemos um modelo ortotópico de avaliação longitudinal do desenvolvimento do tumor de pâncreas e progressão. Cinética de crescimento do tumor primário são reprodutíveis (Figura 3) e podem ser não-invasiva monitorizada utilizando imagem de bioluminescência de células marcadas com luciferase, por exemplo, para análises da resposta do tumor a novas terapêuticas anti-cancro do pâncreas. Consistente com a doença humana, o modelo mostra invasão pancreático local <stron...

O câncer de pâncreas é a quarta principal causa de morte relacionada ao câncer, com uma taxa de sobrevida em 5 anos de 4-6%. 1,2 Apenas 15% dos pacientes são diagnosticados logo no início da doença para ser elegível para a cirurgia, e os tumores se repetir em&gt; 80% dos pacientes. 3,4 gemcitabina é utilizada para o tratamento do adenocarcinoma pancreático, no entanto, quimiorresistência é comum e muitas vezes a droga tem pouco impacto na sobrevivência global. cinco novas estratégias farmacológicas para o tratamento de cancro pancreático são urgentemente necessárias. Seu desenvolvimento depende melhorou significativamente a compreensão das principais etapas de progressão da doença que podem ser sensíveis à intervenção terapêutica. Modelos ortotópico de câncer de pâncreas emular os principais aspectos da doença humana, tornando-as ferramentas ideais para o estudo da biologia do câncer de pâncreas. 6-9 Em contraste com os ensaios in vitro com células de comportamento de células de câncer de pâncreas umad subcutânea modelos in vivo de câncer de pâncreas, os modelos ortotópico permitir a investigação das interações de células tumorais com o microambiente pancreático. A cinética de progressão da doença são altamente reprodutível em modelos ortotópico e ocorrem ao longo de um curto espaço de tempo (semanas), o que os torna adequados para testes pré-clínicos de novas terapias. Isto está em contraste com os modelos transgénicos, onde o início da doença ocorre ao longo de um período mais e mais variáveis ​​de tempo (meses a 1 ano). 10 Quando utilizado com linhas de células mais agressivas, os modelos ortotópicos de cancro do pâncreas têm padrões de metástases espontâneas semelhantes aos observados em pacientes. 8 Expressão de genes repórter bioluminescentes como vaga-lume luciferase facilita o acompanhamento longitudinal do crescimento tumoral, disseminação metastática, recorrência e resposta à terapêutica 6,11. Aqui nós descrevemos um modelo ortotópico de câncer pancreático que utiliza Matrigel para entrega localizada de células e em imagiologia in vivo de bioluminescência para monitorização não invasiva da progressão do tumor. Este modelo ortotópico de câncer pancreático permite análises não invasivas de progressão da doença e resposta a intervenções terapêuticas em modelos singênicos ou heteróloga.

<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; O câncer de pâncreas tem uma taxa de sobrevivência de cinco anos extremamente pobre de 4-6%. Novas opções terapêuticas são urgentemente necessárias e dependem de uma melhor compreensão da biologia do câncer de pâncreas. Para entender melhor a interação das células cancerosas com o microambiente pancreático, demonstramos um modelo ortotópico de câncer pancreático que permite o monitoramento não-invasivo de progressão do cancro. Células de câncer pancreático luciferase marcadas são ressuspendidas em Matrigel e entregue na cauda do pâncreas durante a laparotomia. Matrigel solidifica à temperatura do corpo para evitar a fuga das células cancerosas durante a injecção. O crescimento do tumor primário e as metástases para órgãos distantes são monitorizados após a injecção do substrato de luciferase luciferina, usando<em&gt; In vivo</em&gt; Imagiologia de emissão bioluminescência das células cancerosas.<em&gt; In vivo</em&gt; Imagem também pode ser usado para controlar a recorrência após a ressecção do tumor primário. Este modelo ortotópico é adequada tanto para os modelos de xenoenxerto singeneicos e que pode ser utilizado em ensaios pré-clínicos para investigar o impacto das novas terapias anti-cancro no crescimento do tumor pancreático primário e metástases.</p&gt;

Este método descreve um modelo ortotópico de câncer de pâncreas através de procedimentos cirúrgicos, incluindo a indução da anestesia, laparotomia, a injeção de células cancerosas no fechamento Matrigel e abdominal (Figura 1A). As células injectadas formar uma bolha na superfície do pâncreas (Figura 1B). A progressão do cancro do pâncreas podem ser monitorizados de forma não invasiva através de imagiologia in vivo bioluminescência para controlar a pr...

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

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Uma melhor compreensão da biologia do câncer de pâncreas é criticamente necessário para permitir o desenvolvimento de melhores opções terapêuticas para o tratamento de câncer no pâncreas. Para atender a essa necessidade, nós demonstramos um modelo ortotópico de câncer pancreático que permite o monitoramento não-invasivo de progressão do câncer usando<em&gt; In vivo</em&gt; Imagem de bioluminescência.

Modelo ortotópico bioluminescente de progressão do câncer de pâncreas

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

bioluminescent-orthotopic-model-of-pancreatic-cancer-progression

Research

JoVE Journal

Medicine

26.7K visualizações.  Monash University. Uma melhor compreensão da biologia do câncer de pâncreas é criticamente necessário para permitir o desenvolvimento de melhores opções terapêuticas para o tratamento de câncer no pâncreas. Para atender a essa necessidade, nós demonstramos um modelo ortotópico de câncer pancreático que permite o monitoramento não-invasivo de progressão do câncer usando In vivo Imagem de bioluminescência.

Vídeo: 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.

Modelo de tumor hepático murino Bioluminescent

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

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.

Um modelo ortotópico de câncer de bexiga murino

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-fotão imagem de invasão das células tumorais em um modelo de camundongo ortotópico de carcinoma epidermóide oral

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 dupla Substrato Bioluminescent imagem

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.

Radioterapia em uma combinação ortotópico modelo de tumor de cérebro de camundongos

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.

Um modelo de tumor de bexiga ortotópico e Avaliação de Tratamento de sarna intravesical

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.

Procedimentos cirúrgicos para um modelo de rato de Transplante de Fígado parcial ortotópico com reconstrução arterial hepática

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.

Um modelo de cancro da bexiga para Ortotópico Gene Estudos de entrega

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.

Um Modelo murino de próstata humana Metástase Ortotópico

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.